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Credentials

Education ?

Medical School Score Rankings
The University of Texas Southwestern (1998)
Dermatology - Dermatopathology
  •  
Top 25%

Awards & Distinctions ?

Awards  
The Association of Pathology Informatics and CAP Foundation presented the Spring 2003 Informatics Award to Dr. Davie

Publications & Research

Dr. Davie has contributed to 174 publications.
Title Gene Expression Regulation Through 14-3-3 Interactions with Histones and Hdacs.
Date September 2011
Journal Discovery Medicine
Excerpt

14-3-3s are phosphoserine- or phosphothreonine-binding proteins, which are involved in a variety of cellular processes, including gene regulation, differentiation, cell cycle progression, and metabolism. Their mechanism of regulation is typically to induce a conformational change on the target phosphoprotein, which can alter its catalytic activity, localization within the cell, or interactions with other proteins. In addition, 14-3-3s can act as a scaffolding protein, leading to multiprotein complex formation on the target phosphoprotein. As such, 14-3-3s are integrated into a number of important phosphorylation-dependent signaling pathways. In this review, we will examine the interaction of 14-3-3 with chromatin and chromatin modifying enzymes, specifically with phosphorylated histone H3 and histone deacetyltransferases, respectively. Through diverse mechanisms, these interactions directly affect the expression of target genes, many of which are known oncogenes and lead to tumorigenesis and metastasis. Various research studies have indicated that the mechanism of 14-3-3 interaction and its role in transcription is complex and diverse, and likely involving additional components as yet undefined.

Title Selective Association of Peroxiredoxin 1 with Genomic Dna and Cox-2 Upstream Promoter Elements in Estrogen Receptor Negative Breast Cancer Cells.
Date March 2011
Journal Molecular Biology of the Cell
Excerpt

In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen receptor negative (ER-) MDA-MB-231 but not in estrogen receptor positive (ER+) MCF7 breast cancer cells. Indirect immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that PRDX1 association with the transcription factor nuclear factor-kappaB (NF-kappaB) in MDA-MB-231 but not in MCF7 cells contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-kappaB in ER- but not in ER+ breast cancer cells. PRDX1 knockdown attenuated COX-2 expression by reducing NF-kappaB occupancy at its upstream promoter element in MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER- breast cancer cells. Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-kappaB in ER- breast cancer cells.

Title The Role of Sp1 and Sp3 in Normal and Cancer Cell Biology.
Date October 2010
Journal Annals of Anatomy = Anatomischer Anzeiger : Official Organ of the Anatomische Gesellschaft
Excerpt

Sp1 and Sp3 are transcription factors expressed in all mammalian cells. These factors are involved in regulating the transcriptional activity of genes implicated in most cellular processes. Dysregulation of Sp1 and Sp3 is observed in many cancers and diseases. Due to the amino acid sequence similarity of the DNA binding domains, Sp1 and Sp3 recognize and associate with the same DNA element with similar affinity. However, others and our laboratory demonstrated that these two factors possess different properties and exert different functional roles. Both Sp1 and Sp3 can interact with and recruit a large number of proteins including the transcription initiation complex, histone modifying enzymes and chromatin remodeling complexes, which strongly suggest that Sp1 and Sp3 are important transcription factors in the remodeling chromatin and the regulation of gene expression. In this review, the role of Sp1 and Sp3 in normal and cancer cell biology and the multiple mechanisms deciding the functional roles of Sp1 and Sp3 will be presented.

Title Nucleosomal Response, Immediate-early Gene Expression and Cell Transformation.
Date August 2010
Journal Advances in Enzyme Regulation
Title Biotin is Not a Natural Histone Modification.
Date March 2010
Journal Biochimica Et Biophysica Acta
Excerpt

In addition to its role as the cofactor of biotin-dependent carboxylases, biotin has been demonstrated to have a role in cellular processes including transcription and gene silencing. Histones have been proposed to be modified by biotin in a site-specific manner, providing a pathway by which biotin acts as a regulatory molecule for gene expression. However, there is uncertainty whether biotin attachment to histones in vitro can be extrapolated to biotin as a native histone modification. We critically examined a number of methods used to detect biotin attachment on histones, including [(3)H]-biotin uptake, Western blot analysis of histones, and mass spectrometry of affinity purified histone fragments with the objective of determining if the in vivo occurrence of histone biotinylation could be conclusively established. We found for each of these methods that, while biotin could be readily detected on native carboxylases or histones biotinylated in vitro, biotin attachment on native histones could not be detected in cell cultures from various sources. We conclude that biotin is absent in native histones to a sensitivity of at least one part per 100,000, suggesting that the regulatory impact of biotin on gene expression must be through alternate mechanisms.

Title Carcinoma Ex Spiradenoma/cylindroma Confirmed by Immunohistochemical and Molecular Loss-of-heterozygosity Profiling.
Date February 2010
Journal The American Journal of Dermatopathology
Excerpt

We present a case of spiradenoma/cylindroma with admixed carcinoma of unknown origin, resolved using immunohistochemical and molecular loss-of-heterozygosity (LOH) profiling. The patient, a woman in her mid-70s, initially presented with separate mammary (ductal) carcinomas of the right and left breasts that were treated with radical mastectomies. For 9 years, the patient remained disease free until complaining of a slow-growing skin nodule on the lower back that was excised under clinical suspicion of metastatic mammary carcinoma. Histopathological exam revealed a benign eccrine spiradenoma/cylindroma and an intermixed carcinoma, with a differential diagnosis of either primary eccrine carcinoma or mammary carcinoma metastatic to the spiradenoma/cylindroma. Histological features and immunohistochemical staining favored eccrine carcinoma but not unequivocally; therefore, LOH profiles were performed on archival paraffin block tissue from the 3 neoplastic lesions (4 components). The mammary carcinomas showed disparate LOH at 5 of 7 (right breast) and 4 of 7 (left breast) informative genetic loci, establishing these carcinomas as separate primary neoplasms. Both the spiradenoma/cylindroma and eccrine carcinoma revealed no LOH at the tested loci, establishing the unknown carcinoma as an independent carcinoma arising within a spiradenoma/cylindroma. This neoplasm is referred to in the literature as carcinoma ex spiradenoma/cylindroma and spiradenocylindrocarcinoma.

Title H3 Phosphorylation: Dual Role in Mitosis and Interphase.
Date January 2010
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

Chromatin condensation and subsequent decondensation are processes required for proper execution of various cellular events. During mitosis, chromatin compaction is at its highest, whereas relaxation of chromatin is necessary for DNA replication, repair, recombination, and gene transcription. Since histone proteins are directly complexed with DNA in the form of a nucleosome, great emphasis is put on deciphering histone post-translational modifications that control the chromatin condensation state. Histone H3 phosphorylation is a mark present in mitosis, where chromatin condensation is necessary, and in transcriptional activation of genes, when chromatin needs to be decondensed. There are four characterized phospho residues within the H3 N-terminal tail during mitosis: Thr3, Ser10, Thr11, and Ser28. Interestingly, H3 phosphorylated at Ser10, Thr11, and Ser28 has been observed on genomic regions of transcriptionally active genes. Therefore, H3 phosphorylation is involved in processes requiring opposing chromatin states. The level of H3 phosphorylation is mediated by opposing actions of specific kinases and phosphatases during mitosis and gene transcription. The cellular contexts under which specific residues on H3 are phosphorylated in mitosis and interphase are known to some extent. However, the functional consequences of H3 phosphorylation are still unclear.

Title Epigenetic Control.
Date March 2009
Journal Journal of Cellular Physiology
Excerpt

Epigenetics refers to mitotically and/or meiotically heritable variations in gene expression that are not caused by changes in DNA sequence. Epigenetic mechanisms regulate all biological processes from conception to death, including genome reprogramming during early embryogenesis and gametogenesis, cell differentiation and maintenance of a committed lineage. Key epigenetic players are DNA methylation and histone post-translational modifications, which interplay with each other, with regulatory proteins and with non-coding RNAs, to remodel chromatin into domains such as euchromatin, constitutive or facultative heterochromatin and to achieve nuclear compartmentalization. Besides epigenetic mechanisms such as imprinting, chromosome X inactivation or mitotic bookmarking which establish heritable states, other rapid and transient mechanisms, such as histone H3 phosphorylation, allow cells to respond and adapt to environmental stimuli. However, these epigenetic marks can also have long-term effects, for example in learning and memory formation or in cancer. Erroneous epigenetic marks are responsible for a whole gamut of diseases including diseases evident at birth or infancy or diseases becoming symptomatic later in life. Moreover, although epigenetic marks are deposited early in development, adaptations occurring through life can lead to diseases and cancer. With epigenetic marks being reversible, research has started to focus on epigenetic therapy which has had encouraging success. As we witness an explosion of knowledge in the field of epigenetics, we are forced to revisit our dogma. For example, recent studies challenge the idea that DNA methylation is irreversible. Further, research on Rett syndrome has revealed an unforeseen role for methyl-CpG-binding protein 2 (MeCP2) in neurons.

Title Genomic Instability and Histone H3 Phosphorylation Induction by the Ras-mitogen Activated Protein Kinase Pathway in Pancreatic Cancer Cells.
Date December 2008
Journal International Journal of Cancer. Journal International Du Cancer
Excerpt

Activating mutations in K-Ras occur in most pancreatic cancers. We investigated whether genetic changes (K-Ras mutations) in human pancreatic cancer cell lines altered genomic instability and epigenetic events responding to Ras-mitogen activated protein kinase (MAPK) signaling by characterizing 3 human pancreatic cancer cells lines with and without activating mutations in K-Ras. Activation of the Ras-MAPK pathway results in the stimulation of the histone H3 kinase, mitogen and stress activated kinase (MSK) 1, and increased phosphorylation of histone H3 at serine 10 (H3 S10ph). MSK1 and H3 S10ph have roles in neoplastic transformation. We demonstrate that the presence of a K-Ras mutation did not correlate with elevated chromosomal aberrations or increased genomic instability. Although the levels of the epidermal growth factor receptors and MSK were similar, the Ras-MAPK pathway was differentially induced by phorbol esters (12-O tetradecanoylphorbol-13-acetate) or epidermal growth factor, with the response of this signaling pathway being cell-type specific. This response corresponded downstream at the level of chromatin where stimuli-induced elevation of H3 S10ph typically paralleled the increase in phospho-extracellular signal regulated kinase 1/2. Our results present evidence that nonclonal chromosomal aberrations and epigenetic programming responding to stimulation of the Ras-MAPK pathway may be better markers for cancer progression than the upstream mutated oncogenes.

Title Mitotic Partitioning of Transcription Factors.
Date October 2008
Journal Journal of Cellular Biochemistry
Excerpt

Mitosis is a highly orchestrated process involving numerous protein kinases and phosphatases. At the onset of mitosis, the chromatin condensation into metaphase chromosomes is correlated with global phosphorylation of histone H3. The bulk of transcription is silenced while many of the transcription-associated proteins, including transcription and chromatin remodeling factors, are excluded from chromatin, typically as a consequence of their phosphorylation. Components of the transcription machinery and regulatory proteins are recycled and equally partitioned between newly divided cells by mechanisms that may involve microtubules, microfilaments or intermediate filaments. However, as demonstrated in the case of Runx2, a subset of transcription factors involved in lineage-specific control, likely remain associated with their target genes to direct the deposition or removal of epigenetic marks. The displacement and re-entry into daughter cells of transcription and chromatin remodeling factors are temporally defined and regulated. Reformation of daughter nuclei is a critical time to re-establish the proper gene expression pattern. The mechanisms involved in the marking and re-establishment of gene expression has been elucidated for few genes. The elucidation of how the memory of a programmed expression profile is transmitted to daughter cells represents a challenge.

Title Chromatin Organization and Nuclear Microenvironments in Cancer Cells.
Date October 2008
Journal Journal of Cellular Biochemistry
Excerpt

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. However, our knowledge of events resulting in changes in nuclear shape and chromatin organization in cancer cells is limited. Nuclear matrix proteins, which include lamins, transcription factors (Sp1) and histone modifying enzymes (histone deacetylases), and histone modifications (histone H3 phosphorylation) have roles in organizing chromatin in the interphase nucleus, regulating gene expression programs and determining nuclear shape. Histone H3 phosphorylation, a downstream target of the Ras-mitogen activated protein kinase pathway, is involved in neoplastic transformation. This article will review genetic and epigenetic events that alter chromatin organization in cancer cells and the role of the nuclear matrix in determining nuclear morphology.

Title Nuclear Microenvironments and Cancer.
Date October 2008
Journal Journal of Cellular Biochemistry
Excerpt

Nucleic acids and regulatory proteins are architecturally organized in nuclear microenvironments. The compartmentalization of regulatory machinery for gene expression, replication and repair, is obligatory for fidelity of biological control. Perturbations in the organization, assembly and integration of regulatory machinery have been functionally linked to the onset and progression of tumorigenesis. The combined application of cellular, molecular, biochemical and in vivo genetic approaches, together with structural biology, genomics, proteomics and bioinformatics, will likely lead to new approaches in cancer diagnostics and therapy.

Title Effects of the in Vivo Supply of Butyrate on Histone Acetylation of Cecum in Piglets.
Date March 2008
Journal Jpen. Journal of Parenteral and Enteral Nutrition
Excerpt

BACKGROUND: In vitro, butyrate inhibits histone deacetylase and down-regulates expression of cyclin D1. We hypothesized that an increased entry rate of butyrate into the cecal lumen would have similar effects in vivo. METHODS: We used frozen cecal tissue and data from previous studies, one showing that lactulose supplementation caused an increased rate of cecal synthesis of butyrate and decreased cecal cell proliferation and density of clostridia and the other showing that cecal cell proliferation was increased by an exogenous cecal butyrate infusion at a comparable rate. The ratio of acetylated to total histones (AH ratio) and cyclin D1 mRNA expression were measured in cecal tissue. RESULTS: Lactulose supplementation caused a 189% increase in the AH ratio (p = .004), which inversely correlated with cecal cell proliferation (r = -0.782; p = .008). With cecal butyrate infusion, we observed a significant decrease in histone acetylation (p = .02), which also inversely correlated with cecal cell proliferation (r = -0.797; p = .002). Cyclin D1 expression was increased 6.5-fold by lactulose feeding (p = .02) but decreased 50% with cecal butyrate infusion (p = .004). CONCLUSIONS: The effects on histone acetylation of increased "endogenous" butyrate production produced by lactulose feeding, but not exogenous cecal infusion of butyrate, mirror those in vitro. Thus, bacterial production and exogenous infusion of butyrate have opposite effects on histone acetylation and cyclin D1 expression, suggesting that the composition of bacterial flora may play a role in butyrate's in vivo effects on the cell cycle.

Title Competitive Inhibition of Histone Deacetylase Activity by Trichostatin A and Butyrate.
Date March 2008
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

Histone deacetylases (HDACs) play a pivotal role in gene expression through their involvement in chromatin remodeling. The abnormal targeting or retention of HDACs to DNA regulatory regions is observed in many cancers, and hence HDAC inhibitors are being tested as promising anti-tumor agents. The results of previous kinetic studies, characterizing trichostatin A (TSA), as well as butyrate, as HDAC noncompetitive inhibitors, conflict with crystallographic and homology modeling data suggesting that TSA should act as a competitive inhibitor. Our results demonstrate that each of the HDAC inhibitors TSA and butyrate inhibits HDAC activity in a competitive fashion. Co-immunoprecipitation studies show that the inhibition of HDAC1 and HDAC2 activity by TSA does not disturb the extensive level of their association in the human breast cancer cell line MCF-7. Moreover, the inhibition of HDAC activity by TSA does not interfere with the interaction of HDAC1 and HDAC2 with Sin3A, a core component of the Sin3 complex. Thus, repressor complexes such as Sin3, appear to be stable in the presence of TSA. The association of HDAC2 with transcription factor Sp1 is also not affected by TSA.

Title An Integrated Analysis of Genes and Pathways Exhibiting Metabolic Differences Between Estrogen Receptor Positive Breast Cancer Cells.
Date February 2008
Journal Bmc Cancer
Excerpt

BACKGROUND: The sex hormone estrogen (E2) is pivotal to normal mammary gland growth and differentiation and in breast carcinogenesis. In this in silico study, we examined metabolic differences between ER(+)ve breast cancer cells during E2 deprivation. METHODS: Public repositories of SAGE and MA gene expression data generated from E2 deprived ER(+)ve breast cancer cell lines, MCF-7 and ZR75-1 were compared with normal breast tissue. We analyzed gene ontology (GO), enrichment, clustering, chromosome localization, and pathway profiles and performed multiple comparisons with cell lines and tumors with different ER status. RESULTS: In all GO terms, biological process (BP), molecular function (MF), and cellular component (CC), MCF-7 had higher gene utilization than ZR75-1. Various analyses showed a down-regulated immune function, an up-regulated protein (ZR75-1) and glucose metabolism (MCF-7). A greater percentage of 77 common genes localized to the q arm of all chromosomes, but in ZR75-1 chromosomes 11, 16, and 19 harbored more overexpressed genes. Despite differences in gene utilization (electron transport, proteasome, glycolysis/gluconeogenesis) and expression (ribosome) in both cells, there was an overall similarity of ZR75-1 with ER(-)ve cell lines and ER(+)ve/ER(-)ve breast tumors. CONCLUSION: This study demonstrates integral metabolic differences may exist within the same cell subtype (luminal A) in representative ER(+)ve cell line models. Selectivity of gene and pathway usage for strategies such as energy requirement minimization, sugar utilization by ZR75-1 contrasted with MCF-7 cells, expressing genes whose protein products require ATP utilization. Such characteristics may impart aggressiveness to ZR75-1 and may be prognostic determinants of ER(+)ve breast tumors.

Title Phosphorylated Serine 28 of Histone H3 is Associated with Destabilized Nucleosomes in Transcribed Chromatin.
Date December 2007
Journal Nucleic Acids Research
Excerpt

Histone modifications and variants have key roles in the activation and silencing of genes. Phosphorylation of histone H3 at serine 10 and serine 28 is involved in transcriptional activation of genes responding to stress or mitogen-stimulated signaling pathways. The distribution of H3-modified isoforms in G0 phase chicken erythrocyte chromatin was investigated. H3 phosphorylated at serine 28 was found highly enriched in the active/competent gene fractions, as was H3 di- and trimethylated at lysine 4. The H3 variant H3.3 in this chromatin fraction was preferentially phosphorylated at serine 28. Conversely, H3 phosphorylated at serine 10 was present in all chromatin fractions, while H3 dimethylated at lysine 9 was associated with the chromatin-containing repressed genes. H3 phosphorylated at serine 28 was located at the promoter region of the transcriptionally active, but not competent, histone H5 and beta-globin genes. We provide evidence that H3.3 phosphorylated at serine 28 was present in labile nucleosomes. We propose that destabilized nucleosomes containing H3.3 phosphorylated at serine 28 aid in the dynamic disassembly-assembly of nucleosomes in active promoters.

Title Differential Distribution of Unmodified and Phosphorylated Histone Deacetylase 2 in Chromatin.
Date December 2007
Journal The Journal of Biological Chemistry
Excerpt

Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, HDAC2 is found in the Sin3 and NuRD multiprotein complexes, which are recruited to promoters by DNA-binding proteins. In this study, we show that the majority of HDAC2 in human breast cancer cells is not phosphorylated. However, the minor population of HDAC2, preferentially cross-linked to DNA by cisplatin, is mono-, di-, or tri-phosphorylated. Furthermore, HDAC2 phosphorylation is required for formation of Sin3 and NuRD complexes and recruitment to promoters by transcription factors including p53, Rb, YY1, NF-kappaB, Sp1, and Sp3. Unmodified HDAC2 requires linker DNA to associate with chromatin but is not cross-linked to DNA by formaldehyde. We provide evidence that unmodified HDAC2 is associated with the coding region of transcribed genes, whereas phosphorylated HDAC2 is primarily recruited to promoters.

Title Suppression of Dpyd Expression in Rko Cells Via Dna Methylation in the Regulatory Region of the Dpyd Promoter: a Potentially Important Epigenetic Mechanism Regulating Dpyd Expression.
Date September 2007
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

Dihydropyrimidine dehydrogenase (DPD) is one of the factors that determine the efficacy and toxicity of 5-fluorouracil. Variations in DPD activity may result from alterations at the transcriptional level of the DPYD gene. Heterogeneity in DPYD expression has been reported, but the molecular mechanisms responsible for this remain unclear. We investigated methylation of the DPYD promoter as a mechanism for transcriptional regulation of DPYD in the RKO colorectal cancer cell line. We demonstrate that the active transcription machinery for DPYD is present in RKO cells, but promoter binding of Sp1, a transactivator of DPYD, was inhibited, which on subsequent examination was shown to be associated with dense promoter methylation. Treatment with 5-aza-2'-deoxycytidine alone or the combination of 5-aza-2'-deoxycytidine and trichostatin A induced demethylation of the promoter and markedly increased the DPYD mRNA level in RKO cells but not in unmethylated WiDr cells. Furthermore, in vitro methylation of the DPYD promoter decreased promoter activity. These data suggest an important role for methylation in DPYD suppression. The transcriptional suppression of DPYD by methylation may be responsible for the increased 5-fluorouracil sensitivity observed in some patients. This may also provide insight into the mechanism underlying the downregulation of DPYD in some colorectal cancers.

Title Estrogen Receptor-beta Regulates Psoriasin (s100a7) in Human Breast Cancer.
Date September 2007
Journal Breast Cancer Research and Treatment
Excerpt

We have previously observed a paradoxical relationship of the psoriasin/S100A7 gene with estrogen response in-vitro in ERalpha positive cells but its association with ERalpha negative status in-vivo raising the possibility that S100A7 might be regulated by ERbeta in breast cancer. Using doxycycline-inducible ERbeta and ERalpha expressing MCF-7 cells the hypothesis that psoriasin/S100A7 is ERbeta regulated was investigated To explore the relationship between psoriasin/S100A7 and ERbeta expression in-vivo, we also assessed a cohort of 233 ERalpha negative breast tumors using tissue microarrays and immunohistochemistry. Psoriasin/S100A7 was increased by 17beta-estradiol (E2) following ERbeta induction, in several clones of ERbeta over-expressing but not in the original MCF-7 cells, nor clones over-expressing ERalpha. The effect of E2 on psoriasin/S100A7 was inhibited by 4-hydroxytamoxifen and ICI 182780 but not with a selective ERalpha antagonist. An ERbeta selective-agonist but not an ERalpha selective-agonist, induced psoriasin/S100A7. This induction still occurred after stable down-regulation of ERalpha using siRNA in ERbeta inducible cells. E2 increased psoriasin/S100A7 mRNA but cycloheximide treatment inhibited this effect. A relationship between ERbeta and psoriasin/S100A7 was observed in the p53 immunohistochemically negative subset of invasive breast tumors in-vivo (r = 0.225, p = 0.046, n = 79). In conclusion we demonstrate that E2 induction of psoriasin/S100A7 can be specifically regulated through ERbeta in-vitro and associated with ERbeta in-vivo. These data support the hypothesis that psoriasin/S100A7 is specifically regulated by ERbeta activity and could be useful to guide future therapies targeting ERbeta in certain phenotypic subsets of breast cancers in-vivo.

Title Potential Role of Estrogen Receptor Alpha (eralpha) Phosphorylated at Serine118 in Human Breast Cancer in Vivo.
Date January 2007
Journal The Journal of Steroid Biochemistry and Molecular Biology
Excerpt

Post-translational modifications of proteins are known to be important in protein activity and ERalpha is known to be phosphorylated at multiple sites within the protein. The exact function of site-specific phosphorylation in ERalpha is unknown, although several hypotheses have been developed using site-directed mutagenesis and cell culture models. Targeting the ERalpha at the level of such post-translational modification pathways would be a new and exciting approach to endocrine therapy in breast cancer, but adequate knowledge is lacking with regard to the relevance of site-specific phosphorylation in ERalpha in human breast cancer in vivo. Recently, antibodies to P-Serine(118)-ERalpha and P-Serine(167)-ERalpha, two major sites of phosphorylation in ERalpha, have become available and some in vivo data are now available to complement studies in cells in culture. However, the in vivo data are somewhat contradictory and limited by the small cohorts used and the lack of standard well-characterized reagents and protocols.

Title Transcriptional Silencing of the Death Gene Bnip3 by Cooperative Action of Nf-kappab and Histone Deacetylase 1 in Ventricular Myocytes.
Date January 2007
Journal Circulation Research
Excerpt

Earlier we identified a survival role for NF-kappaB in ventricular myocytes, however, the underlying mechanism was undefined. In this report we provide new mechanistic evidence that the hypoxia-inducible death factor BNIP3 is transcriptionally silenced by NF-kappaB through a mechanism that involves the cooperative actions of HDAC1. Activation of the NF-kappaB signaling pathway in ventricular myocytes suppressed basal and hypoxia-inducible BNIP3 gene activity. Basal Bnip3 gene expression was increased in cells derived from p65(-/-) deficient mice. The histone deacetylase (HDAC) inhibitor Trichostatin A (TSA 10 nM) suppressed the inhibitory actions of NF-kappaB on Bnip3 gene transcription. Basal and hypoxia- induced Bnip3 transcription was repressed by wild type but not a catalytically inactive mutant of HDAC1. Immunoprecipitation assays verified interaction of HDAC1 with wild type p65 NF-kappaB and mutations of p65 defective for transactivation in ventricular myocytes. Deletion analysis revealed canonical NF-kappaB elements within the Bnip3 promoter to be important for repression of Bnip3 gene expression by HDAC1. Further, the ability of HDAC1 to repress Bnip3 gene transcription was lost in cells derived from p65(-/-) deficient mice but was restored by repletion of p65 NF-kappaB into p65(-/-) cells. Mutations of p65 NF-kappaB defective for DNA binding but not for transactivation abrogated the inhibitory actions of HDAC1 on the Bnip3 gene transcription. Together, our findings provide new mechanistic insight into the cytoprotective actions conferred by NF-kappaB that extend to the active transcriptional repression of the death factor Bnip3 through a mechanism that is mutually dependent on HDAC-1.

Title Estrogen Receptor-alpha Phosphorylated at Ser118 is Present at the Promoters of Estrogen-regulated Genes and is Not Altered Due to Her-2 Overexpression.
Date December 2006
Journal Cancer Research
Excerpt

Detection of estrogen receptor (ER)-alpha phosphorylated at Ser(118) (P-Ser(118)-ER-alpha) may be an indicator of an intact ligand-dependent ER-alpha in breast tumors in vivo and may predict responsiveness to endocrine therapy. The current study addresses whether P-Ser(118)-ER-alpha is functionally involved in ER target gene transcription and if this is modulated by HER-2 overexpression. Using chromatin immunoprecipitation analysis, P-Ser(118)-ER-alpha was found associated with the promoters of several estrogen-regulated genes in MCF-7 breast cancer cells 30 minutes following estrogen treatment. Coactivators AIB1 and p300 were coimmunoprecipitated with P-Ser(118)-ER-alpha following estrogen treatment. The overexpression of HER-2 protein in MCF-7 cells did not affect estrogen induction of phosphorylation of Ser(118) or its presence at the promoters of several estrogen-regulated genes. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) pathway, had no effect on P-Ser(118)-ER-alpha. The lack of effect of HER-2 overexpression on P-Ser(118)-ER-alpha expression in cell models is supported by similar levels of expression of P-Ser(118)-ER-alpha in ER(+)/HER-2-overexpressing and ER(+)/HER-2(-) breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], and IkappaB kinase-alpha (IKK-alpha; BAY-11-7082), we show that IKK-alpha, but not Cdk7, is at least in part involved in estrogen-mediated phosphorylation at Ser(118) in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser(118)-ER-alpha in estrogen-regulated signaling and do not support the hypothesis that resistance of breast tumors to tamoxifen therapy involves ligand independent activation of ER-alpha due to constitutive phosphorylation of Ser(118) by constitutive activation of MAPK pathway.

Title The Role of Sp1 and Sp3 in the Constitutive Dpyd Gene Expression.
Date September 2006
Journal Biochimica Et Biophysica Acta
Excerpt

Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in the 5-fluorouracil (5-FU) catabolic pathway, has been implicated as one of the factors determining the efficacy and toxicity of the anticancer agent 5-FU. Studies have attributed variation in DPD activity partially to alterations at the transcriptional level of DPYD gene. We investigated the transcription factors implicated in the constitutive expression of DPYD by utilizing a 174-bp fragment of the DPYD promoter region in which three consensus Sp protein binding sites (SpA, SpB and SpC) were predicted. The binding of Sp1 and Sp3 transcription factors to this region was detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. By ectopically expressing human Sp1 and Sp3 in Sp-deficient Drosophila S2 cells, we demonstrated that Sp1 is a strong activator, while Sp3 by its own is a weak activator of the DPYD promoter. Moreover, Sp3 may serve as a competitor of Sp1, thus decreasing the Sp1 induced promoter activity. SpA, SpB and SpC sites are all Sp1 inducible. In the full activation of the DPYD promoter in human cell lines, the SpB site is essential; the SpC site works cooperatively with SpB, while SpA has minor promoter activity. These studies provide further insight into the molecular mechanisms underlying the heterogeneity of DPD activity, and may facilitate the efficacy and safety of 5-FU-based chemotherapy.

Title Sp1 and Sp3 Foci Distribution Throughout Mitosis.
Date July 2006
Journal Journal of Cell Science
Excerpt

The mammalian transcription factors Sp1 and Sp3 compete for the same DNA binding sites but play different roles in the regulation of expression of numerous genes. It is known that, in the interphase nucleus, Sp1 and Sp3 are organized into distinct foci. In this study, we show that throughout the mitotic process, while being displaced from the condensed chromosomes and dispersed throughout the cell, Sp1 and Sp3 maintain their separate punctate distributions. In metaphase, both Sp1 and Sp3 foci show a high degree of colocalization with microfilaments, suggesting that F-actin is involved in the organization of Sp1 and Sp3 foci during mitosis. Constant Sp1 and Sp3 levels were observed during mitosis, signifying a recovery of the pre-existing Sp1 and Sp3 population in newly formed nuclei. In late telophase, Sp1 and Sp3 are equally segregated between daughter cells, and their subnuclear organization as distinct foci is restored in a sequential fashion with Sp3 regrouping into the newly formed nuclei prior to Sp1. Both Sp1 and Sp3 return to the nuclei ahead of RNA polymerase II. Our results support a model in which entry of Sp1, Sp3 and RNA polymerase II into the newly formed nuclei is an ordered process.

Title Chromatin Modification of the Trefoil Factor 1 Gene in Human Breast Cancer Cells by the Ras/mitogen-activated Protein Kinase Pathway.
Date July 2006
Journal Cancer Research
Excerpt

Histone H3 phosphorylation is a downstream response to activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. This modification is thought to have a role in chromatin remodeling and in the initiation of gene transcription. In MCF-7 breast cancer cells, we observed that phosphorylated histone H3 (phospho-H3) at Ser(10) but not Ser(28) increased with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) treatment. Although phosphorylated extracellular signal-regulated kinase 1/2 levels in these cells cultured under estradiol deplete and replete conditions displayed no change, a significant induction was observed after TPA treatment. Furthermore, whereas both estradiol and TPA increased trefoil factor 1 (TFF1) mRNA levels in these cells, only TPA-induced and not estradiol-induced TFF1 expression was inhibited by the H3 kinase mitogen and stress activated protein kinase (MSK) inhibitor H89 and MAPK kinase inhibitor UO126, showing the involvement of the Ras/MAPK following TPA induction. Mutation of the activator protein 1 (AP-1) binding site abrogated the TPA-induced transcriptional response of the luciferase reporter gene under the control of the TFF1 promoter, showing the requirement for the AP-1 site. In chromatin immunoprecipitation assays, estradiol treatment resulted in the association of the estrogen receptor-alpha (ERalpha) and acetylated H3 with the TFF1 promoter. The levels of phospho-H3 and MSK1 associated with the TFF1 promoter were moderately increased. In the presence of TPA, whereas ERalpha was not bound to the promoter, a strong association of acetylated and/or phospho-H3, MSK1, and c-Jun was observed. These results show that although both stimuli lead to TFF1 gene activation, estradiol and TPA exert their effects on TFF1 gene expression by different mechanisms.

Title Phosphorylation of Histones by Tissue Transglutaminase.
Date May 2006
Journal The Journal of Biological Chemistry
Excerpt

Tissue transglutaminase 2 (TG2) has recently been shown to have intrinsic serine/threonine kinase activity. Since histones are known to be cross-linked by TG2, we investigated whether histones are also substrates for TG2 kinase activity. TG2 was able to phosphorylate H1, H2A, H2B, H3, and H4 histones in vitro. Using peptide substrates and phosphospecific antibodies we demonstrated that TG2 phosphorylated Ser(10) in H3 and that this phosphorylation was reduced by acetylation, whereas phosphorylation of Ser(10) by TG2 enhanced acetylation. Furthermore we demonstrated that exogenous TG2 phosphorylated H1 and H3 in nucleosome preparations. We examined the abundance of TG2 in DNA-associated proteins from MCF-7 cells treated with phorbol ester (TPA) and 17beta-estradiol (E2). TG2 abundance was significantly reduced in E2-treated cells and enhanced in TPA-treated cells. In summary we have demonstrated that TG2 is able to phosphorylate purified histone proteins, and H3 and H1 in chromatin preparations, and it is associated with chromatin in breast cancer cells. These studies suggest a novel role for TG2 in the regulation of chromatin structure and function.

Title The Ras-mapk Signal Transduction Pathway, Cancer and Chromatin Remodeling.
Date March 2006
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway results in a multitude of events including expression of the immediate-early genes, c-fos and c-myc. Downstream targets of this stimulated pathway are the mitogen- and stress-activated protein kinases (MSK) 1 and 2, which are histone H3 kinases. In chromatin immunoprecipitation assays, it has been shown that the mitogen-induced phosphorylated H3 is associated with the immediate-early genes and that MSK1/2 activity and H3 phosphorylation have roles in chromatin remodeling and transcription of these genes. In oncogene-transformed fibroblasts in which the Ras-MAPK pathway is constitutively active, histone H1 and H3 phosphorylation is increased and the chromatin of these cells has a more relaxed structure than the parental cells. In this review we explore the deregulation of the Ras-MAPK pathway in cancer, with an emphasis on breast cancer. We discuss the features of MSK1 and 2 and the impact of a constitutively activated Ras-MAPK pathway on chromatin remodeling and gene expression.

Title Histone H4-k16 Acetylation Controls Chromatin Structure and Protein Interactions.
Date February 2006
Journal Science (new York, N.y.)
Excerpt

Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.

Title Abnormalities of Chromatin in Tumor Cells.
Date February 2006
Journal Exs
Excerpt

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. Tumorigenesis involves a series of poorly understood morphological changes that lead to the development of hyperplasia, dysplasia, in situ carcinoma, invasive carcinoma, and in many instances finally metastatic carcinoma. Nuclei from different stages of disease progression exhibit changes in shape and the reorganization of chromatin, which appears to correlate with malignancy. Multistep tumorigenesis is a process that results from alterations in the function of DNA. These alterations result from stable genetic changes, including those of tumor suppressor genes, oncogenes and DNA stability genes, and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. DNA methylation and histone modifications are two epigenetic mechanisms that are altered in cancer cells. The impact of genetic (e.g., mutations in Rb and ras family) and epigenetic alterations with a focus on histone modifications on chromatin structure and function in cancer cells are reviewed here.

Title Differential Intranuclear Organization of Transcription Factors Sp1 and Sp3.
Date January 2006
Journal Molecular Biology of the Cell
Excerpt

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that activate or repress the expression of a variety of genes and are thought to compete for the same DNA binding site. We used indirect immunofluorescence microscopy and image deconvolution to show that Sp1 and Sp3 are organized into distinct nonoverlapping domains in human breast and ovarian cells. Domains of Sp1 and Sp3 infrequently associate with sites of transcription. Sp3 partitions with the tightly bound nuclear protein fraction of hormone responsive MCF-7 breast cancer cells, whereas only a subpopulation of Sp1 is found in that fraction. Both Sp1 and Sp3 are bound to the nuclear matrix, and the nuclear matrix-associated sites of Sp1 and Sp3 are different. Indirect immunofluorescence studies demonstrate that Sp1 and Sp3 associate with histone deacetylases 1 and 2 and with the estrogen receptor alpha, albeit at low frequencies in MCF-7 cells. Chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that although both Sp1 and Sp3 bind to the estrogen-responsive trefoil factor 1 promoter in MCF-7 cells, they do not occupy the same promoter. Our results demonstrate the different features of Sp1 and Sp3, providing further evidence that Sp3 is not a functional equivalent of Sp1.

Title Histone Modifications As a Platform for Cancer Therapy.
Date October 2005
Journal Journal of Cellular Biochemistry
Excerpt

Tumorigenesis and metastasis are a progression of events resulting from alterations in the processing of the genetic information. These alterations result from stable genetic changes (mutations) involving tumor suppressor genes and oncogenes (e.g., ras, BRAF) and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. Mutations of genes coding for proteins that directly or indirectly influence epigenetic processes will alter the cell's gene expression program. Epigenetic mechanisms often altered in cancer cells are DNA methylation and histone modifications (acetylation, methylation, phosphorylation). This article will review the potential of these reversible epigenetic processes as targets for cancer therapies.

Title Inducible Upregulation of Oestrogen Receptor-beta1 Affects Oestrogen and Tamoxifen Responsiveness in Mcf7 Human Breast Cancer Cells.
Date September 2005
Journal Journal of Molecular Endocrinology
Excerpt

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.

Title Stimulation of the Ras-mapk Pathway Leads to Independent Phosphorylation of Histone H3 on Serine 10 and 28.
Date June 2005
Journal Oncogene
Excerpt

The Ras-mitogen activated protein kinase (Ras-MAPK) pathway plays an integral role in the formation of human malignancies. Stimulation of this pathway results in phosphorylation of histone H3 at serines 10 and 28 and expression of immediate-early genes. Phosphorylated (serine 10) H3, which is also acetylated on lysine 14, is associated with immediate-early genes. In this report, we investigated the relationship between these two H3 phosphorylation events in parental and ras-transformed fibroblasts. Immunoblot analyses of two-dimensional gel patterns demonstrated that all three H3 variants were phosphorylated after stimulation of the Ras-MAPK pathway and during mitosis. Following stimulation of the Ras-MAPK pathway, H3 phosphorylated on serines 10 and 28 was excluded from regions of highly condensed chromatin and was present in increased levels in ras-transformed cells. Although H3 phosphorylated at serine 10 or 28 was dynamically acetylated, H3 phosphorylated at serine 28 had a higher steady state of acetylation than that of H3 phosphorylated at serine 10. When visualized with indirect immunofluorescence, most foci of phosphorylated serine 28 H3 did not co-localize with foci of H3 phosphorylated on serine 10 or phosphoacetylated on serine 10 and lysine 14, suggesting that these two phosphorylation events act separately to promote gene expression.

Title Elevated Expression of the Estrogen Receptor Prevents the Down-regulation of P21waf1/cip1 in Hormone Dependent Breast Cancer Cells.
Date March 2005
Journal Journal of Cellular Biochemistry
Excerpt

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.

Title Gene Regulation by Sp1 and Sp3.
Date February 2005
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

The Sp family of transcription factors is united by a particular combination of three conserved Cys2His2 zinc fingers that form the sequence-specific DNA-binding domain. Within the Sp family of transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. They can bind and act through GC boxes to regulate gene expression of multiple target genes. Although Sp1 and Sp3 have similar structures and high homology in their DNA binding domains, in vitro and in vivo studies reveal that these transcription factors have strikingly different functions. Sp1 and Sp3 are able to enhance or repress promoter activity. Regulation of the transcriptional activity of Sp1 and Sp3 occurs largely at the post-translational level. In this review, we focus on the roles of Sp1 and Sp3 in the regulation of gene expression.

Title Mitogen- and Stress-activated Protein Kinase 1 Activity and Histone H3 Phosphorylation in Oncogene-transformed Mouse Fibroblasts.
Date February 2005
Journal Cancer Research
Excerpt

Activation of the Ras-Raf-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-ERK signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinase 1 (MSK1). This activation of MSK1 leads to a rapid phosphorylation of histone H3 at Ser(10). Previously, we had demonstrated that Ser(10) phosphorylated H3 was elevated in Ciras-3 (c-Ha-ras-transformed 10T12) mouse fibroblasts and that H3 phosphatase activity was similar in Ciras-3 and 10T12 cells. Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Ciras-3 cells relative to these activities in the parental 10T12 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T12 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the Ras-mitogen-activated protein kinase signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in these cells.

Title Estrogen Regulation of Trefoil Factor 1 Expression by Estrogen Receptor Alpha and Sp Proteins.
Date January 2005
Journal Experimental Cell Research
Excerpt

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.

Title Measurement of Histone Acetyltransferase and Histone Deacetylase Activities and Kinetics of Histone Acetylation.
Date June 2004
Journal Methods (san Diego, Calif.)
Excerpt

Dynamic histone acetylation has a role in chromatin remodeling and in the regulation of transcription. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) catalyze reversible histone acetylation. HATs and HDACs exist as multiprotein complexes that have coactivator and corepressor activities, respectively. The steady-state level of acetylation at a chromatin site is determined by the local net activities of these enzymes. Here we describe methods to isolate different subcellular fractions (cytosol, nuclei, tightly bound nuclear, loosely bound nuclear, immunoprecipitated multiprotein complexes, and nuclear matrix) to determine the subcellular distribution of HAT and HDAC activities. Procedures to assay the activities of these enzymes and to measure the kinetics of histone acetylation and deacetylation are presented.

Title Chromatin Immunoprecipitation: a Tool for Studying Histone Acetylation and Transcription Factor Binding.
Date June 2004
Journal Methods (san Diego, Calif.)
Excerpt

The function of a protein in gene expression can often be explained, in part, by the location of that protein along a specific gene sequence. In recent years, the chromatin immunoprecipitation (ChIP) assay has been developed to study the association of proteins located within 2 A of DNA such as transcription factors and modified histones. Numerous important findings have been published using the ChIP assay and many questions about transcription have been answered. In this article, we present the ChIP assay currently used in our lab and discuss the various ways to optimize this assay for one's own use.

Title Identification of a Direct Dlx Homeodomain Target in the Developing Mouse Forebrain and Retina by Optimization of Chromatin Immunoprecipitation.
Date May 2004
Journal Nucleic Acids Research
Excerpt

Understanding homeobox gene specificity and function has been hampered by the lack of proven direct transcriptional targets during development. Dlx genes are expressed in the developing forebrain, retina, craniofacial structures and limbs. Dlx1/Dlx2 double knockout mice die at birth with multiple defects including abnormal forebrain development and decreased Dlx5 and Dlx6 expression. We have successfully applied chromatin immunoprecipitation (ChIP) to identify a direct transcriptional target of DLX homeoproteins from embryonic tissues in vivo. We optimized cross-linking conditions to enrich for protein-DNA complexes, then using specific high affinity DLX antibodies captured immunoenriched DLX genomic DNA transcriptional targets. DLX homeobox proteins bind differentially to the Dlx5/Dlx6 intergenic enhancer in newborn retina (DLX2) and embryonic striatum (DLX1, DLX2) in situ. Reporter gene assays demonstrated the functional significance of the binding of DLX proteins to this regulatory element, confirmed in vitro by electrophoretic mobility shift assays, using tissue extracts or recombinant DLX proteins. ChIP provides the best approach to identify direct Dlx homeoprotein targets from developing tissues in situ. The use of this technology will advance our understanding of Dlx gene function in the vertebrate in vivo and can be applied to examine targets of other homeobox genes and other classes of transcription factors.

Title The Many Roles of the Transcriptional Regulator Ctcf.
Date March 2004
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

The nuclear factor CTCF was first identified as one of the factors binding to the regulatory regions of the c-myc gene. Further study of this protein revealed roles in transcriptional repression, insulator function, and imprinting genetic information. Recent studies have provided new insight into the mechanism through which this factor acts at various levels of gene regulation.

Title Inhibition of Histone Deacetylase Activity by Butyrate.
Date October 2003
Journal The Journal of Nutrition
Excerpt

This article reviews the effects of the short-chain fatty acid butyrate on histone deacetylase (HDAC) activity. Sodium butyrate has multiple effects on cultured mammalian cells that include inhibition of proliferation, induction of differentiation and induction or repression of gene expression. The observation that butyrate treatment of cells results in histone hyperacetylation initiated a flurry of activity that led to the discovery that butyrate inhibits HDAC activity. Butyrate has been an essential agent for determining the role of histone acetylation in chromatin structure and function. Interestingly, inhibition of HDAC activity affects the expression of only 2% of mammalian genes. Promoters of butyrate-responsive genes have butyrate response elements, and the action of butyrate is often mediated through Sp1/Sp3 binding sites (e.g., p21(Waf1/Cip1)). We demonstrated that Sp1 and Sp3 recruit HDAC1 and HDAC2, with the latter being phosphorylated by protein kinase CK2. A model is proposed in which inhibition of Sp1/Sp3-associated HDAC activity leads to histone hyperacetylation and transcriptional activation of the p21(Waf1/Cip1) gene; p21(Waf1/Cip1) inhibits cyclin-dependent kinase 2 activity and thereby arrests cell cycling. Pending the cell background, the nonproliferating cells may enter differentiation or apoptotic pathways. The potential of butyrate and HDAC inhibitors in the prevention and treatment of cancer is presented.

Title The Insulator Binding Protein Ctcf Associates with the Nuclear Matrix.
Date September 2003
Journal Experimental Cell Research
Excerpt

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.

Title Chd1 Associates with Ncor and Histone Deacetylase As Well As with Rna Splicing Proteins.
Date September 2003
Journal Biochemical and Biophysical Research Communications
Excerpt

CHD1 is one of a family of nuclear proteins containing two chromodomains, a SWI/SNF-like helicase/ATPase domain and a DNA binding domain. We found that CHD1 co-immunoprecipitates with histone deacetylase (HDAC) activity and that CHD1 also associates with NCoR, a transcriptional corepressor, in yeast two-hybrid and in vitro pull-down assays. NCoR is known to associate with HDACs to effect its repressive activity, suggesting that the predicted chromatin remodeling activity of CHD1 plays a role in this repression. Yeast two-hybrid assays also showed that CHD1 interacts with splicing proteins mKIAA0164, Srp20, and SAF-B. Splicing assays show that CHD1 overexpression can affect alternative splicing. These results suggest that CHD1 may function in both chromatin mediated transcriptional repression and RNA splicing.

Title Msk1 and Msk2 Mediate Mitogen- and Stress-induced Phosphorylation of Histone H3: a Controversy Resolved.
Date August 2003
Journal Science's Stke : Signal Transduction Knowledge Environment
Excerpt

It is well established that mitogen- and stress-activated signal transduction pathways result in the rapid phosphorylation (Ser10 and Ser28) and acetylation of mammalian histone H3 associated with immediate-early genes. However, the prerequisite of H3 phosphorylation for the acetylation event and the identity of the mitogen-activated H3 kinase as RSK2 or MSK1 were controversial. A recent study with mouse embryonic fibroblasts lacking MSK1 and/or MSK2 demonstrated that MSK2 and MSK1 were the stimulus-induced H3 kinases and that neither of these enzyme activities was required for acetylation of H3 bound to immediate-early genes to occur.

Title Histone H1(s)-3 Phosphorylation in Ha-ras Oncogene-transformed Mouse Fibroblasts.
Date January 2003
Journal Oncogene
Excerpt

Phosphorylation of linker histone H1(S)-3 (previously named H1b) and core histone H3 is elevated in mouse fibroblasts transformed with oncogenes or constitutively active mitogen-activated protein kinase (MAPK) kinase (MEK). H1(S)-3 phosphorylation is the only histone modification known to be dependent upon transcription and replication. Our results show that the increased amounts of phosphorylated H1(S)-3 in the oncogene Ha-ras-transformed mouse fibroblasts was a consequence of an elevated Cdk2 activity rather than the reduced activity of a H1 phosphatase, which our studies suggest is PP1. Induction of oncogenic ras expression results in an increase in H1(S)-3 and H3 phosphorylation. However, in contrast to the phosphorylation of H3, which occurred immediately following the onset of Ras expression, there was a lag of several hours before H1(S)-3 phosphorylation levels increased. We found that there was a transient increase in the levels of p21(cip1), which inhibited the H1 kinase activity of Cdk2. Cdk2 activity and H1(S)-3 phosphorylated levels increased after p21(cip1) levels declined. Our studies suggest that persistent activation of the Ras-MAPK signal transduction pathway in oncogene-transformed cells results in deregulated activity of kinases phosphorylating H3 and H1(S)-3 associated with transcribed genes. The chromatin remodelling actions of these modified histones may result in aberrant gene expression.

Title The Estrogen Receptor: More Than the Average Transcription Factor.
Date December 2002
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

The human estrogen receptor is a steroid nuclear receptor found in breast cancer and a variety of other tissues. Located in the nucleus, it can exist either loosely or tightly associated with the nuclear matrix depending on whether or not it is bound to ligand. When bound to ligand, it is responsible for the transcriptional regulation of estrogen-responsive genes through recruitment of coactivators and corepressors of transcription. The estrogen receptor is also capable of ligand-independent transcriptional activation via the mitogen-activated protein kinase pathway. Ligands have been implicated in the regulation of estrogen receptor levels via changing the levels and stability of estrogen receptor mRNA and protein. The resulting levels of estrogen receptor and the type of ligand bound to it have a direct impact on the transcription of estrogen-responsive genes.

Title Characterization of Stably Transfected Fusion Protein Gfp-estrogen Receptor-alpha in Mcf-7 Human Breast Cancer Cells.
Date December 2002
Journal Journal of Cellular Biochemistry
Excerpt

Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.

Title The Transcriptional Repressor Sp3 is Associated with Ck2-phosphorylated Histone Deacetylase 2.
Date November 2002
Journal The Journal of Biological Chemistry
Excerpt

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogen-regulated genes.

Title Napp2, a Peroxisomal Membrane Protein, is Also a Transcriptional Corepressor.
Date June 2002
Journal Genomics
Excerpt

Nuclear factor-erythroid number 2 (NF-E2) is a positive regulatory, DNA binding transcription factor for gene expression in erythroid and megakaryocytic cells. To further understand the mechanisms of NF-E2 function, we used expression cloning to identify coregulators interacting with the erythroid-specific subunit of NF-E2, p45. We have isolated a protein, NAPP2, which contains an aspartic-acid- and glutamic-acid-rich region and a nuclear localization signal. The gene encoding NAPP2, PEX14, is located on chromosome 1p36 and is ubiquitously expressed. The domains of interaction in vitro and in vivo between p45 and NAPP2 were mapped by a yeast two-hybrid system and cotransfection experiments. In mammalian cell culture, ectopically expressed NAPP2 inhibited p45-directed transcriptional activation. Furthermore, NAPP2 functions as a corepressor and interacts specifically with histone deacetylase l (HDAC1), but not HDAC2 or HDAC3. NAPP2 is thus potentially a negative coregulator of NF-E2. NAPP2 is identical to PEX14, an integral membrane protein essential for protein docking onto the peroxisomes. These studies have identified a novel, bifunctional protein capable of acting as a transcriptional corepressor and a polypeptide transport modulator. They also suggest that NF-E2 may function both positively and negatively in the transcription regulation of specific erythroid and megakaryocytic genes.

Title Isolation of Transcriptionally Active Chromatin from Human Breast Cancer Cells Using Sulfolink Coupling Gel Chromatography.
Date March 2002
Journal Journal of Cellular Biochemistry
Excerpt

The process of transcription unfolds the nucleosome. The unfolded nucleosome structure will be maintained as long as the histones are in a highly acetylated state. Typically the cysteine residue at position 110 of histone H3 is buried in the interior of the nucleosome. However, the transcribed unfolded nucleosome has its H3 cysteine exposed, offering a tag to isolate and study transcribed nucleosomes. In this study, we applied Sulfolink Coupling Gel chromatography to isolate unfolded nucleosomes from estrogen dependent human cancer T5 cells. Inhibition of histone deacetylase activity did not enhance the yield of unfolded nucleosomes from these cells. We show that the estrogen receptor and c-myc transcribed DNA sequences are associated with unfolded nucleosomes. In chromatin immunoprecipitation (ChIPs) assays, we found that the coding regions of the estrogen receptor and c-myc genes are bound to highly acetylated H3 and H4 in cultured T5 Cells. We conclude that in cultured T5 breast cancer cells H3 and H4 are in highly acetylated states maintaining the unfolded structure of the transcribed nucleosome and facilitating subsequent rounds of elongation.

Title Ser-10 Phosphorylation of Histone H3 and Immediate Early Gene Expression in Oncogene-transformed Mouse Fibroblasts.
Date February 2002
Journal Cancer Research
Excerpt

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) pathway by growth factors, phorbol esters, and oncoproteins results in the phosphorylation of histone H3. Rsk-2 and MSK1 have been reported to be H3 kinases activated by the Ras-MAPK signal transduction pathway. In this study, we used inhibitors of Rsk-2 and MSK1 to decide which of these kinases was responsible for the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of H3 in 10T(1/2) and Ciras-3 (H-ras-transformed 10T(1/2)) mouse fibroblasts. These studies demonstrated that MSK1, but not Rsk-2, was the H3 kinase activated in these cells. Furthermore, assays with Rsk-2 showed that this kinase phosphorylates H2B but not H3 in vitro. H89, a potent MSK1 inhibitor, prevented TPA induction of H3 phosphorylation and diminished the TPA-induced expression of the c-fos and urokinase plasminogen activator genes. We propose that persistent activation of the Ras-MAPK pathway and MSK1 resulting in the elevation of phosphorylated H3 levels may contribute to the aberrant gene expression observed in the oncogene-transformed cells.

Title Effect of Estradiol on Histone Acetylation Dynamics in Human Breast Cancer Cells.
Date January 2002
Journal The Journal of Biological Chemistry
Excerpt

Histone acetylation plays an important role in remodeling chromatin structure, facilitating nuclear processes such as transcription. We investigated the effect of estradiol on global histone acetylation in hormone-responsive human breast cancer cells. Pulse-chase experiments and immunoblot analyses of dynamically acetylated histones show that estradiol rapidly increases histone acetylation in estrogen receptor (ER)-positive, hormone-dependent T5, but not in ER-negative, hormone-independent MDA MB 231 breast cancer cells. The effect of estradiol on the rates of histone acetylation and deacetylation in T5 cells was determined. We found that estradiol increased the level of acetylated histones by reducing the rate of histone deacetylation, whereas the rate of histone acetylation was not altered. Enzymatic assays and immunoblot analyses of cell fractions showed that estradiol did not affect the level, subnuclear distribution, or activity of class I and II histone deacetylases. However, estradiol did alter the intranuclear distribution of ER and histone acetyltransferases, with both becoming tightly bound in the nucleus and associated with the nuclear matrix. We propose that, following the association of ER with nuclear matrix sites, ER alters the balance of histone acetyltransferases and histone deacetylases at these sites and the dynamics of acetylation of histones associated with transcriptionally active and competent chromatin.

Title Dynamically Acetylated Histone Association with Transcriptionally Active and Competent Genes in the Avian Adult Beta-globin Gene Domain.
Date October 2001
Journal The Journal of Biological Chemistry
Excerpt

In chicken immature erythrocytes, class 1 acetylated histones are rapidly tri- and tetra-acetylated and rapidly deacetylated. Class 2 acetylated H3 and H4 are rapidly acetylated to mono- and di-acetylated isoforms and slowly deacetylated. Our previous studies suggested that class 1 acetylated histones were primarily associated with transcriptionally active DNA (beta(A)-globin) but not competent DNA (epsilon-globin). Chromatin salt solubility (chromatin fiber oligomerization) is directly influenced by hyperacetylation. In this study we investigated the association of class 1 histones with beta(A)- and epsilon-globin DNA by measuring their loss of solubility rates in 150 mm NaCl and 3 mm MgCl(2) as a function of hyperacetylated histone deacetylation. Expressed and competent chromatin was associated with class 1 acetylated histones. As most active chromatin and hyperacetylated histones are associated with the low salt-insoluble residual nuclear material containing the nuclear matrix, we investigated whether hyperacetylated histones are bound to the beta(A)- and epsilon-globin DNA in this fraction. In chromatin immunoprecipitation assays, we found that the beta(A)- and epsilon-globin coding regions are bound to hyperacetylated H3 and H4. Our observations are consistent with a model in which nuclear matrix-associated histone acetyltransferases and deacetylases mediate a dynamic attachment between active and competent chromatin and the nuclear matrix.

Title Regulation of Neuronal Traits by a Novel Transcriptional Complex.
Date September 2001
Journal Neuron
Excerpt

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Title Signal Transduction Pathways and Chromatin Structure in Cancer Cells.
Date August 2001
Journal Journal of Cellular Biochemistry. Supplement
Excerpt

Molecular mechanisms controlling gene expression include cell shape, mechanical and chemical signal transduction pathways, chromatin remodeling, and DNA methylation. In this article, we will review the contribution of these molecular mechanisms and structural alterations in the malignant transformation of cells. The mechanical signaling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. The cytoskeleton array is a dynamic system that transmits signals from the cell exterior to nuclear DNA. The composition and function of this mechanical signaling pathway is altered in cancer cells. Chemical signaling pathways such as the Ras/mitogen-activated protein kinase (MAPK) pathway stimulate the activity of kinases that modify transcription factors, histones, and chromatin remodeling factors. Oncoproteins deregulating this signaling pathway set in motion a series of events that cumulate to chromatin remodeling and aberrant gene expression. J. Cell. Biochem. Suppl. 35:27-35, 2000.

Title An Essential Role for Mad Homology Domain 1 in the Association of Smad3 with Histone Deacetylase Activity*.
Date July 2001
Journal The Journal of Biological Chemistry
Excerpt

The Smads are a family of sequence-specific DNA-binding proteins that modulate transcription in response to transforming growth factor beta (TGFbeta) by recruiting transcriptional activators like the histone acetyltransferase, p300/CBP, or repressors like the histone deacetylase, HDAC1, to TGFbeta target genes. The association of Smads and HDAC1 is mediated in part by direct binding of Smads to the HDAC1-associated proteins, TG-interacting factor, c-ski, and SnoN. Although ectopic expression of these proteins inhibits Smad-activated transcription, the contribution of histone deacetylase enzymatic activity to transcriptional repression by TGFbeta is unknown. Here, the biological requirements for the interaction between Smads and endogenous histone deacetylase activity are investigated. We identify residues in Mad homology domain 1 of Smad3 that are required for association with histone deacetylase activity. An amino acid change at one of these critical residues does not disrupt the association of Smad3 with c-ski, SnoN, and transforming growth-interacting factor but does abrogate the ability of Smad3 to repress transcription. These findings indicate that the association of Smad3 and histone deacetylase activity relies on additional protein mediators that make contact with Smad3 at its amino terminus. Moreover, these data suggest that the suppressive effect of Smad3 on transcription is dependent upon its association with histone deacetylase enzymatic activity.

Title Control of Chromatin Remodeling.
Date April 2001
Journal Critical Reviews in Eukaryotic Gene Expression
Excerpt

Chromatin structure has a pivotal role in the regulation of gene expression. Transcriptional activation or the repression of a gene require the recruitment of multiple chromatin remodeling complexes. Chromatin remodeling complexes modulate the higher order structure of chromatin, facilitate or hinder the binding of transcription factors, and aid in or prevent the establishment of a transcriptional preinitiation complex. Two types of chromatin remodeling complexes have been extensively studied--ATP-dependent chromatin remodeling complexes and histone-modifying enzymes--which include histone acetyltransferases, histone deacetylases, and histone kinases. Transcriptional activators and repressors are responsible for recruitment of one or more of these large, multisubunit chromatin remodeling complexes. In this review, the features of the chromatin remodeling complexes and the modes of their recruitment are presented.

Title Altered Profiles in Nuclear Matrix Proteins Associated with Dna in Situ During Progression of Breast Cancer Cells.
Date March 2001
Journal Cancer Research
Excerpt

Nuclear matrix proteins (NMPs) show promise as informative biomarkers in following the pathogenesis of breast cancer. The nuclear matrix is a dynamic RNA-protein network involved in the organization and expression of chromatin. Cisplatin, which preferentially cross-links nuclear matrix proteins to DNA in situ, may be used to identify NMPs that organize and/or regulate the processing of DNA. In this study, we analyzed the nuclear matrix proteins from an estrogen receptor-positive breast cancer cell line panel consisting of MCF-7, MIII, LCC1, and LCC2 cell lines. This cell line panel reflects the stages of malignant progression in breast cancer. Proteins isolated from nuclear matrices and proteins cross-linked to nuclear DNA in situ with cisplatin were analyzed by two-dimensional gel electrophoresis. Specific changes in nuclear matrix proteins bound to nuclear DNA were identified. In concordance with estrogen independence and antiestrogen insensitivity, a loss in cisplatin cross-linking of specific NMPs to nuclear DNA was observed. Our results suggest that progression of breast cancer is accompanied by a reorganization of chromosomal domains, which may lead to alterations in gene expression.

Title Cug-initiated Fgf-2 Induces Chromatin Compaction in Cultured Cardiac Myocytes and in Vitro.
Date March 2001
Journal Journal of Cellular Physiology
Excerpt

Fibroblast growth factor-2 (FGF-2) is a mitogen found in CUG-initiated 21-25 kDa ("hi") or AUG-initiated 16-18 kDa ("lo") forms. Previously we demonstrated that "hi"-but not "lo"-FGF-2 caused a distinct nuclear phenotype characterized by apparently condensed chromatin present as separate clumps in the nucleus of cardiac myocytes. In this manuscript we investigated whether these effects were related to apoptosis or mitosis and whether they reflected a direct effect of "hi" FGF-2 on chromatin. Myocytes overexpressing "hi" FGF-2 and presenting the clumped chromatin phenotype: (i) were not labeled above background with antibodies to phosphorylated histones H1 and H3 used as indicators of mitotic chromatin condensation; (ii) did not stain positive for TUNEL; (iii) their nuclear lamina, visualized by anti-laminB immunofluorescence, appeared intact; (iv) neither caspase inhibitors, nor Bcl-2 or "lo" FGF-2 overexpression prevented the manifestation of the compacted nuclear phenotype. Purified recombinant "hi" FGF-2 was more potent than "lo" FGF-2 in promoting the condensation/aggregation of chick erythrocyte chromatin partially reconstituted with histone H1 in vitro. We conclude that the DNA phenotype induced by "hi" FGF-2 in cardiac myocytes likely reflects a direct effect on chromatin structure that does not require the engagement of mitosis or apoptosis. By affecting chromatin compaction "hi" FGF-2 may contribute to the regulation of gene expression.

Title Expression of E1 Component of Human Branched-chain Alpha-keto Acid Dehydrogenase Complex in Escherichia Coli by Cotransformation with Chaperonins Groel and Groes.
Date January 2001
Journal Methods in Enzymology
Title Production of Recombinant Mammalian Holo-e2 and E3 and Reconstitution of Functional Branched-chain Alpha-keto Acid Dehydrogenase Complex with Recombinant E1.
Date January 2001
Journal Methods in Enzymology
Title Drosophila C-terminal Binding Protein Functions As a Context-dependent Transcriptional Co-factor and Interferes with Both Mad and Groucho Transcriptional Repression.
Date January 2001
Journal The Journal of Biological Chemistry
Excerpt

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. We show here that dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. We find that mSin3A-histone deacetylase complexes are altered in the presence of dCtBP and that dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, we propose that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function.

Title Rapid Induction of Histone Hyperacetylation and Cellular Differentiation in Human Breast Tumor Cell Lines Following Degradation of Histone Deacetylase-1.
Date January 2001
Journal The Journal of Biological Chemistry
Excerpt

Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells. Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death. The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine. We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.

Title Signal Transduction Pathways and the Modification of Chromatin Structure.
Date October 2000
Journal Progress in Nucleic Acid Research and Molecular Biology
Excerpt

Mechanical and chemical signaling pathways are involved in transmitting information from the exterior of a cell to its chromatin. The mechanical signaling pathway consists of a tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and karyoskeleton. The tissue matrix system governs cell and nuclear shape and forms a structural and functional connection between the cell periphery and chromatin. Further, this mechanical signaling pathway has a role in controlling cell cycle progression and gene expression. Chemical signaling pathways such as the Ras/mitogen-activated protein kinase (MAPK) pathway can stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. Activation of the Ras/MAPK pathway results in the alteration of chromatin structure and gene expression. The tissue matrix and chemical signaling pathways are not independent and one signaling pathway can affect the other. In this chapter, we will review chromatin organization, histone variants and modifications, and the impact that signaling pathways have on chromatin structure and function.

Title The Human Factors Yy1 and Lsf Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Via Recruitment of Histone Deacetylase 1.
Date August 2000
Journal Journal of Virology
Excerpt

Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

Title Tamoxifen-bound Estrogen Receptor (er) Strongly Interacts with the Nuclear Matrix Protein Het/saf-b, a Novel Inhibitor of Er-mediated Transactivation.
Date April 2000
Journal Molecular Endocrinology (baltimore, Md.)
Excerpt

The estrogen receptor (ER) is a ligand-dependent transcription factor that acts in a cell- and promoter-specific manner. Evidence suggests that the activity of the ER can be regulated by a number of other stimuli (e.g. growth factors) and that the effects of the ER are modulated by nuclear factors termed coregulators. While the interplay among these factors may in part explain the pleiotropic effects elicited by the ER, there are several other less well described mechanisms of control, such as interactions with the nuclear matrix. Here we report that the nuclear matrix protein/scaffold attachment factor HET/SAF-B is an ER-interacting protein. ER and HET/SAF-B interact in in vitro binding assays, with HET binding to both the ER DNA-binding domain and the hinge region. Coimmunoprecipitation experiments reveal that HET/SAF-B and ER associate in cell lines in the presence or absence of estradiol, but binding is increased by the antiestrogen tamoxifen. HET/SAF-B enhances tamoxifen antagonism of estrogen-induced ER-mediated transactivation, but at high concentrations can inhibit both estrogen and tamoxifen-induced ER activity. HET/SAF-B-mediated repression of ER activity is dependent upon interaction with the ER-DBD. While the existence of high-affinity binding sites for the ER in the nuclear matrix has been known for some time, we now provide evidence of a specific nuclear matrix protein binding to the ER. Furthermore, our data showing that HET/SAF-B binds to ER particularly strongly in the presence of tamoxifen suggests that it may be important for the antagonist effect of tamoxifen.

Title Nuclear Matrix Proteins Associated with Dna in Situ in Hormone-dependent and Hormone-independent Human Breast Cancer Cell Lines.
Date February 2000
Journal Cancer Research
Excerpt

The nuclear matrix is a dynamic RNA-protein complex that organizes chromatin and regulates nuclear DNA metabolism. Nuclear matrix proteins informative in the diagnosis of cancer have been identified. Here, the nuclear matrix breast cancer proteins (NMBCs) cross-linked to nuclear DNA in situ with cisplatin in human breast cancer cell lines were analyzed by two-dimensional gel electrophoresis. We identified NMBCs that were differentially associated with nuclear DNA of hormone-dependent and -independent breast cancer cell lines. Three DNA cross-linked NMBCs were found to be exclusive to estrogen receptor-positive, hormone-dependent breast cancer cells, whereas two NMBCs were observed only in estrogen receptor-negative, hormone-independent breast cancer cells. Changes in these NMBCs were observed when hormone-dependent breast cancer cells became hormone independent. Furthermore, we show that the intermediate filament protein vimentin is associated with the nuclear DNA of MDA-MB-231 breast cancer cells, an estrogen receptor-negative, hormone-independent breast cancer cell line with high metastatic potential. These nuclear matrix DNA-binding proteins may play important roles in breast tumorigenesis.

Title Control of Histone Modifications.
Date February 2000
Journal Journal of Cellular Biochemistry
Excerpt

A role for histone modifications in transcription processes and the remodeling of chromatin structure has been established. This review highlights the recent advances made in studies on histone acetyltransferases, histone deacetylases, histone kinases, and protein phosphatases, as well as their roles in transcriptional activation and repression. Coactivators with histone acetyltransferase activity stimulate transcription, whereas corepressors with histone deacetylase activity repress transcription. Families of histone acetyltransferases and deacetylases have been identified. We have learned that their substrates are not limited to histones but also include transcription factors and architectural proteins. Studies on the composition of multiprotein complexes with histone acetyltransferase or histone deacetylase have revealed mechanisms by which these complexes are recruited to specific genomic sites that are transcriptionally active, silenced, or being repaired. A new and exciting development, presented in this review, is the role of signal transduction pathways in the phosphorylation of histone H3 and the expression of immediate-early genes. J. Cell. Biochem. Suppls. 32/33:141-148, 1999.

Title Role of Covalent Modifications of Histones in Regulating Gene Expression.
Date January 2000
Journal Gene
Excerpt

DNA is organized into a hierarchy of structures, resulting in the level of compaction required to pack 2m of DNA into a nucleus with a diameter of 10 micrometer. The orderly packaging of DNA in the nucleus plays an important role in the functional aspects of gene regulation. A small percentage of chromatin is made available to transcription factors and the transcription machinery, while the remainder of the genome is in a state that is essentially invisible to the RNA polymerases. Modification of histones has a key role in altering chromatin higher order structure and function. In this review, we will present the latest developments in the study of histone modifications (ubiquitination, acetylation, methylation, and phosphorylation) and the enzymes involved in these processes.

Title Organization of Chromatin in Cancer Cells: Role of Signalling Pathways.
Date November 1999
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.

Title Increased Ser-10 Phosphorylation of Histone H3 in Mitogen-stimulated and Oncogene-transformed Mouse Fibroblasts.
Date September 1999
Journal The Journal of Biological Chemistry
Excerpt

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

Title Purification and Characterization of Chicken Erythrocyte Histone Deacetylase 1.
Date May 1999
Journal Biochemistry
Excerpt

Histone acetylation is involved in nuclear processes requiring chromatin remodeling. In chicken erythrocytes, DNA replication has ceased, and active reversible histone acetylation is restricted to transcriptionally active/competent chromatin domains. In this study, we set out to identify and purify the erythroid histone deacetylase responsible for catalyzing dynamic acetylation of transcriptionally active chromatin. Histone deacetylase purified from chicken erythrocytes had a molecular mass of 66 kDa. Complementary DNA encoding the chicken histone deacetylase was cloned from erythrocytes, and analysis of the derived amino acid sequence showed the chicken histone deacetylase to be the chicken homologue of mammalian HDAC1. Purified chicken erythrocyte HDAC1 deacetylated the four core histones, with a preference for H3. We present evidence that chicken HDAC1 is a metalloenzyme, the activity of which is lost when incubated with zinc chelators. In Western blot analysis with anti-HDAC1 antibodies, we found that most erythrocyte HDAC1 is associated with the low-salt insoluble chromatin fraction and, to a lesser extent, with 150 mM NaCl-soluble oligo- and polynucleosomes. The distribution of HDAC1 in erythrocyte chromatin parallels that of dynamically acetylated class 1 histones. Further, we show that HDAC1 is associated with the erythroid nuclear matrix and that the enzyme is bound to nuclear DNA in situ. We propose that in addition to catalyzing dynamic acetylation of transcribed chromatin, the enzyme has a role in the organization of nuclear DNA.

Title Regulation and Regulatory Parameters of Histone Modifications.
Date April 1999
Journal Journal of Cellular Biochemistry. Supplement
Excerpt

Histone acetylation and phosphorylation destablizes nucleosome and chromatin structure. Relaxation of the chromatin fiber facilitates transcription. Coactivator complexes with histone acetyltransferase activity are recruited by transcription factors bound to enhancers or promoters. The recruited histone acetyltransferases may acetylate histone or nonhistone chromosomal proteins, resulting in the relaxation of chromatin structure. Alternatively, repressors recruit corepressor complexes with histone deacetylase activity, leading to condensation of chromatin. This review highlights the recent advances made in our understanding of the roles of histone acetyltransferases, histone deacetylases, histone kinases, and protein phosphatases in transcriptional activation and repression. Exciting reports revealing mechanistic connections between histone modifying activities and the RNA polymerase II machinery, the coupling of histone deacetylation and DNA methylation, the possible involvement of histone deacetylases in the organization of nuclear DNA, and the role of chromatin modulators in oncogenesis are discussed.

Title Direct Visualization of the Human Estrogen Receptor Alpha Reveals a Role for Ligand in the Nuclear Distribution of the Receptor.
Date March 1999
Journal Molecular Biology of the Cell
Excerpt

The human estrogen receptor alpha (ER alpha) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER- (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER- but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

Title Eto, a Target of T(8;21) in Acute Leukemia, Interacts with the N-cor and Msin3 Corepressors.
Date December 1998
Journal Molecular and Cellular Biology
Excerpt

t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.

Title Chromatin, Nuclear Matrix and the Cytoskeleton: Role of Cell Structure in Neoplastic Transformation (review).
Date December 1998
Journal International Journal of Oncology
Excerpt

Aberrant nuclear and cellular structures are hallmarks of malignant transformation. Thus it is not surprising that the three-dimensional structure of the cell both affects and is affected by changes in gene expression. Here we review the role of the cytoskeleton, nuclear matrix, and chromatin structure in the genesis of cancer. The shape of a cell is governed by a dynamic tissue matrix, which includes extracellular matrix, cytoskeleton and nuclear matrix. Mechanical and chemical signals are transmitted to the nucleus, resulting in alterations in the three-dimensional chromatin organization of genes. The signal transduction pathways affect histone modifications, such as acetylation and phosphorylation, resulting in a relaxed chromatin structure observed in oncogene-transformed cells.

Title Estrogen Regulates the Association of Intermediate Filament Proteins with Nuclear Dna in Human Breast Cancer Cells.
Date November 1998
Journal The Journal of Biological Chemistry
Excerpt

In a previous study we showed that the levels of the intermediate filament proteins, cytokeratins 8, 18, and 19, in the nuclear matrix-intermediate filament (NM-IF) fraction from the hormone-dependent and estrogen receptor (ER)-positive human breast cancer cell line T-47D5 were regulated by estrogens. In contrast, estrogens did not regulate the cytokeratins in the NM-IF fraction of the hormone-independent and ER-positive cell line, T5-PRF. In this study, human breast cancer cells were treated with cis-diamminedichloroplatinum to cross-link protein to nuclear DNA in situ, and proteins bound to DNA were isolated. We show that cytokeratins 8, 18, and 19 of T-47D5 and T5-PRF were associated with nuclear DNA in situ. The levels of the cytokeratins 8, 18, and 19 bound to nuclear DNA or associated with the cytoskeleton of T-47D5 human breast cancer cells decreased when estrogens were depleted or the pure antiestrogen ICI 164,384 was added. In contrast, the cytokeratin levels associated with nuclear DNA or cytoskeleton were not significantly affected by estrogen withdrawal or antiestrogen administration in T5-PRF cells. These observations suggest that estrogen regulates the organization of nuclear DNA by rearrangement of the cytokeratin filament network in hormone-dependent, ER-positive human breast cancer cells and that this regulation is lost in hormone-independent, ER-positive breast cancer cells.

Title Sap30, a Component of the Msin3 Corepressor Complex Involved in N-cor-mediated Repression by Specific Transcription Factors.
Date August 1998
Journal Molecular Cell
Excerpt

The transcriptional corepressor mSin3 is found in a large multiprotein complex containing the histone deacetylases HDAC1 and HDAC2, in addition to at least five tightly associated polypeptides. We have cloned and characterized a novel component of the mSin3 complex, SAP30, SAP30 binds to mSin3 and is capable of mediating transcriptional repression via histone deacetylases. SAP30 also binds the N-CoR corepressor and is required for N-CoR-mediated repression by antagonist-bound estrogen receptor and the homeodomain protein Rpx, as well as N-CoR suppression of transactivation by the POU domain protein Pit-1. However, SAP30 is not required for N-CoR-mediated repression by unliganded retinoic acid receptor or thyroid hormone receptor, suggesting that SAP30 is involved in the functional recruitment of the mSin3-histone deacetylase complex to a specific subset of N-CoR corepressor complexes.

Title Ras-associated Nuclear Structural Change Appears Functionally Significant and Independent of the Mitotic Signaling Pathway.
Date August 1998
Journal Journal of Cellular Biochemistry
Excerpt

An altered nuclear morphology has been previously noted in association with Ras activation, but little is known about the structural basis, functional significance, signaling pathway, or reproducibility of any such change. We first tested the reproducibility of Ras-associated nuclear change in a series of rodent fibroblast cell lines. After independently developing criteria for recognizing Ras-associated nuclear change in a Papanicolaou stained test cell line with an inducible H(T24)-Ras oncogene, two cytopathologists blindly and independently assessed 17 other cell lines. If the cell lines showed Ras-associated nuclear change, a rank order of increasing nuclear change was independently scored. Ras-associated nuclear changes were identified in v-Fes, v-Src, v-Mos, v-Raf, and five of five H(T24)-Ras transfectants consisting of a change from a flattened, occasionally undulating nuclear shape to a more rigid spherical shape and a change from a finely textured to a coarse heterochromatic appearance. Absent or minimal changes were scored in six control cell lines. The two cytopathologists' independent morphologic rank orders were similar (P < .0002). The mitogen signaling pathway per se does not appear to transduce the change since no morphologic alterations were identified in cell lines with activations of downstream components of this pathway--MAPKK or c-Myc--and the rank orders did not correlate with markers of mitotic rate (P > .11). The rank order correlated closely with metastatic potential (P < .0014 and P < .0003) but not with histone H1 composition or global nuclease sensitivity. Based on published studies of five of the cell lines, there may be a correlation between increases in certain nuclear matrix proteins and the Ras-associated nuclear change.

Title In Situ Cross-linking by Cisplatin of Nuclear Matrix-bound Transcription Factors to Nuclear Dna of Human Breast Cancer Cells.
Date August 1998
Journal Cancer Research
Excerpt

Cisplatin is an antitumor drug that is used to treat several types of cancers. In this study, we analyzed the proteins that were cross-linked to DNA in situ in MCF-7 human breast cancer cells incubated with cisplatin. We show that cisplatin cross-links nuclear matrix proteins to DNA. In immunoblotting experiments, we found that nuclear matrix-associated transcription factors and cofactors (estrogen receptor, HET/SAF-B, hnRNP K, and histone deacetylase 1) were cross-linked to nuclear DNA. These transcription factors and cofactors have essential roles in the regulation of genes involved in the proliferation of breast cancer cells and in the organization and structure of chromatin. We applied a novel protocol to demonstrate that the nuclear matrix-bound transcription factors/cofactors were cross-linked to DNA fragments attached to the nuclear matrix. These results suggest that the cross-linking of nuclear matrix-associated transcription factors and cofactors to DNA may be one of the mechanisms by which cisplatin inhibits transcription and replication processes.

Title Covalent Modifications of Histones: Expression from Chromatin Templates.
Date August 1998
Journal Current Opinion in Genetics & Development
Excerpt

Recent advances highlight the involvement of histone acetyltransferases in transcriptional activation and histone deacetylases in transcriptional repression. Transcription factors loaded onto regulatory DNA elements may recruit either coactivators with histone acetyltransferase activity or corepressors associated with histone deacetylases. The recruited enzymes may either acetylate or deacetylate proximal nucleosomal histones or nonhistone chromosomal proteins.

Title Histone Acetylation is Required to Maintain the Unfolded Nucleosome Structure Associated with Transcribing Dna.
Date July 1998
Journal The Journal of Biological Chemistry
Excerpt

Nucleosomes associated with transcribing chromatin of mammalian cells have an unfolded structure in which the normally buried cysteinyl-thiol group of histone H3 is exposed. In this study we analyzed transcriptionally active/competent DNA-enriched chromatin fractions from chicken mature and immature erythrocytes for the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with [3H]iodoacetate. In mature and immature erythrocytes, the active DNA-enriched chromatin fractions are associated with histones that are rapidly highly acetylated and rapidly deacetylated. When histone deacetylation was prevented by incubating cells with histone deacetylase inhibitors, sodium butyrate or trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected in the soluble chromatin and nuclear skeleton-associated chromatin of immature, but not mature, erythrocytes. We did not find thiol-reactive nucleosomes in active DNA-enriched chromatin fractions of untreated immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to maintain, the unfolded structure of transcribing nucleosomes.

Title Impaired Assembly of E1 Decarboxylase of the Branched-chain Alpha-ketoacid Dehydrogenase Complex in Type Ia Maple Syrup Urine Disease.
Date June 1998
Journal The Journal of Biological Chemistry
Excerpt

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1alpha (45.5 kDa) and two E1beta (37.5 kDa) subunits forming an alpha2 beta2 tetramer. In patients with type IA maple syrup urine disease, the E1alpha subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1alpha missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)6-E1alpha and untagged E1beta subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two 35S-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin. The nine E1alpha maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1beta in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-alpha and R220W-alpha), moderately slow (G245R-alpha), slow (G204S-alpha, A240P-alpha, F364C-alpha, Y368C-alpha, and Y393N-alpha), and no (T265R-alpha) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 degreesC (in the case of T265R-alpha), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as alpha2 beta2 tetramers. In contrast, a subset of E1alpha missense mutations caused the occurrence of exclusive alphabeta dimers (Y393N-alpha and F364C-alpha) or of both alpha2beta2 tetramers and lower molecular weight species (Y368C-alpha and T265R-alpha). Thermal denaturation at 50 degreesC indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min-1), with the T265R-alpha mutant E1 most severely affected (rate constant, 4.45 min-1). The results establish that the human E1alpha mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-alpha, have no effect on E1 subunit assembly. The T265R-alpha mutation adversely impacts both E1alpha folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native alpha2 beta2 structure.

Title Ubiquitination of Histone H3 in Elongating Spermatids of Rat Testes.
Date June 1998
Journal The Journal of Biological Chemistry
Excerpt

Because of the potential role of histone ubiquitination in altering chromatin structure, we characterized the levels of ubiquitination of specific histones in meiotic and postmeiotic germ cells in rat testes by two-dimensional gel electrophoresis. The levels of the major ubiquitinated histone forms, mono- and poly-ubiquitinated H2A, were highest in the pachytene spermatocyte stage, declined thereafter through the round spermatid stage, and reached their lowest levels in elongating spermatids. Three additional ubiquitinated histone species, besides H2A, were detected using anti-ubiquitin antibodies specifically in the fraction enriched in elongating spermatids. Based on their electrophoretic mobilities, they corresponded to uH3, uTH3, and uH2B. Polyubiquitinated forms of these proteins were also observed. The identity of these proteins was confirmed by immunoblotting with anti-H3 antisera and by differential extraction of the proteins from the nucleus with increasing salt concentrations. This is the first report of ubiquitination of H3 in vivo. We speculate that its ubiquitination could loosen the nucleosome structure in preparation for histone removal, be a consequence of nucleosome relaxation or disruption caused by other means, or target H3 for degradation.

Title Estrogen Receptor Diminishes Dna-binding Activities of Chicken Gata-1 and Caccc-binding Proteins.
Date February 1998
Journal Dna and Cell Biology
Excerpt

The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes.

Title Rapid Deubiquitination of Nucleosomal Histones in Human Tumor Cells Caused by Proteasome Inhibitors and Stress Response Inducers: Effects on Replication, Transcription, Translation, and the Cellular Stress Response.
Date January 1998
Journal Biochemistry
Excerpt

The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.

Title Isolation and Characterization of Cdnas Corresponding to an Additional Member of the Human Histone Deacetylase Gene Family.
Date December 1997
Journal The Journal of Biological Chemistry
Excerpt

Several human cDNAs encoding a histone deacetylase protein, HDAC3, have been isolated. Analysis of the predicted amino acid sequence of HDAC3 revealed an open reading frame of 428 amino acids with a predicted molecular mass of 49 kDa. The HDAC3 protein is 50% identical in DNA sequence and 53% identical in protein sequence compared with the previously cloned human HDAC1. Comparison of the HDAC3 sequence with human HDAC2 also yielded similar results, with 51% identity in DNA sequence and 52% identity in protein sequence. The expressed HDAC3 protein is functionally active because it possesses histone deacetylase activity, represses transcription when tethered to a promoter, and binds transcription factor YY1. Similar to HDAC1 and HDAC2, HDAC3 is ubiquitously expressed in many different cell types.

Title Novel Nuclear Matrix Protein Het Binds to and Influences Activity of the Hsp27 Promoter in Human Breast Cancer Cells.
Date December 1997
Journal Journal of Cellular Biochemistry
Excerpt

Since the small heat shock protein hsp27 enhances both growth and drug resistance in breast cancer cells, and is a bad prognostic factor in certain subsets of breast cancer patients, we have characterized the transcriptional regulation of hsp27, with the long-term goal of targeting its expression clinically. The majority of the promoter activity resides in the most proximal 200 bp. This region contains an imperfect estrogen response element (ERE) that is separated by a 13-bp spacer that contains a TATA box. Gel-shift analysis revealed the binding of a protein (termed HET for Hsp27-ERE-TATA-binding protein) to this region that was neither the estrogen receptor nor TATA-binding protein. We cloned a complete cDNA (2.9 kb) for HET from an MCF-7 cDNA library. To confirm the identity of the HET clone, we expressed a partial HET clone as a glutathione S-transferase fusion protein, and showed binding to the hsp27 promoter fragment in gel-retardation assays. The HET clone is almost identical to a recently published scaffold attachment factor (SAF-B) cloned from a HeLa cell cDNA library. Scaffold attachment factors are a subset of nuclear matrix proteins (NMP) that interact with matrix attachment regions. Analyzing how HET could act as a regulator of hsp27 transcription and as a SAF/NMP, we studied its subnuclear localization and its effect on hsp27 transcription in human breast cancer cells. We were able to show that HET is localized in the nuclear matrix in various breast cancer cell lines. Furthermore, in transient transfection assays using hsp27 promoter-luciferase reporter constructs, HET overexpression resulted in a dose-dependent decrease of hsp27 promoter activity in several cell lines.

Title Nuclear Matrix, Dynamic Histone Acetylation and Transcriptionally Active Chromatin.
Date October 1997
Journal Molecular Biology Reports
Excerpt

The nuclear matrix, the RNA-protein skeleton of the nucleus, has a role in the organization and function of nuclear DNA. Nuclear processes associated with the nuclear matrix include transcription, replication and dynamic histone acetylation. Nuclear matrix proteins, which are tissue and cell type specific, are altered with transformation and state of differentiation. Transcription factors are associated with the nuclear matrix, with the spectra of nuclear matrix bound factors being cell type specific. There is compelling evidence that the transcription machinery is anchored to the nuclear matrix, and the chromatin fiber is spooled through this complex. Transcriptionally active chromatin domains are associated with dynamically acetylated histones. The energy exhaustive process of dynamic histone acetylation has several functions. Acetylation of the N-terminal tails of the core histones alters nucleosome and higher order chromatin structure, aiding transcriptional elongation and facilitating the binding of transcription factors to nucleosomes associated with regulatory DNA sequences. Histone acetylation can manipulate the interactions of regulatory proteins that bind to the N-terminal tails of the core histones. Lastly, dynamic acetylation may contribute to the transient attachment of transcriptionally active chromatin to the nuclear matrix. Reversible histone acetylation is catalyzed by histone acetyltransferase and deacetylase, enzymes associated with the nuclear matrix. The recent isolation and characterization of histone acetyltransferase and deacetylase reveals that these enzymes are related to transcriptional regulators, providing us with new insights about how these enzymes are targeted to nuclear matrix sites engaged in transcription.

Title Nuclear Matrix Proteins in Well and Poorly Differentiated Human Breast Cancer Cell Lines.
Date August 1997
Journal Journal of Cellular Biochemistry
Excerpt

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type-specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1-5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators.

Title Histone Deacetylases Associated with the Msin3 Corepressor Mediate Mad Transcriptional Repression.
Date June 1997
Journal Cell
Excerpt

Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B. Sin3p, the S. cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2. Here, we demonstrate that mSin3A and HDAC1/2 are associated in vivo. HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression. In addition, Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity. Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression. We propose that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription.

Title A Complex Containing N-cor, Msin3 and Histone Deacetylase Mediates Transcriptional Repression.
Date May 1997
Journal Nature
Excerpt

Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.

Title Histone H1b Phosphorylation is Dependent Upon Ongoing Transcription and Replication in Normal and Ras-transformed Mouse Fibroblasts.
Date May 1997
Journal The Journal of Biological Chemistry
Excerpt

We have previously shown that mouse phosphorylated histone H1b (pH1b) was localized near nuclear sites that contained splicing factors. This observation suggested to us that pH1b was associated with transcribing chromatin. Here we investigated the relationship between phosphorylation of H1b and transcription. We demonstrate that treatment of normal or ras-transformed mouse fibroblasts with the transcription inhibitor actinomycin D for 70 min results in a dramatic decrease in the level of pH1b. Similar results were observed when transcription was inhibited by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). When DRB was removed, the level of pH1b was restored after 2 h. Treatment of the cells with aphidicolin, a potent inhibitor of replication, resulted in a marked decrease in the level of pH1b after 30 min. This is the first report showing a dependence of histone modification upon ongoing transcription and replication. Inhibition of transcription or replication may hinder accessibility of H1b to the H1 kinase, supporting the idea that pH1b is associated with transcribing chromatin. Phosphorylation of H1b may be required to maintain a more decondensed chromatin structure to facilitate transcription and replication processes.

Title Developmental Changes in Transcription Factors Associated with the Nuclear Matrix of Chicken Erythrocytes.
Date March 1997
Journal Journal of Cellular Biochemistry
Excerpt

The nuclear matrix has roles in organizing nuclear DNA and in controlling transcription. Transcription factors are associated with the nuclear matrix, with the spectra of transcription factors differing from one cell type to another. In this study we identified the transcription factors and enzymes functioning in the regulation of gene expression that were associated with nuclear matrix and nonmatrix nuclear fractions in erythrocytes isolated from chick embryos at different stages of development, anemic and normal adult birds. We found that the primitive erythroid nuclear matrix had the greatest histone deacetylase activity and highest levels of several transcription factors, including GATA-1, CACCC-binding proteins, and NF1. These transcription factors have key roles in erythroid-specific gene expression. The levels of these transcription factors were lower in the nonmatrix and matrix fractions isolated from definitive erythrocytes. For primitive and definitive erythrocytes, the level of CACCC-binding proteins in the nuclear matrix fraction was greater than that of Sp1. The relative levels of these transcription factors were reversed in the nonmatrix fraction. Casein kinase II was not found in erythroid nuclear matrices. The observed erythroid lineage specific alterations in erythroid nuclear matrix transcription factor composition and abundance may be involved in erythroid-specific gene expression.

Title Estrogen Regulation of Nuclear Matrix-intermediate Filament Proteins in Human Breast Cancer Cells.
Date March 1997
Journal Journal of Cellular Biochemistry
Excerpt

The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T-47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix-intermediate filament fraction (NM-IF) of cells grown in estrogen-replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen-depleted conditions. Replacing estrogen in the medium of acute estrogen-depleted cells restored expression of these proteins. T-47D5 cells that are chronically depleted of estrogen (T5-PRF) are estrogen-nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5-PRF cells with estrogen did not lead to further up-regulation of these proteins. Treating T-47D5 cells in estrogen-replete conditions with the antiestrogens 4-hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long-term chronic estrogen-depleted T-47D5 cells. In conclusion, we have identified NM-IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells.

Title Altered Nuclear Matrix Protein Profiles in Oncogene-transformed Mouse Fibroblasts Exhibiting High Metastatic Potential.
Date January 1997
Journal Cancer Research
Excerpt

The nuclear matrix provides the structural support of the nucleus and is involved in various cellular functions of the nucleus. Nuclear matrix proteins, which are both tissue and cell type specific, are altered with transformation and state of differentiation. Here, nuclear matrix protein profiles of oncogene-transformed mouse fibroblasts with various degrees of metastatic activity were analyzed using two-dimensional gel electrophoresis. This study shows that as the metastatic potential increases, similar nuclear matrix protein profiles are associated with each increase regardless of transformation agent.

Title Changes in the Nuclear Matrix of Chicken Erythrocytes That Accompany Maturation.
Date January 1997
Journal The Biochemical Journal
Excerpt

The protein composition and structure of nuclear matrices isolated from adult chicken immature and mature erythrocytes were analysed. Visualization of nuclear matrices by electron microscopy showed that immature-erythrocyte nuclear matrices had internal structures, while most mature-erythrocyte nuclear matrices did not. Both mature- and immature-erythrocyte nuclear matrices were surrounded by a fibrous network of intermediate filaments. Two-dimensional gel electrophoretic analysis of proteins obtained from fractionated nuclear matrices led to the assignment of the proteins as components of the nuclear porelamina, internal matrix, or cytoskeleton. Common and different proteins belonging to one of the three groups were identified in nuclear matrices of immature and mature erythrocytes. Investigation of the partitioning of histone deacetylase activity, an enzyme associated with the internal matrix, among the erythroid nuclear matrix fractions provided evidence that mature- and immature-erythrocyte nuclear matrices have internal structures. However, the activity of histone deacetylase and level of internal matrix proteins from mature-erythrocyte nuclear matrices were less than those from immature-erythrocyte matrices. The low levels of nuclear RNA and internal matrix proteins may account for lack of visual evidence for an internal matrix in mature erythrocytes.

Title Histone Modifications, Chromatin Structure, and the Nuclear Matrix.
Date December 1996
Journal Journal of Cellular Biochemistry
Excerpt

The nuclear matrix has a role in the organization and function of nuclear DNA. A combination of stable and transient interactions between chromatin and the nuclear matrix is involved in organizing DNA within the nucleus. DNA sequences (matrix attachment regions) at the base of a loop bind to nuclear matrix proteins and arrange the nuclear DNA into chromatin loop domains. Multiple, transient interactions between the nuclear matrix and transcriptionally active chromatin are thought to be responsible for the insoluble feature of transcriptionally active chromatin. Current evidence suggests that histone acetyltransferase, histone deacetylase (enzymes that catalyze rapid histone acetylation and deacetylation), transcription factors, and the transcription machinery mediate the transient attachments between nuclear matrix and active chromatin. Highly acetylated core histones, which are associated with transcriptionally active DNA, are also ubiquitinated and phosphorylated. Recent studies show that specific H1 subtypes and their phosphorylated isoforms are localized in centers of RNA splicing in the nucleus. The implications of these findings and the impact of the histone modifications on the nuclear-organization of chromatin are discussed.

Title Novel Dnase I Hypersensitive Sites in the 3'-flanking Region of the Human C-myc Gene.
Date September 1996
Journal Dna and Cell Biology
Excerpt

DNase I hypersensitivity regions correlate with genetic regulatory loci and binding sites for sequence-specific DNA-binding proteins. We present data supporting the presence of novel DNase 1 hypersensitive sites (which we have designated sites VI-IX) in both the body of the human c-myc gene downstream from exon 2 and the 3'-flanking region of the c-myc gene in HL-60 cells. All of these novel DH sites are markedly decreased when HL-60 cells are treated with either dimethyl sulfoxide (DMSO) or retinoic acid. Moreover, a similar pattern of DNase I hypersensitive sites in this region of c-myc was present in MCF-7 human breast cancer cells growing in culture. Our results suggest a potential role for these sites in transcriptional regulation of the human c-myc gene.

Title Analysis of Human Breast Cancer Nuclear Proteins Binding to the Promoter Elements of the C-myc Gene.
Date September 1996
Journal Journal of Cellular Biochemistry
Excerpt

The expression of the c-myc gene is essential for the proliferation of both hormone-dependent and -independent human breast cancer cells. The regulation of c-myc gene expression in MCF-7 (hormone-dependent, estrogen-receptor (ER)-positive) and MDA MB 231 (hormone-independent, ER-negative) human breast cancer cells differs, with the c-myc gene of MCF-7 but not MDA MB 231 cells being regulated at the transcriptional level by estrogen. We have shown previously that the DNAase I hypersensitive (DH) sites in the c-myc chromatin of hormone-dependent and -independent human breast cancer cells were similar, with the exception of DH site II2. DH site II2, which maps near the P0 promoter, was less sensitive in hormone-dependent than in hormone-independent cells. As DH sites generally indicate the presence of sequence-specific DNA-binding proteins, we undertook a study to identify the nuclear proteins isolated from MCF-7 and MDA MB 231 cells that bound to the P0 and P2 promoter regions of the c-myc gene in vitro. The studies presented here provide evidence that Sp1 and/or Sp1-like proteins bind to the P0 and P2 promoter regions of the c-myc gene of MCF-7 and MDA MB 231 cells. Furthermore, evidence is presented for the presence of several previously unidentified sequence-specific DNA-binding proteins binding to these promoters. The DNA-binding activities of these latter proteins differed in the nuclear extracts of the MCF-7 and MDA MB 231 human breast cancer cells.

Title Properties of Chicken Erythrocyte Histone Deacetylase Associated with the Nuclear Matrix.
Date August 1996
Journal The Biochemical Journal
Excerpt

Histone H2B is deacetylated more rapidly than H3 and H4 in chicken immature erythrocytes. Histone deacetylase from chicken immature erythrocytes was partially purified, and the histone specificities of the multiple histone deacetylase forms were determined. Ion-exchange (Q-Sepharose) and gel-exclusion (Superdex 200) chromatography of extracts from erythrocyte nuclei showed two forms (HD1 and HD2) of histone deacetylase. HD1, with a molecular mass of about 55 kDa, preferred free H3-H4 relative to H2A-H2B, while HD2, with a molecular mass of approx. 220 kDa, had a slight preference for H3-H4. HD1 and HD2 differed in pH- and ion-strength-dependence. HD2 dissociated into HD1 when treated with 1.6 M NaCl or when applied to a Q-Sepharose column. The enzymic properties of nuclear-matrix-bound histone deacetylase showed a striking difference from that of HD1 and HD2, particularly in its strong preference for H2A-H2B. Treatment of the nuclear matrix with 1.6 M NaCl and 1% 2-mercaptoethanol solubilized histone deacetylase which chromatographed as 400 and 220 kDa forms on a Superdex 200 column. The solubilized enzyme retained its histone preference for H2A-H2B. Chromatography of the nuclear-matrix-derived enzyme on Q-Sepharose yielded one peak of enzyme activity with chromatographic properties and histone specificities similar to those of HD1. These results provide support for the active form of the enzyme in situ being a high-molecular mass complex associated with proteins that are components of the nuclear matrix. Substrate preference of the enzyme is governed by the proteins associated with the histone deacetylase.

Title In Situ Footprinting of Chicken Histone H5 Gene in Mature and Immature Erythrocytes Reveals Common Factor-binding Sites.
Date June 1996
Journal Chromosoma
Excerpt

In vitro DNAase I footprinting and gel mobility shift assays have shown that the activities of several nuclear factors (GATA-1, Sp1) that bind to the promoter and downstream enhancer regions of the chicken histone H5 gene are reduced in mature erythrocytes relative to those in immature erythrocytes. In this study we investigated site occupancy in the promoter and downstream enhancer regions of the H5 gene in mature and immature erythrocytes. The ligation-mediated polymerase chain reaction was used to detect DNAase I footprints generated in situ. Most of the sites that bound to Sp1 and/or Sp1-like proteins and GATA-1 in the promoter and enhancer were occupied in situ in mature and immature erythrocytes. However, the level of protection at Sp1/Sp-1-like binding sites in the H5 enhancer region of mature erythroid cells was generally less than that observed for immature cells, suggesting that for any given mature cell not all of the Sp1/Sp1-like binding sites are occupied. Nevertheless, the results of this study suggest that the enhancer and promoter of the H5 gene in mature erythrocytes should be functional, agreeing with nuclear run-on studies showing transcriptional activity of the H5 gene in mature permeabilized cells.

Title The Nuclear Matrix and the Regulation of Chromatin Organization and Function.
Date March 1996
Journal International Review of Cytology
Excerpt

Nuclear DNA is organized into loop domains, with the base of the loop being bound to the nuclear matrix. Loops with transcriptionally active and/or potentially active genes have a DNase I-sensitive chromatin structure, while repressed chromatin loops have a condensed configuration that is essentially invisible to the transcription machinery. Core histone acetylation and torsional stress appear to be responsible for the generation and/or maintenance of the open potentially active chromatin loops. The transcriptionally active region of the loop makes several dynamic attachments with the nuclear matrix and is associated with core histones that are dynamically acetylated. Histone acetyltransferase and deacetylase, which catalyze this rapid acetylation and deacetylation, are bound to the nuclear matrix. Several transcription factors are components of the nuclear matrix. Histone acetyltransferase, deacetylase, and transcription factors may contribute to the dynamic attachment of the active chromatin domains with the nuclear matrix at sites of ongoing transcription.

Title Fibroblasts Transformed by Combinations of Ras, Myc and Mutant P53 Exhibit Increased Phosphorylation of Histone H1 That is Independent of Metastatic Potential.
Date February 1996
Journal Febs Letters
Excerpt

H1 histones play an important role in regulating higher order structure of chromatin and are potential regulators of gene expression. H1s are phosphorylated, a modification which alters their interaction with DNA. We measured the abundance of three phosphorylated H1 subtypes in mouse fibroblasts transformed by combinations of ras, myc and mutant p53 which differ in metastatic potential. We found that there is an increase in phosphorylation of H1 subtypes in fibroblasts transformed with ras, myc and mutant p53. This increase was found to correlate with cellular transformation but not with induction of the metastatic phenotype.

Title Histones of Chlamydomonas Reinhardtii. Synthesis, Acetylation, and Methylation.
Date December 1995
Journal Plant Physiology
Excerpt

Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a single threonine as residue 28 instead of the serine28-alanine29 sequence, present in all other known plant and animal H3 histones.

Title Expression and Characterization of Branched-chain Alpha-ketoacid Dehydrogenase Kinase from the Rat. Is It a Histidine-protein Kinase?
Date September 1995
Journal The Journal of Biological Chemistry
Excerpt

The recombinant rat branched-chain alpha-ketoacid dehydrogenase kinase has been amplified from rat kidney cDNA, based on the previously reported rat cDNA sequence (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 13127-13130). This kinase was expressed in Escherichia coli as a fusion protein with bacterial maltose-binding protein (MBP). Expression was improved by overexpression of chaperonins GroEL and GroES. The MBP-kinase, when reconstituted with lipoylated recombinant E2 (dihydrolipoyl transacylase), catalyzed phosphorylation of recombinant E1 (branched-chain alpha-ketoacid decarboxylase) with a kcat of 28.5 nmol of phosphate/min/nmol of MBP-kinase at 25 degrees C. Recombinant MBP-kinase alone demonstrated a slow rate of autophosphorylation with a kcat of 3.25 pmol of phosphate/min/nmol of kinase at 25 degrees C. Serine 22 of the kinase was identified as the possible site of autophosphorylation by Edman microsequencing analysis. Autophosphorylated kinase cannot transfer phosphate to E1, indicating that autophosphorylation of kinase is not an intermediate in ATP-dependent phosphorylation of E1. Therefore, despite the reported sequence similarity to prokaryotic histidine protein kinases, the mitochondrial rat branched-chain alpha-ketoacid dehydrogenase kinase apparently does not phosphorylate E1 via a histidine-mediated phosphotransfer reaction. Significant corrections to the published cDNA sequence of rat branched-chain alpha-ketoacid dehydrogenase kinase are included.

Title Increased Phosphorylation of Histone H1 in Mouse Fibroblasts Transformed with Oncogenes or Constitutively Active Mitogen-activated Protein Kinase Kinase.
Date September 1995
Journal The Journal of Biological Chemistry
Excerpt

We compared the nucleosomal organization, histone H1 subtypes, and histone H1 phosphorylated isoforms of ras-transformed and parental 10T1/2 mouse fibroblasts. In agreement with previous studies, we found that ras-transformed mouse fibroblasts have a less condensed chromatin structure than normal fibroblasts. ras-transformed and parental 10T1/2 cells had similar amounts of H1 subtypes, proteins that have a key role in the compaction of chromatin. However, labeling studies with 32P and Western blot experiments with an antiphosphorylated H1 antibody show that interphase ras-transformed cells have higher levels of phosphorylated H1 isoforms than parental cells. G1/S phase-arrested ras-transformed cells had higher amounts of phosphorylated H1 than G1/S phase-arrested parental cells. Mouse fibroblasts transformed with fes, mos, raf, myc, or constitutively active mitogen-activated protein (MAP) kinase kinase had increased levels of phosphorylated H1. These observations suggest that increased phosphorylation of H1 is one of the consequences of the persistent activation of the mitogen-activated protein kinase signal transduction pathway. Indirect immunofluorescent studies show that phosphorylated H1b is localized in centers of RNA splicing in the nucleus, suggesting that this modified H1 subtype is complexed to transcriptionally active chromatin.

Title Molecular Basis of Maple Syrup Urine Disease and Stable Correction by Retroviral Gene Transfer.
Date July 1995
Journal The Journal of Nutrition
Excerpt

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex. This results in the accumulation of the branched-chain amino acids (BCAA) and branched-chain alpha-keto acids (BCKA), which often produce severe neurological damage and mental retardation. The present studies focus on mutations in the E1 alpha gene of the BCKAD complex and their effects on the assembly of the E1 decarboxylase component of the enzyme complex. We have developed an efficient histidine-tagged bacterial expression system that allows the folding and assembly of E1 alpha and E1 beta subunits into the E1 heterotetramer (alpha 2 beta 2) in the presence of overexpressed chaperonins GroEL and GroES. The results of pulse-chase experiments with this bacterial expression system showed that a majority of the 15 known E1 alpha mutations, including the prevalent Y393N of Mennonite MSUD patients, decrease the rate of association of normal E1 beta with mutant E1 alpha. This results in limited or no assembly of mutant E1. It is concluded that the carboxy-terminal region of the E1 alpha subunit encoded by exons 7-9 is important for subunit interaction. To stably correct MSUD, we have developed a retroviral vector that contains a normal E1 alpha precursor complementary DNA. Transduction of cultured lymphoblasts from a Mennonite MSUD patient with this recombinant retroviral vector completely restored the rate of decarboxylation of BCKA. The normal decarboxylation activity in transduced MSUD cells remained stable without antibiotic selection during the 14-week study.(ABSTRACT TRUNCATED AT 250 WORDS)

Title Molecular and Biochemical Basis of Intermediate Maple Syrup Urine Disease. Occurrence of Homozygous G245r and F364c Mutations at the E1 Alpha Locus of Hispanic-mexican Patients.
Date April 1995
Journal The Journal of Clinical Investigation
Excerpt

Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.

Title Differential Compaction of Transcriptionally Competent and Repressed Chromatin Reconstituted with Histone H1 Subtypes.
Date March 1995
Journal Biochimica Et Biophysica Acta
Excerpt

Chromatin fragments stripped of H1 histones regain the ability to form higher order structures and aggregates in 0.15 M NaCl following reconstitution with histone H1. However, transcriptionally competent chromatin fragments are resistant to chicken erythrocyte H1/H5 histone-induced 0.15 M NaCl aggregation/precipitation. In this study, we investigated the ability of stripped chromatin fragments reconstituted with one of four histone H1 subtypes (chicken erythrocyte H1, H5, trout liver H1a, H1b) at various stoichiometries to form salt precipitable higher order structures. Our results provide evidence that chicken erythrocyte histone H1 was more effective than histone H5 and trout liver histone H1b better than H1a in forming higher order structures. None of the histone H1 subtypes could render transcriptionally competent chromatin fragments insoluble in 0.15 M NaCl. These results are consistent with the ideas that the histone H1 subtypes differ in their capacities to compact chromatin fiber, and that the alterations in the structure of transcriptionally competent nucleosomes interfere with the capacity of all H1 subtypes to form higher order structures.

Title Nuclear Factor 1 is a Component of the Nuclear Matrix.
Date October 1994
Journal Journal of Cellular Biochemistry
Excerpt

Chicken histone H5 is an H1-like linker histone that is expressed only in nucleated erythrocytes. The histone H5 promoter has binding sites for Sp1 (a high affinity site) and UPE-binding protein, while the 3' erythroid-specific enhancer has binding sites for Sp1 (one moderate and three weak affinity), GATA-1, and NF1. In this study we investigated whether trans-acting factors that bind to the chicken histone H5 promoter or enhancer are associated with adult chicken immature and mature erythrocyte nuclear matrices. We show that NF1, but not Sp1, GATA-1, or UPE-binding protein, is associated with the internal nuclear matrices of these erythroid cells. Further, we found that a subset of the NF1 family of proteins is bound to the mature erythrocyte nuclear matrix. These results suggest that in chicken erythrocytes NF1 may mediate an interaction between the histone H5 enhancer and the erythroid internal nuclear matrix. NF1 was also present in the internal nuclear matrices of chicken liver and trout liver. The observations of this study provide evidence that NF1 may have a role in a variety of cell types in targeting specific DNA sequences to the nuclear matrix.

Title Multiple Functions of Dynamic Histone Acetylation.
Date October 1994
Journal Journal of Cellular Biochemistry
Excerpt

Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear functions, including replication, transcription, and RNA splicing. It is becoming increasingly evident that nuclear processes are localized to distinct regions in the nucleus. For example, transcriptionally active genes and RNA transcripts are found in discrete transcription foci. Current evidence suggests that nuclear matrix-bound transcriptionally active DNA sequences are in nucleosomes with dynamically acetylated histones. Histone acetylation, which precedes transcription, alters nucleosome and chromatin structure, decondensing the chromatin fibre and making the nucleosomal DNA accessible to transcription factors. Histone acetyltransferase and histone deacetylase, which catalyze this rapid acetylation and deacetylation, are associated with the internal nuclear matrix. We hypothesize that these enzymes play a role in maintaining the association of the active chromatin domains with the internal nuclear matrix at sites of ongoing transcription.

Title Histone Acetyltransferase is Associated with the Nuclear Matrix.
Date October 1994
Journal The Journal of Biological Chemistry
Excerpt

Only a small fraction of the adult chicken erythrocyte histones is involved in dynamic acetylation. We have reported previously that the rapidly acetylated and deacetylated H4 histones are primarily associated with the transcriptionally active DNA-enriched chromatin fragments that remain attached to the residual nuclear material following micrococcal nuclease digestion and chromatin solubilization. Furthermore, this nuclear fraction contained most of the histone deacetylase activity. In this study we show that the bulk of the nuclear histone acetyltransferase activity is located with the insoluble residual nuclear material. We demonstrate that in vitro the enzymes associated with the residual nuclear material catalyze reversible acetylation when the endogenous histones of the nuclear skeleton-bound chromatin fragments are used as substrate. Nuclear matrices isolated from adult chicken immature erythrocyte and trout liver nuclei had 60-76% of the nuclear histone acetyltransferase activity. Procedures that solubilized the internal nuclear matrix also resulted in the release of the enzyme from the nuclear matrix. Together, our observations suggest that histone acetyltransferase and deacetylase are associated with the internal nuclear matrix, and one of the functions of these enzymes may be to mediate a dynamic attachment between transcriptionally active chromatin and the nuclear matrix.

Title Inhibition of Transcription Selectively Reduces the Level of Ubiquitinated Histone H2b in Chromatin.
Date September 1994
Journal Biochemical and Biophysical Research Communications
Excerpt

The effect of inhibiting transcription and/or replication on the steady state levels of the ubiquitinated histone isoforms was investigated. We show that treatment of chinese hamster ovary (CHO), monkey kidney (COS), human endometrial carcinoma (Hec-50 and Ishikawa) cells with actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the levels of ubiquitinated (u) H2B, but not uH2A, uH2A.Z, polyubiquitinated H2A or a novel ubiquitinated histone species, in the chromatin of these cells. The level of the ubiquitinated histones was not affected when synthesis of DNA was arrested. These results show that, in general, maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription.

Title In Vitro Reconstitution of the 24-meric E2 Inner Core of Bovine Mitochondrial Branched-chain Alpha-keto Acid Dehydrogenase Complex: Requirement for Chaperonins Groel and Groes.
Date August 1994
Journal Biochemistry
Excerpt

We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain alpha-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn.HCl). The denatured E2c monomers (apparent M(r) = 27,000) were diluted 100-fold at 25 degrees C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn.HCl at 25 degrees C. The E2c trimers with an apparent M(r) of 84,000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn.HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins.(ABSTRACT TRUNCATED AT 250 WORDS)

Title Molecular Chaperones: Heat-shock Proteins, Foldases, and Matchmakers.
Date August 1994
Journal The Journal of Laboratory and Clinical Medicine
Excerpt

The term molecular chaperone includes a large family of unrelated proteins that comprise both stress-inducible and constitutive molecules. In recent years, molecular chaperones or heat-shock proteins (Hsps) have emerged as fundamentally important topics in cell biology. Living organisms respond to stressful conditions, such as heat shock, by rapidly producing a relatively small class of specific proteins that function to stabilize cellular components against stress. Hsps or molecular chaperones function by preventing misfolding of newly synthesized proteins, escorting proteins targeted for other cellular compartments, and modulating or regulating proteins involved in cell growth and differentiation. Clearly these proteins are of enormous importance to cellular function but of even more importance in helping to fight disease. From heart tissue protection after coronary thrombosis to the regulation of tumor suppressor activity to the use of these molecules as new diagnostic and therapeutic agents, molecular chaperones offer scientists many potential rewards. This review provides an insider's peek at molecular chaperones--a most indispensable set of molecules for cell growth, survival, and regulation.

Title Transcription Factor Gata-1-multiprotein Complexes and Chicken Erythroid Development.
Date May 1994
Journal Febs Letters
Excerpt

The chicken erythrocyte transcription factor, GATA-1, is associated with several non-DNA binding proteins. We show that GATA-1 multiprotein complexes exist in primitive and definitive erythrocytes. These complexes bind to GATA motifs of the rho-globin promoter and histone H5 enhancer with high affinity, and to the chicken beta-globin promoter specialized TATA element and enhancer GATA with low affinity. The low affinity beta-globin TATA element would allow basal transcription factors to displace the GATA-1 multiprotein complex. Further, our results suggest that rho-globin promoter's low affinity Sp1 binding site and reduced levels of Sp1 in definitive cells prevent its expression in these cells.

Title Effects of Histone Acetylation, Ubiquitination and Variants on Nucleosome Stability.
Date February 1994
Journal The Biochemical Journal
Excerpt

The properties of the nucleosomes of a salt-soluble, transcriptionally active gene-enriched fraction of chicken erythrocyte chromatin were evaluated by hydroxyapatite dissociation chromatography. We have demonstrated previously that the salt-soluble, transcriptionally active gene-enriched polynucleosomes are enriched in dynamically acetylated and ubiquitinated histones, and in an atypical U-shaped nucleosome that possessed about 20% less protein than a typical nucleosome. Further, newly synthesized histones H2A and H2B exchange preferentially with the nucleosomal histones H2A and H2B of this salt-soluble chromatin fraction. Analysis of the histones eluting from the hydroxyapatite-bound chromatin demonstrated that hyperacetylated and ubiquitinated (u), including multi-ubiquitinated, H2A-H2B.1 dimers dissociated at lower concentrations of NaCl than unmodified dimers or dimers with histone variants H2A.Z and/or H2B.2. Cross-linking studies revealed that at least 50% of uH2B.1 was paired with uH2A. uH2A-uH2B.1 dimers dissociated at lower NaCl concentrations than H2A-uH2B.1 dimers. Hyperacetylated histone (H3-H4)2 tetramers also eluted at lower concentrations of NaCl than unmodified tetramers. Our results support the idea that acetylation and ubiquitination of histones H2A and H2B.1 increase the lability of H2A-H2B.1 dimers in transcriptionally active nucleosomes. In contrast, our observations suggest that histone variants H2A.Z and H2B.2. stabilize the association of the H2A-H2B dimer in nucleosomes. The elevated lability of the H2A-H2B dimer may facilitate processes such as the exchange of these dimers with newly synthesized histones, the elongation process of transcription and transcription factor binding.

Title Repression of Histone H5 Gene Expression in Chicken Mature Erythrocytes is Correlated with Reduced Dna-binding Activities of Transcription Factors Sp1 and Gata-1.
Date October 1993
Journal Febs Letters
Excerpt

During the final stages of erythroid maturation, the expression of the chicken histone H5 gene ceases. The histone H5 promoter has binding sites for Sp1 and UPE-binding protein. The 3' histone H5 enhancer has binding sites for Sp1, GATA-1 and NF1. Here, we show that the DNA-binding activities of transcription factors Sp1 and GATA-1 is reduced 5- to 10-fold in mature cells, while the activities of UPE-binding protein and NF1 remain the same in mature and immature erythrocytes. The reduced activities of Sp1 and GATA-1 may contribute to the inactivation of the histone H5 gene in mature erythrocytes.

Title Molecular Cloning and Cdna Sequence Analysis of Coho Salmon Stanniocalcin.
Date May 1993
Journal Molecular and Cellular Endocrinology
Excerpt

Stanniocalcin (STC) (formerly known as both teleocalcin and hypocalcin) is an anti-hypercalcemic, glycoprotein hormone that is produced by the corpuscles of Stannius (CS), endocrine glands that are confined to bony fishes. The hormone has a unique amino acid sequence and exists as a disulfide-linked homodimer in the native state. In previous studies, we have described the purification and characterization of two salmon STCs, and examined the regulation of hormone secretion in response to calcium using both in vitro and in vivo model systems. This report describes the molecular cloning and cDNA sequence analysis of a coho salmon STC messenger RNA (mRNA) from a salmon CS lambda gt10 cDNA library. The STC mRNA in salmon is approximately 2 kilobases in length and encodes a primary translation product of 256 amino acids. The first 33 residues comprise the prepro region of the hormone, whereas the remaining 223 residues make up the mature form of the hormone. One N-linked, glycosylation consensus sequence was identified in the protein coding region as well as an odd number of half cysteine residues, the latter of which would allow for interchain bonding or dimerization of monomeric subunits. In addition, three sites were identified in the mature protein core of STC (two dibasic, one tribasic) that may be acted upon by endopeptidases to produce truncated forms of the hormone. In support of this latter possibility, Western blot analysis revealed multiple molecular weight forms of sTC within salmon glands.

Title C-myc Gene Chromatin of Estrogen Receptor Positive and Negative Breast Cancer Cells.
Date May 1993
Journal Molecular and Cellular Endocrinology
Excerpt

Expression of the c-myc protooncogene is estrogen regulated in estrogen receptor (ER) positive, hormone-dependent human breast cancer cells, but it is constitutively active in ER negative, hormone-independent breast cancer cells. To determine whether these differences are reflected in c-myc chromatin, DNase I hypersensitive sites (DHS) were mapped. Six DHS were detected in all cell lines studied, with DHS 3(2) being more prominent than DHS 3(1). The accessibility of DHS 2 was markedly greater in ER negative cells than in ER positive cells, and this relative accessibility remained unchanged when cells were grown in estrogen free medium. DHS 2, 3(1) and 3(2) map near the P0, P1 and P2 promoters, respectively. An analysis of promoter usage demonstrated that P2 was the preferred promoter. Thus, the differences in the accessibility of DHS 2 in c-myc chromatin of ER positive and negative cells likely reflects alterations in DNA-protein interactions in this region.

Title Nuclear Matrix Proteins Bind Very Tightly to Specific Regions of the Chicken Histone H5 Gene.
Date February 1993
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

The nuclear matrix is operationally defined as the structure remaining after nuclease-digested nuclei are extracted with high concentrations of salt. The nuclear matrix is thought to have a role in organizing higher order chromatin into loop domains. We determined whether specific regions of the histone H5 gene were very tightly bound to protein of erythrocyte and liver nuclear matrices in vitro. We demonstrate that DNA fragments spanning sequences 5' to the promoter and the 3' enhancer region of the histone H5 gene, but not DNA fragments spanning the promoter, were very tightly bound to protein of nuclear matrices of erythrocytes and liver. The nuclear matrix consists of internal nuclear matrix and nuclear pore-lamina complex. Recently, we demonstrated that histone deacetylase could be used as a marker enzyme of the internal nuclear matrix. We demonstrate that nuclear pore-lamina complex preparations that were depleted of histone deacetylase activity, and thus of internal nuclear matrix, retained the protein that bound very tightly to the beta-globin and histone H5 enhancers. These results provide evidence that specific regions of the histone H5 gene are very tightly bound to nuclear pore-lamina complex protein.

Title Analysis of Erythroid Nuclear Proteins Binding to the Promoter and Enhancer Elements of the Chicken Histone H5 Gene.
Date February 1993
Journal Nucleic Acids Research
Excerpt

The chicken erythroid proteins binding to the histone H5 5' promoter and 3' erythroid-specific enhancer regions were identified. In DNase I footprinting and gel mobility shift experiments with immature adult erythrocyte nuclear extracts, we have demonstrated the binding of proteins to the GC-box, a high affinity Sp1 binding site, and to the upstream promoter element. We have previously demonstrated that a multisubunit complex containing the transcription factor GATA-1 was associated with the enhancer. Here, we show that the enhancer region also has four Sp1 binding sites (one medium and three weak affinity, one of which may also bind the CACCC factor), a potential NF-E4 binding site, and a binding site for a NF1-like factor. The results of gel mobility-shift and competition experiments provide evidence that the Sp1 binding sites are associated with a high molecular mass (greater than 450 kDa), Sp1 containing protein complex. We propose that Sp1 multimers bound at the promoter and enhancer interact to mediate the juxta-positioning of the enhancer and promoter elements, bringing the GATA-1 multisubunit complex next to the initiation site. The GATA-1 complex may contribute to the protein-protein interactions between the enhancer and promoter.

Title Expression and Assembly of a Functional E1 Component (alpha 2 Beta 2) of Mammalian Branched-chain Alpha-ketoacid Dehydrogenase Complex in Escherichia Coli.
Date September 1992
Journal The Journal of Biological Chemistry
Excerpt

We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1 alpha and E1 beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1 alpha subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide. When co-expressed, the MBP-E1 alpha fusion and E1 beta subunits were shown to co-purify as a MBP-E1 component that exhibited both E1 activity and binding competence for recombinant branched-chain E2 component. In contrast, in vitro mixing of individually expressed MBP-E1 alpha and E1 beta did not result in assembly or produce E1 activity. Following proteolytic removal of the affinity ligand and linker peptide with Factor Xa, a recombinant E1 species was eluted from a Sephacryl S-300HR sizing column as an enzymatically active 160-kDa species. The latter showed 1:1 subunit stoichiometry, which was consistent with an alpha 2 beta 2 structure. The recovery of this 160-kDa recombinant E1 species (estimated at 0.07% of total lysate protein) was low, with the majority of the recombinant protein lost as insoluble aggregates. Our findings suggest that the concurrent expression of both E1 alpha and E1 beta subunits in the same cellular compartment is important for assembly of both subunits into a functional E1 alpha 2 beta 2 heterotetramer. By using this co-expression system, we also find that the E1 alpha missense mutation (Tyr-393----Asn) characterized in Mennonites with maple syrup urine disease prevents the assembly of soluble E1 heterotetramers.

Title Western Blotting and Immunochemical Detection of Histones Electrophoretically Resolved on Acid-urea-triton- and Sodium Dodecyl Sulfate-polyacrylamide Gels.
Date August 1992
Journal Analytical Biochemistry
Excerpt

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.

Title Chaperonins Groel and Groes Promote Assembly of Heterotetramers (alpha 2 Beta 2) of Mammalian Mitochondrial Branched-chain Alpha-keto Acid Decarboxylase in Escherichia Coli.
Date August 1992
Journal The Journal of Biological Chemistry
Excerpt

We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase (E1) in Escherichia coli. The mature E1 alpha subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1 beta on the same vector in ES- and EL- mutant strains. Only small or trace amounts of active E1 component were obtained. Cotransformation of the ES- mutant host with a second vector overexpressing groEL and groES resulted in a greater than 500-fold increase in E1-specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the content of both MBP-E1 alpha and E1 beta polypeptides was markedly increased in the presence of overexpressed chaperonin proteins. The time course studies showed that the increase in E1-specific activity and subunit levels correlated with the increase in groEL and groES until the concentration of the chaperonins reached a saturating level in the cell. The functional MBP-E1 fusion protein from ES- double transformants were purified by amylose resin affinity chromatography. The MBP moiety was removed by subsequent digestion with Factor Xa endoprotease, followed by Sephacryl S-300HR chromatography. It was found that E1 alpha and E1 beta assembled into an active 160-kDa species, which was consistent with the alpha 2 beta 2 structure of E1. The present results demonstrate that chaperonins groEL and groES promote folding and assembly of heterotetrameric proteins of mammalian mitochondrial origin.

Title Acetylation and Methylation of Histones H3 and H4 in Chicken Immature Erythrocytes Are Not Directly Coupled.
Date July 1992
Journal Biochemical and Biophysical Research Communications
Excerpt

The relationship between histone methylation and dynamic histone acetylation was investigated. Previously, we demonstrated in chicken erythrocytes that dynamically acetylated histones H3 and H4 of transcriptionally active gene chromatin were selectively methylated. However, methylation of these histones was not dependent upon their acetylated states. Here, we tested the hypothesis that methylation tags these histones for participation in dynamic acetylation. Using an inhibitor of protein methylation, adenosine dialdehyde, we show that the processes of histone methylation and dynamic acetylation are not directly coupled. Our results suggest that the selective methylation of dynamically acetylated chromatin reflects features of the organization of transcriptionally active gene chromatin.

Title Multisubunit Erythroid Complexes Binding to the Enhancer Element of the Chicken Histone H5 Gene.
Date June 1992
Journal The Biochemical Journal
Excerpt

We identified the factor(s) that bind to the chicken erythroid-cell-specific histone H5 enhancer region which is located on the 3' end of the gene. In DNAase I footprinting and u.v. cross-linking experiments with nuclear extracts from adult chicken immature erythrocytes, we determined that the trans-acting factor GATA-1 was the predominating protein interacting with the histone H5 enhancer. GATA-2 and GATA-3 were not detected. In contrast, gel-mobility-shift assays and competition experiments demonstrated that several specific complexes formed with the histone H5 enhancer region. Gel-mobility-shift assays with 23 bp oligonucleotides containing the GATA-binding site (AGATAA) of the histone H5 enhancer or of the beta-globin enhancer showed that the GATA sequence was sufficient for the formation of at least five complexes. Diagonal mobility-shift assays demonstrated that multisubunit complexes were forming with the GATA-1 protein. Our interpretation of the results is that GATA-1 interacts with a protein of approx. 105 kDa which, in turn, can associate with protein or protein complexes of approx. 26 kDa, 146 kDa and a protein(s) of molecular mass greater than 450 kDa. The different multisubunit complexes formed via the trans-acting factor GATA-1 may impart different transcriptional responses to the promoter and enhancer elements of the histone H5 and globin genes.

Title Colonic Aberrant Crypt Foci Are Associated with Increased Expression of C-fos: the Possible Role of Modified C-fos Expression in Preneoplastic Lesions in Colon Cancer.
Date June 1992
Journal Carcinogenesis
Excerpt

Aberrant crypt foci represent the earliest detectable lesions of colon cancer. The expression of c-fos in these aberrant crypts was determined by in situ hybridization and immunohistochemistry. These lesions were induced in the colonic epithelium with azoxymethane using the rat model system. Expression of c-fos was markedly increased in the aberrant colonic crypts. Increases of approximately 60 and approximately 70% in the proportion of epithelial cells labelled were observed in the early and advanced aberrant crypts respectively. This was found to be statistically significant (P less than 0.001). Consistent with cell proliferation patterns in the colonic crypts, the epithelial cells of the lower crypt had greater levels of c-fos mRNA than those of the upper part of the crypts. In addition, immunohistochemical staining with c-fos polyclonal antibodies demonstrated increased levels of c-fos protein in aberrant crypts. This combined approach using in situ hybridization and immunohistochemistry has shown that increased c-fos expression at the RNA level in these lesions is associated with increased amounts of c-fos protein. Since c-fos has been implicated in the process of cell proliferation and differentiation, modifications in its expression may be significant to understanding the mechanisms whereby preneoplastic lesions transform to neoplastic lesions in colon cancer.

Title Intracellular Histamine and Liver Regeneration: High Affinity Binding of Histamine to Chromatin, Low Affinity Binding to Matrix, and Depletion of a Nuclear Storage Pool Following Partial Hepatectomy.
Date June 1992
Journal Biochemical and Biophysical Research Communications
Excerpt

We have demonstrated in rat hepatocytes that 3H-histamine binds specifically to novel low (microM) and high (nM) affinity sites, designated "HIC" to denote their intracellular location. Low affinity HIC sites are associated with microsomes, while both low and high affinity HIC sites are associated with the nucleus. A growth-regulatory action of intracellular histamine at HIC, independent of the rise in cytosolic calcium, has been demonstrated in mitogen-stimulated lymphocytes. We now report that the high affinity HIC sites in liver cell nuclei are associated exclusively with chromatin, while only low affinity sites are found in the residual material containing the nuclear matrix. Moreover, hepatocyte nuclei contain histamine (approximately 1 ng/mg protein), unaffected by incubation for up to 18 hours with the histidine decarboxylase inhibitor, alpha-FMH, suggesting a slow rate of turnover typical of a storage pool. A decrease in nuclear histamine parallels a rise in DNA synthesis in the first 24 hours after partial hepatectomy. Our findings support a role for a nuclear pool of pre-formed histamine in the mediation of liver regeneration.

Title Expression of Rainbow Trout Apolipoprotein A-i Genes in Liver and Hepatocellular Carcinoma.
Date May 1992
Journal Journal of Lipid Research
Excerpt

Screening for genes overexpressed in trout aflatoxin B1-induced hepatocellular carcinomas resulted in the isolation of cDNA sequences of two apolipoprotein A-Is, apoA-I-1 and apoA-I-2. The levels of apoA-I-1 and -2 mRNAs of liver and tumor were quite different. ApoA-I-1 mRNA was the major species in liver, while apoA-I-1 and -2 mRNAs were present at similar levels in several tumors. This elevated level of apoA-I-2 mRNA was observed in seven different tumors, suggesting that the overexpression of apoA-I-2 was a general feature of aflatoxin B1-induced liver tumors. Hybridization to genomic DNA demonstrated that trout has two different apoA-I genes which is in contrast to other vertebrates which have one gene coding for apoA-I. Liver apoA-I-1 and -2 cDNA clones specified the same amino acid sequence as the tumor apoA-I-1 and -2 cDNA clones. Analysis of the cDNA-derived amino acid sequences showed that trout apoA-I-1 and -2, like human apoA-I, consist largely of multiple 22 amino acid repeats having the potential to generate an amphipathic alpha-helix. The similarity of the repeat pattern in trout and human apoA-Is suggests that all the internal repeats in these sequences arose before the fish-mammal split, some 400 million years ago.

Title Nuclear Distribution of Histone Deacetylase: a Marker Enzyme for the Internal Nuclear Matrix.
Date May 1992
Journal Biochimica Et Biophysica Acta
Excerpt

Nuclear matrins are proteins that localize to the internal nuclear matrix. In a previous study, we reported that histone deacetylase is a component of the internal matrix, suggesting that histone deacetylase is a nuclear matrin. Here, we demonstrate that the majority of the histone deacetylase activity is associated with the internal nuclear matrices of chicken and trout liver. Thus, the association of the histone deacetylase with the internal nuclear matrix is neither tissue- nor species-specific. Using histone deacetylase as a marker enzyme for the partitioning of the internal nuclear matrix during nuclear fractionations, we show that in contrast to the internal nuclear matrices of trout liver, trout hepatocellular carcinoma and chicken liver, the stability of the chicken erythrocyte internal nuclear matrix is temperature-dependent. Our results support a model that has the histone deacetylase mediating transient interactions between the internal nuclear matrix and chromatin regions undergoing dynamic acetylation, for example transcriptionally active chromatin regions.

Title Cloning and Expression in Escherichia Coli of Mature E1 Beta Subunit of Bovine Mitochondrial Branched-chain Alpha-keto Acid Dehydrogenase Complex. Mapping of the E1 Beta-binding Region on E2.
Date February 1992
Journal The Journal of Biological Chemistry
Excerpt

A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha-keto acid dehydrogenase complex was isolated from a lambda ZAP expression library. The bovine E1 beta cDNA is 1,393 base pairs in length. It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues. The calculated molecular mass of the mature bovine E1 beta subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04. The mature bovine E1 beta subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG). When expression was induced by IPTG at 37 degrees C, the soluble recombinant E1 beta subunit existed as a single high molecular weight form (Mr congruent to 3.5 x 10(5)), which sedimented during sucrose gradient ultracentrifugation at 2 x 10(5) x g. However, lowering the induction temperature to 25 degrees C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1 beta protein. The low molecular weight form (Mr congruent to 9.1 x 10(4)) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins. The results showed that the E1 beta subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain. In contrast, the recombinant E1 alpha subunit did not bind the E2 component. The data suggest an apparent binding order of E2-E1 beta-E1 alpha, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M.L., Xu, W.-X., Oliver, R.M., Wall, J.S., Hainfeld, J.F., Mullinax, T.R., and Reed, L.J. (1989) Biochemistry 28, 6816-6821). The relatively inaccessible topology of E1 beta may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex.

Title Characterization and Chromatin Distribution of the H1 Histones and High-mobility-group Non-histone Chromosomal Proteins of Trout Liver and Hepatocellular Carcinoma.
Date January 1992
Journal The Biochemical Journal
Excerpt

The H1 histones serve as general repressors of gene expression by inducing the formation of a compact chromatin structure, whereas the high-mobility-group (HMG) non-histone chromosomal proteins have roles in maintaining the structure and function of transcriptionally active chromatin. The distribution of the H1 histone subtypes and HMG proteins among various trout tissues (liver, hepatocellular carcinoma, testis and erythrocyte) was determined. Histone H1b was present in the chromatin of liver, but not in the chromatin of hepatocellular carcinoma, testis or erythrocyte. Nuclease-resistant regions of liver chromatin had elevated levels of histone H1b. Histone H1b was isolated, and the N-terminal amino acid sequence of histone H1b was found to be highly similar to that of mammalian histone H1(0) and duck H5. HMG proteins T1, T2, T3, H6, C, D and F were associated with liver and hepatocellular-carcinoma chromatin, with hepatocellular carcinoma containing higher levels of HMG T1 and F. Testis and erythrocyte had HMG T2 and H6 as their predominant HMG proteins. Most of the HMG H6 of hepatocellular carcinoma, but not of liver, was located in a chromatin fraction that was soluble at physiological ionic strength and enriched in transcriptionally active DNA. These alterations in the chromatin distribution and content of hepatocyte HMG proteins and H1 histone subtypes may contribute to aberrant hepatocyte gene expression in the hepatocellular carcinoma.

Title Histone Deacetylase is a Component of the Internal Nuclear Matrix.
Date December 1991
Journal The Journal of Biological Chemistry
Excerpt

In chicken immature erythrocytes, approximately 4% of the modifiable histone lysine sites participate in active acetylation. There are two categories of actively acetylated histone H4. Although both are acetylated at the same rate (t1/2 = 12 min), one is acetylated to the tetraacetylated form and is rapidly deacetylated (class 1), and the other is acetylated to mono- and diacetylated forms and is slowly deacetylated (class 2). We show that the chromatin distribution of the class 1 labeled tetraacetylated H4 species paralleled that of the transcriptionally active DNA sequences. For example, the chromatin fragments of the insoluble nuclear material contained 76% of the active DNA and 74% of the labeled tetraacetylated H4. Class 2 labeled acetylated H4 species were found in repressed chromatin and were enriched in active/competent gene-enriched chromatin fragments. The majority of the histone deacetylase activity (75-80%) was located with the insoluble residual nuclear material. Further, approximately 40-50% of the enzyme activity was associated with nuclear matrices prepared by two methods using high salt and intermediate/high salt extraction. Histone deacetylase was solubilized by extracting the nuclear matrices with high salt and 2-mercaptoethanol, a procedure that generates nuclear pore-lamina complexes. These results demonstrate that histone deacetylase is a component of the internal nuclear matrix.

Title The Antiproliferative Potency of Histamine Antagonists Correlates with Inhibition of Binding of [3h]-histamine to Novel Intracellular Receptors (hic) in Microsomal and Nuclear Fractions of Rat Liver.
Date July 1991
Journal Agents and Actions. Supplements
Excerpt

Previously, we identified in rat liver microsomes, low (microM) affinity histamine receptors (HIC), associated with antiestrogen binding sites (AEBS). N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a potent AEBS ligand, is a specific HIC antagonist. Through binding HIC, newly-formed intracellular histamine mediates, and DPPE inhibits, human platelet aggregation. We now provide evidence that histamine, mobilized from cytoplasmic stores, is a mediator of the mitogenic response to concanavalin A in mouse spleen cells. DNA synthesis and intracellular histamine levels are decreased over time by the histidine decarboxylase inhibitor, alpha-fluoromethylhistidine. For DPPE, H1 and H2 antagonists, rank order of potency to inhibit [3H]-histamine binding to HIC in rat liver microsomes correlates with antiproliferative potency. DPPE also competes for [3H]-histamine binding at low and high affinity sites in rat liver nuclei (IC50 approximately 2 microM). Thus, histamine may mediate growth through two intracellular subtypes of HIC.

Title Timing of the Appearance of Ubiquitinated Histones in Developing New Macronuclei of Tetrahymena Thermophila.
Date July 1991
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inert micronucleus. During vegetative growth, macronuclear histones H2A and H2B and micronuclear H2A are ubiquitinated. Despite differences in function, macro- and micro-nuclei are related. During conjugation (the sexual phase of the life cycle in Tetrahymena), postzygotic division products of micronuclei give rise to new micro- and macro-nuclei. Using an anti-ubiquitin antibody in Western blotting experiments, we determined the levels of ubiquitinated histones in new macro- and micro-nuclei at various times during conjugation. Very soon after the second postzygotic division (approximately 8 h) when new macronuclei begin to synthesize RNA, ubiquitinated H2B and polyubiquitinated H2A are present. At this time micronuclei have only low levels of ubiquitinated H2A. During later stages of conjugation (15 h), the level of polyubiquitinated H2A decreases, while ubiquitinated H2B increases in developing new macronuclei, attaining levels of ubiquitinated H2B approaching that of parental macronuclei. Ubiquitinated histones are not detectable in the 15-h micronuclei. These results show that ubiquitination of H2B coincides with the transformation of an inert germinal nucleus into that of a transcriptionally active somatic nucleus, suggesting that ubiquitinated H2B has a role in maintaining the transcriptionally active chromatin state.

Title Dynamically Acetylated Histones of Chicken Erythrocytes Are Selectively Methylated.
Date March 1991
Journal The Biochemical Journal
Excerpt

The relationship between histone acetylation and methylation in chicken immature erythrocytes was investigated. Previous studies have shown that transcriptionally active/competent gene-enriched chromatin fragments are enriched in newly methylated histones H3 and H4. Moreover, newly methylated histone H4 is hyperacetylated. Here, we show that dynamically acetylated histone H4 is selectively engaged in ongoing methylation. While sodium butyrate (an inhibitor of histone deacetylase) does not inhibit ongoing histone methylation, it does affect the acetylation state of newly methylated histone H4 when chicken immature erythrocytes are incubated in its presence or absence. Only one rate of acetylation of labelled newly methylated unacetylated histone H4 with a t1/2 of 8 min is observed. Previous studies have shown that the solubility of transcriptionally active/competent gene chromatin fragments in 0.15 M-NaCl is dependent upon the level of acetylated histone species, with induction of hyperacetylation increasing the solubility of this gene chromatin. Here, we show that the low salt solubility of chromatin fragments associated with newly methylated histones H3 and H4 is also dependent upon the level of acetylated histones. These results provide further support for the hypothesis that histones participating in ongoing methylation are associated with transcriptionally active/competent chromatin and suggest that the processes of histone H4 methylation and dynamic acetylation are partially coupled in terminally differentiated erythrocytes.

Title Ultrastructure of Transcriptionally Competent Chromatin.
Date January 1991
Journal Nucleic Acids Research
Excerpt

We have examined a salt-soluble, transcriptionally competent gene-enriched fraction of chicken erythrocyte chromatin and compared it to bulk chromatin using the unique microanalytical capabilities of Electron Spectroscopic Imaging (ESI). The salt-soluble fraction is enriched 48 fold in beta-globin gene sequences and is also enriched in histones that are post-synthetically modified, including acetylation and ubiquitination. Differences between the two fractions are also apparent in the distribution of the two major forms of nucleoprotein structures, including (1) particles which present a circular profile and possess protein and DNA content nearly identical to that of the canonical nucleosome and account for 89% of particles in the bulk fraction but account for only 66% of the particles in the competent fraction, and (2) u-shaped particles which possess about 20% less protein mass than particles of circular profile and are about 10x more prevalent in the transcriptionally competent fraction than in the bulk. Additionally, elongated particles with protein and DNA content similar to the u-shaped objects are also seen in the competent fraction.

Title Nucleosomal Histones of Transcriptionally Active/competent Chromatin Preferentially Exchange with Newly Synthesized Histones in Quiescent Chicken Erythrocytes.
Date November 1990
Journal The Biochemical Journal
Excerpt

The incorporation of newly synthesized histones among various chromatin fraction isolated from non-replicating cell-cycle-phase-Go chicken immature erythrocytes was investigated. We find that newly synthesized erythroid-specific histone Hl variant H5, is incorporated randomly into chromatin. In contrast, newly synthesized nucleosomal histones H2A, H2A.Z, H2B, H3.3, and H4 are preferentially found in a fraction that is highly enriched in active/competent gene chromatin fragments and depleted in repressed gene chromatin. Moreover, ubiquitinated species of histones H2A and H2B and hyperacetylated species of H4 and H2B, which are complexed to active DNA, are labelled. These observations provide evidence that newly synthesized histones preferentially exchange with the nucleosomal histones of transcriptionally active/component chromatin domains. The results of this study suggest that nucleosomes of active chromatin may be inherently less stable than bulk nucleosomes in vivo and have implications for chromatin remodelling.

Title Level of Ubiquitinated Histone H2b in Chromatin is Coupled to Ongoing Transcription.
Date August 1990
Journal Biochemistry
Excerpt

The relationship between transcription and ubiquitination of the histones was investigated. Previous studies have shown that ubiquitinated (u) histone H2B and, to a lesser extent, mono- and polyubiquitinated histone H2A are enriched in transcriptionally active gene-enriched chromatin fractions. Here, we show that treatment of T-47D-5 human breast cancer cells with actinomycin D (10 micrograms/mL) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the level of uH2B, but not uH2A, uH2A.Z, or polyubiquitinated H2A, in chromatin. Treatment of the cells with low levels (0.04 micrograms/mL) of actinomycin D slightly reduced the level of uH2B, suggesting that inhibition of ribosomal RNA synthesis does not have a profound effect on the level of uH2B in chromatin. The level of the ubiquitinated histones was not affected by treating the cells with inhibitors of DNA synthesis (sodium butyrate or aphidicolin), but heat-shock treatment resulted in the loss of all the monoubiquitinated histone species. These results demonstrate that maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription, particularly the synthesis of hnRNA. Thus, histone H2B would be ubiquitinated when the nucleosome was opened during transcription. Ubiquitination of histone H2B may impede nucleosome refolding, facilitating subsequent rounds of transcription.

Title Transposition of the Abl Proto-oncogene in Philadelphia--negative Chronic Myeloid Leukaemia and Acute Lymphocytic Leukaemia.
Date August 1990
Journal Cytobios
Excerpt

Transposition of the abl gene was demonstrated in seven Philadelphia (Ph'-) negative patients with either chronic myeloid leukaemia (CML) or acute lymphocytic leukaemia (ALL) by chromosomal in situ hybridization. In six out of seven CML patients and one out of two ALL patients, a significant accumulation of abl hybridization grains was localized to chromosome 22. Hybridization with both abl and sis resulted in the consistent formation of double hybridization events on chromosome 22. Transposition of abl was not apparent in one patient with Ph'-negative CML and in one patient with Ph'-negative ALL. The data suggest that transposition of abl to chromosome 22 is a feature of a particular subgroup of Ph'-negative leukaemias.

Title Histone Acetylation Alters the Capacity of the H1 Histones to Condense Transcriptionally Active/competent Chromatin.
Date May 1990
Journal The Journal of Biological Chemistry
Excerpt

The relationship between histone acetylation and the capacity of H1 histones to cause the 0.15 M NaCl-induced aggregation/precipitation of transcriptionally active/competent gene chromatin fragments was investigated. Previous studies have shown that transcriptionally active/competent, but not repressed, gene chromatin polynucleosomes, which were isolated from chicken erythrocytes, remained soluble in 0.15 M NaCl after being reconstituted with H1 histones. This result suggested that some component of the active/competent gene nucleosome altered the capacity of the H1 histones to condense the chromatin fiber. Recently, Hebbes et al. (Hebbes, T.R., Thorne, A.W., and Crane-Robinson, C. (1988) EMBO J. 7, 1395-1402) demonstrated directly that active, but not repressed, gene chromatin of chicken erythroid cells contain high levels of acetylated histones. Here, we show that the solubility of active/competent gene chromatin fragments in 0.15 M NaCl is dependent on the level of acetylated histone species, with induction of hyperacetylation increasing the solubility of this gene chromatin. Also, we show that lowering the levels of the acetylated histone forms reduces the ability of the active/competent gene chromatin fragments to resist exogenously added H1-histone-induced 0.15 M NaCl aggregation/precipitation. These results suggest that histone acetylation alters the capacity of the H1 histones to form compact higher order chromatin structures such that active/competent gene chromatin is maintained in a less folded state than the bulk of chromatin.

Title Chromatin Structure of Erythroid-specific Genes of Immature and Mature Chicken Erythrocytes.
Date February 1990
Journal The Biochemical Journal
Excerpt

The beta-globin and histone H5 genes are transcriptionally active in immature chicken erythrocytes and potentially active in mature erythrocytes. In both immature and mature erythrocytes, the majority of these erythroid-specific gene sequences are located in two chromatin fractions: the low-salt-insoluble residual nuclear material and the 0.15 M-NaCl-soluble oligo- and poly-nucleosomes. These salt-soluble chromatin fragments are enriched in hyperacetylated species of H4 and H2B, ubiquitinated and polyubiquitinated species of H2A and H2B and are depleted of linker histones H1 and H5. The competent, transcriptionally inactive embryonic epsilon-globin gene, which is part of the DNAase I-sensitive beta-globin domain, is highly enriched in the 0.15 M-NaCl-soluble polynucleosome fraction but not in the insoluble nuclear material. The repressed vitellogenin gene shows no enrichment in either of these fractions. These results suggest that only those genes that are expressed or have the potential for expression are enriched in the low-salt-insoluble nuclear material of immature or mature erythrocytes. The enrichment of active genes in the low-salt-insoluble residual nuclear material of immature erythrocytes is not dependent on on-going transcription, the presence of RNA or changes in the amount of acetylated histone species. Our results are consistent with the hypothesis that active and potentially active genes are insoluble because of the presence of preinitiation transcription complexes.

Title Distribution of Methylated Histones and Histone Methyltransferases in Chicken Erythrocyte Chromatin.
Date December 1989
Journal The Journal of Biological Chemistry
Excerpt

The relationship between histone methylation and the transcriptionally active chromatin state was investigated. Immature chicken erythrocytes, which were obtained from the peripheral blood of anemic birds, were incubated with L-[methyl-3H]methionine and cycloheximide. Under these conditions histones H3 and H4 are methylated. The erythrocyte nuclei were incubated with micrococcal nuclease, and the chromatin fragments were fractionated according to their solubility in EDTA and 0.15 M NaCl. Chromatin fractions, which were enriched in transcriptionally active genes, were enriched in methylated histones. Moreover, the acetylated species of histones H3 and H4, which are complexed with active genes (Hebbes, T. R., Thorne, A. W., and Crane-Robinson, C. (1988) EMBO J. 7, 1395-1402), were preferentially methylated. The methylation of these histones was not dependent on ongoing transcription. The distribution of histone H3 methyltransferase activity among the various chromatin regions was also studied. This enzyme activity was greatest for the chromatin fragments that were enriched in active/competent genes. However, our results suggest that histone H3 methyltransferase is bound to the nucleosome. The enzyme, which may be localized in the active gene chromatin domains, may ensure that the histones associated with active genes are methylated. Histone methylation, which has a slow turnover rate, may contribute to the maintenance of the transcriptionally active chromatin state.

Title Regulation of a Bifunctional Mrna Results in Synthesis of Secreted and Nuclear Probasin.
Date November 1989
Journal Proceedings of the National Academy of Sciences of the United States of America
Excerpt

Probasin, a rat prostatic protein, is statistically related to members of a protein family that includes serum, cellular, and nuclear proteins. In vivo, probasin appears both in the secretions and in the nucleus of prostatic epithelial cells. Using primer extension and S1 nuclease protection assays we detected only one probasin mRNA. Thus, the localization of this protein to two separate cellular regions must be encoded by this one mRNA. Furthermore, in vitro translation of synthetic probasin mRNA demonstrated that a protein containing a signal peptide and a protein lacking a signal peptide were synthesized by initiation at different AUG codons. These data are consistent with a mechanism of translational regulation of a eukaryotic bifunctional mRNA.

Title Structure of Polyubiquitinated Histone H2a.
Date June 1989
Journal Biochemistry
Excerpt

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.

Title Ubiquitinated Histone H2b is Preferentially Located in Transcriptionally Active Chromatin.
Date June 1989
Journal Biochemistry
Excerpt

Using an anti-ubiquitin antibody in Western blotting experiments, we detected polyubiquitinated species of histones H2A, H2A.Z, and H2B in histone preparations of bovine thymus, chicken erythrocyte, and Tetrahymena macro- and micronuclei. Histone H2A had the greatest level of polyubiquitinated species, with tetra- to hexaubiquitinated forms of this histone being observed. The fraction of bovine thymus and chicken erythrocyte chromatin enriched in transcriptionally active gene sequences was enriched in mono- and polyubiquitinated species of histones H2A, H2B, and H2A.Z, especially in the ubiquitinated forms of histone H2B. Histones H2A and H2B were ubiquitinated in the transcriptionally active Tetrahymena macronucleus, with monoubiquitinated (u) H2B being the predominant ubiquitinated histone species. Ubiquitinated forms of histones H2A and H2B were found in transcriptionally inert micronuclei, but at lower levels than seen in macronuclear histones. Also, the level of micronuclear uH2A was greater than that of uH2B which may be from macronuclei that contaminate the preparation. These results indicate that the mono- and polyubiquitinated species of histone H2B are preferentially located in transcriptionally active chromatin regions. Ubiquitinated histone H2A is located in both expressed and repressed chromatin domains, but expressed chromatin is enriched in mono- and polyubiquitinated forms of this histone. These observations are consistent with the hypothesis that ubiquitinated histones have a role maintaining the structure of transcriptionally active chromatin.

Title The Protamine Gene Chromatin in Developing Trout Testis Exists in an Altered State.
Date February 1989
Journal Biochimica Et Biophysica Acta
Excerpt

Micrococcal nuclease was used to probe the nucleosomal organization of the rainbow trout germ-line-specific protamine multi-gene family in testis and erythrocytes. In erythrocyte chromatin, the repressed protamine genes show a distinct nucleosomal repeat pattern. However, in early-stage testis chromatin, where the protamine genes are expressed, they lack a distinct nucleosomal repeat pattern, indicating that the disrupted chromatin structure is related to their transcriptional activity. Micrococcal nuclease-digested testis and erythrocyte chromatin was separated into soluble and insoluble fractions. Transcriptionally active/competent genes of testis that had been labeled by nuclear nick-translation were enriched in the low-salt eluted, micrococcal nuclease-sensitive chromatin fraction. This fraction was not enriched in protamine DNA sequences. In testis, but not erythrocytes, protamine DNA sequences were slightly enriched in chromatin that fractionated with insoluble nuclear material, suggesting that transcriptionally active protamine gene chromatin has an insoluble character. Since the different protamine genes may not be simultaneously expressed, our results show the distribution of both transcriptionally active and inactive protamine genes. However, our observations indicate that the active germ-line-specific protamine gene chromatin shares several, but not all, of the features associated with other active tissue-specific genes.

Title Gene-specific Differences in the Aflatoxin B1 Adduction of Chicken Erythrocyte Chromatin.
Date January 1989
Journal Cancer Research
Excerpt

Mature and immature chicken erythrocyte nuclei were treated with activated aflatoxin B1 (2,3-dichloroaflatoxin B1), producing covalently bound DNA adducts. This reaction produces alkali-labile sites in the DNA which can be identified by using a variation of the Maxam-Gilbert sequencing procedure. We determined the aflatoxin B1 accessibility of defined regions of the erythroid genome by using different specific probes and monitoring the disappearance of similar-sized fragments generated by restriction enzyme digestion. The genes studied were the erythroid-specific beta-globin and histone H5 genes, which are potentially active in mature erythroid nuclei and transcriptionally active in immature erythrocytes, and the vitellogenin and ovalbumin genes, which are both transcriptionally inactive in these cells. The beta-globin and histone H5 genes were more accessible than the repressed vitellogenin and ovalbumin genes to aflatoxin B1 modification in mature and immature erythroid chromatin. Micrococcal nuclease was used to probe the nucleosomal organization of active (beta-globin and histone H5) and repressed (vitellogenin and ovalbumin) genes in chicken erythrocytes. The vitellogenin and ovalbumin genes show a canonical nucleosome repeat pattern in mature and immature chicken erythrocyte nuclei. In contrast, the beta-globin and histone H5 genes lack a distinct nucleosomal repeat pattern in these cells. These results support the hypothesis that transcriptionally active genes are preferentially accessible to carcinogen modification because of their disrupted chromatin structure.

Title Bovine Thymus Satellite I Dna Sequences, Which Are Highly Methylated, Are Preferentially Located in H1-rich Chromatin.
Date September 1988
Journal Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
Excerpt

The distribution of 5-methylcytosine among H1-rich and -poor bovine thymus chromatin regions was determined. 5-Methylcytosine was enriched in H1-rich chromatin regions, with linker and nucleosomal DNA containing similar amounts of this modified base. Satellite I DNA sequences, which constitute 5-7% of the genome and are highly methylated, were preferentially localized among H1-rich chromatin regions, in accordance with the distribution of 5-methylcytosine. In contrast to the satellite I DNA sequences, prothrombin (a single copy DNA sequence) was localized among both H1-rich and -poor chromatin regions. The results of this study are consistent with the hypothesis that DNA methylation has a role in modulating the structure of chromatin.

Title Erythroid-specific Gene Chromatin Has an Altered Association with Linker Histones.
Date August 1988
Journal Nucleic Acids Research
Excerpt

The chromatin of several genes was assayed for sensitivity to DNAase I and for solubility as polynucleosomes in 0.15 M NaCl. The degree of solubility of chromatin fragments as polynucleosomes in 0.15 M NaCl correlates well with the sensitivity to DNAase I for several genes. Chromatin of repressed, housekeeping and erythroid-specific genes can be distinguished as distinct groups by the degree to which they display these properties. NaCl precipitation of chromatin fragments stripped and then reconstituted with varying quantities of H1 and H5 (linker) histones indicate that the polynucleosomes of erythroid-specific genes have altered interaction with these histones. Linker histones interacted with bulk chromatin and in the chromatin of the repressed ovalbumin and vitellogenin genes to form salt precipitable structures. Chromatin of erythroid-specific genes (histone H5 and beta-globin) as well as that of the histone H2A.F gene was resistant to linker histone induced precipitation.

Title Changes in the Histone H2a Variant H2a.z and Polyubiquitinated Histone Species in Developing Trout Testis.
Date December 1987
Journal Biochemistry
Excerpt

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.

Title Reduced Levels of Histones H1o and H1b, and Unaltered Content of Methylated Dna in Rainbow Trout Hepatocellular Carcinoma Chromatin.
Date November 1987
Journal Cancer Research
Excerpt

The levels of histone subtypes and DNA methylation of aflatoxin-induced rainbow trout hepatocellular carcinoma and adult liver nuclei were compared. The hepatocellular carcinoma nuclei were enriched in the ubiquitinated species of histone H2A and depleted in histones H1o and H1b. The 5-methylcytosine content and methylation patterns of the vitellogenin genes and the transcriptionally inactive TPG-3 protamine gene were not altered in the trout hepatocellular carcinoma DNA. Thus, undermethylation of DNA is not a general feature of chemically induced tumors in vivo.

Title The Ubiquitinated Histone Species Are Enriched in Histone H1-depleted Chromatin Regions.
Date October 1987
Journal Biochimica Et Biophysica Acta
Excerpt

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.

Title Dna Methylation Pattern and Restriction Endonuclease Accessibility in Chromatin of a Germ-line Specific Gene, the Rainbow Trout Protamine Gene.
Date June 1987
Journal Nucleic Acids Research
Excerpt

The chromatin structure of a germ-line specific gene, the TPG-3 gene, one of the rainbow trout protamine genes was analyzed in various tissues. The protamine genes are expressed in early stage testis but not in late stage testis, liver or erythrocyte. Five potential CpG methylation sites in the coding and flanking regions of the TPG-3 protamine gene were monitored in early and late stage testis, nucleoprotamine, liver and erythrocyte. In all cases the patterns of methylation were identical with only one CpG site at position -740 being methylated. Thus, the methylation pattern of this protamine gene remained the same independently of the expression of the gene. Two Msp I sites at positions -293 and/or -275 and +155 were accessible to the enzyme in the TPG-3 chromatin of early stage testis. Since the Msp I site at position -293 and/or -275 was also present in the TPG-3 chromatin of liver, only the site at position +155 within the transcribed region correlated with the expression of the protamine gene.

Title Selective Solubilization of Beta-globin Oligonucleosomes at Low Ionic Strength.
Date May 1987
Journal Biochemistry
Excerpt

We [Rocha, E., Davie, J.R., van Holde, K.E., & Weintraub, H. (1984) J. Biol. Chem. 259, 8558-8563] have previously reported that the transcriptionally competent beta-globin gene domain is selectively enriched in chromatin fractions eluted with solutions of approximately physiological ionic strength from micrococcal nuclease digested mature chicken erythrocyte nuclei. In this report, we demonstrate that beta-globin chromatin is eluted as oligonucleosomes while vitellogenin, a transcriptionally inactive gene, is eluted as mononucleosomes as is the bulk of sequences found in this fraction. Following removal of the salt, the eluted chromatin was made 100 mM KCl and separated into aggregation-prone and aggregation-resistant fractions. Globin sequences were present in both fractions and had the greatest enrichment in the aggregation-prone fraction which contained H1 and H5, H1 being more abundant. A procedure is presented in which H1 is selectively removed from the erythrocyte nuclei. Following the selective removal of H1 and subsequent fractionation, globin but not vitellogenin oligonucleosomes were present in the aggregation-resistant chromatin fraction. The results indicate the beta-globin domain is a mosaic of aggregation-resistant and aggregation-prone regions with the latter being associated with H1 and H5. Vitellogenin sequences were associated principally with aggregation-prone regions complexed with H5.

Title Chicken Erythrocyte Polynucleosomes Which Are Soluble at Physiological Ionic Strength and Contain Linker Histones Are Highly Enriched in Beta-globin Gene Sequences.
Date April 1987
Journal Nucleic Acids Research
Excerpt

Mature chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength are enriched in beta-globin DNA sequences. Vitellogenin chromatin, which is not expressed in this tissue, is found in aggregation prone, salt insoluble chromatin. There is a direct correlation between the size of soluble fragments and the degree of globin gene enrichment, with the largest fragments being most highly enriched. The highly globin enriched (about 50 fold) polynucleosomes contain significantly elevated levels of acetylated histones H4, H2A.Z, and H2B, and ubiquitinated (prefix "u") histones H2A and H2B (with a significant relative increase of uH2B over uH2A). These polynucleosomes were complexed with histones H1 and H5 but at a lower level than that found in unfractionated chromatin.

Title The Nonhistone Chromosomal Protein, H2a-specific Protease, is Selectively Associated with Nucleosomes Containing Histone H1.
Date September 1986
Journal The Journal of Biological Chemistry
Excerpt

We have determined the distribution of the nucleosomal bound nonhistone chromosomal protein, H2A-specific protease, in calf thymus and liver chromatin. The protease was unevenly distributed in chromatin with domains containing histone H1 being selectively complexed with the enzyme. Moreover, the protease had a preference for the less compact chromatin domains enriched in the H1 subtypes H1a and -c. We have demonstrated that ubiquitinated H2A is a substrate of the H2A-specific protease and that the enzyme is a serine protease which can be inactivated with protease inhibitors only after it is released from the nucleosome. Possible functions of the protease in modulating chromatin structure are discussed.

Title Peptide Mapping of Basic Proteins by Proteolysis in Acetic Acid/urea-minislab Polyacrylamide Gels.
Date June 1985
Journal Analytical Biochemistry
Excerpt

A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications.

Title Efficient Method for Visualization and Isolation of Proteins Resolved in Polyacrylamide Gels.
Date November 1984
Journal Journal of Chromatography
Excerpt

Polyacrylamide gel electrophoresis is a popular method used to purify proteins for reconstitution experiments, amino acid composition and sequence determinations. In this report we describe methods that will be of general use in the isolation and characterization of proteins and the benefits of substituting boric acid for glycine in the electrophoresis tray buffers. We also describe how proteins resolved in a variety of gel systems (including those containing sodium dodecyl sulfate) may be rapidly visualized with 8-anilino-1-naphthalene sulfonic acid and efficiently transferred to a second gel for two-dimensional gel analysis, or isolated by electroelution for subsequent characterization.

Title Differential Salt Fractionation of Active and Inactive Genomic Domains in Chicken Erythrocyte.
Date August 1984
Journal The Journal of Biological Chemistry
Excerpt

We have utilized the Sanders salt fractionation technique (Sanders, M. M. (1978) J. Cell Biol. 79, 97-109) to analyze the products of micrococcal nuclease digestion of adult chicken erythrocyte nuclei. By dot-blot hybridization with specific gene probes, it is found that nucleosomes from the globin gene domain, including a region extending to about 10 kilobase pairs 5' to the beta p gene are selectively enriched in the fractions eluted at low salt. In contrast, a single copy sequence located at about 10 kilobase pairs 5' to the beta p gene was concentrated in the less salt-soluble fractions. The vitellogenin and ovalbumin genes, which are never expressed in erythroid tissues, are also concentrated in the less salt-soluble fractions. Some more generally expressed genes (histone H4, thymidine kinase) appear to be more uniformly distributed. The low salt fractions are depleted in H1/H5, enriched in high mobility group 14 and 17, and contain somewhat more highly acetylated histones.

Title 5-methylcytosine is Not Detectable in Saccharomyces Cerevisiae Dna.
Date July 1984
Journal Molecular and Cellular Biology
Excerpt

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.

Title Two-dimensional Gel Systems for Rapid Histone Analysis for Use in Minislab Polyacrylamide Gel Electrophoresis.
Date August 1982
Journal Analytical Biochemistry
Title Ultrastructural Organization of Yeast Chromatin.
Date June 1982
Journal The Journal of Cell Biology
Excerpt

The ultrastructural organization of yeast chromatin was examined in Miller spread preparations of samples prepared from spheroplasts or isolated nuclei of Saccharomyces cerevisiae. Micrographs from preparations dispersed in 1 mM Tris (pH 7.2) illustrate that the basic chromatin fiber in yeast exists in two ultrastructurally distinct conformations. The majority (up to 95%) of the chromatin displays a beaded nucleosomal organization, although adjacent nucleosomes are separated by internucleosomal linkers of variable lengths. Ribonucleoprotein (RNP) fibrils are only occasionally associated with chromatin displaying the conformation. The remaining 5-10% of the chromatin appears to be devoid of discrete nucleosomes and has a smooth contour with a fiber diameter of 30-40 A. Transcriptional units, including putative ribosomal precursor RNA genes, defined by the presence of nascent RNP fibrils are restricted to chromatin displaying this smooth morphology. Chromatin released from nuclei in the presence of 5 mM Mg++ displays higher-order chromatin fibers, 200-300 A in diameter, these fibers appear to be arranged in a manner than reflects the two forms of the basic chromatin fiber.

Title Chemical Composition of Nucleosomes Among Domains of Calf Thymus Chromatin Differing in Micrococcal Nuclease Accessibility and Solubility Properties.
Date January 1982
Journal The Journal of Biological Chemistry
Excerpt

Calf thymus chromatin was fractionated by the Sanders' procedure ((1978) J. Cell Biol. 79, 97-109). this procedure involves sequential elutions of micrococcal nuclease-digested nuclei with buffers of increasing ionic strength. Through the use of the nuclei nick translation technique of Levitt et al. (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) which specifically labels the transcriptionally competent regions of the chromosome, the lowest salt eluted, micrococcal nuclease-sensitive chromatin fraction, was found to be enriched in transcriptionally competent chromatin. This chromatin fraction contained approximately equimolar amounts of the core histones and low amounts of histone H1. In addition, this fraction was enriched both in the acetylated species of histone H4 and in the high mobility group (HMG) proteins 14 and 17, but it was depleted in 5-methylcytosine. As the ionic strength of the elution buffers increased, chromatin fractions from less micrococcal nuclease-sensitive chromatin domains were eluted. The nuclease-insensitive fractions were enriched in the unacetylated species of histone H4, 5-methylcytosine, and histone H1. Although these fractions had a smaller proportion of nucleosomes containing HMG-14 and HMG-17, they contained about 50% of the total HMG-14 and HMG-17 population.

Title Histone Modifications in the Yeast S. Cerevisiae.
Date November 1981
Journal Nucleic Acids Research
Excerpt

The content of the acetylated histone species associated with the highly transcriptionally active chromatin of yeast was examined. We found yeast chromatin to contain very high levels of the acetylated species for histones H3, H4 and possibly the H2B variants, H2B-1 and H2B-2. Sixty-three percent of the histone H4 species was represented by the di-, tri- and tetra-acetylated forms. These results make yeast chromatin among the most highly acetylated of any chromatins reported thus far. In addition, the results are consistent with the idea that hyperacetylation of histones allows chromatin to be transcribed at an increased rate.

Title Dnase I Sensitive Chromatin is Enriched in the Acetylated Species of Histone H4.
Date July 1980
Journal Febs Letters
Title Acetylated Histone H4 is Preferentially Associated with Template-active Chromatin.
Date December 1978
Journal Proceedings of the National Academy of Sciences of the United States of America
Excerpt

Chromatin from trout testis at an early stage of development was digested with DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase; EC 3.1.4.6), and the solubilized products were fractionated into Mg2+-soluble and -insoluble components. An examination of the histones from these fractions by one- and two-dimensional polyacrylamide gels showed that the highly acetylated species of histone H4 (di-, tri-, and tetra-acetylated) were associated mainly with the Mg2+-soluble material. Digestion of this chromatin fraction with pancreatic ribonuclease converted more than half of it to an insoluble state, and the acetylated H4 remained associated with the precipitated fraction. No changes in the other histones were noted, but two other basic proteins were also found to be associated with the Mg2+-soluble fraction. Since this fraction is enriched in transcribing gene sequences, it is concluded that the histone H4 of active genes is present in a highly acetylated state.

Title Sodium Butyrate Inhibits Histone Deacetylation in Cultured Cells.
Date September 1978
Journal Cell
Excerpt

Sodium butyrate in millimolar concentrations causes an accumulation of acetylated histone species in a variety of vertebrate cell lines. In all lines tested, butyrate caused hyperacetylation of H3 and H4, and in rat IRC8 cells, H2A and H2B were also affected. In Friend erythroleukemic cells, butyrate also induces the synthesis of a nonhistone chromosomal protein, IP25. butyrate does not affect the rate of histone acetylation in cell-free extracts of nuclei of Friend cells. Rather, this fatty acid inhibits histone deacetylation. Cell-free extracts of either control cells or butyrate-grown cells contain comparable levels of histone-deacetylating activity. This in vitro activity is inhibited by the addition of butyrate to the extracts. Thus butyrate appears to be an inhibitor of histone deacetylases both in vivo and in vitro.

Title Chromatin Subunits Contain Normal Levels of Major Acetylated Histone Species.
Date October 1977
Journal The Journal of Biological Chemistry
Excerpt

Chromatin subunits or nucleosomes prepared by micrococcal nuclease digestion of nuclei from trout testis have been examined for the presence of in vivo modified histone species. Both monomers and multimers are labeled when testis cells are incubated in the presence of [14C]acetate, but the level of radioactivity in the monomer fraction is 10 to 20% higher than in the multimer fraction. This difference is attributed to the trimming of nucleotides from the monomer DNA, associated with the loss of H1. The specific activities of the [14C]acetate label in histones H2A, H2B, H3, and H4 in monomer particles are similar to their respective values in whole chromatin. Starch gel electrophoresis of histone fractions derived from monomeric nucleosomes revealed the presence of monoacetylated and phosphorylated species of H2A, monoacetylated species of H2B and H3, and mono-, di-, tri-, and tetraacetylated species of H4. No differences in the content of these species between monomeric nucleosomes and whole chromatin could be discerned.

Title Nuclear Organization and Chromatin Dynamics--sp1, Sp3 and Histone Deacetylases.
Date
Journal Advances in Enzyme Regulation
Title Association of Sp3 and Estrogen Receptor Alpha with the Transcriptionally Active Trefoil Factor 1 Promoter in Mcf-7 Breast Cancer Cells.
Date
Journal Journal of Cellular Biochemistry
Excerpt

To further explore the role of Sp1 and Sp3 in the estrogen regulated TFF1 gene transcription, chromatin immunoprecipitation (ChIP) assay was used to determine the association of estrogen receptor alpha (ERalpha), Sp1 and Sp3 with the endogenous trefoil factor 1 (TFF1) gene promoter in MCF-7 breast cancer cells. ERalpha and serine 5 phosphorylated RNA polymerase II, the form of RNA polymerase II associated with transcription initiation, were recruited to the TFF1 gene promoter following estrogen addition to MCF-7 cells cultured under estrogen deplete conditions. Both Sp1 and Sp3 were bound to the TFF1 gene promoter before and after estrogen treatment. Using the re-ChIP assay, we demonstrate that either Sp1 or Sp3 but not both bind to a TFF1 promoter. The co-occupancy of ERalpha and Sp1 on TFF1 promoter remains at similar level with and without estrogen, while that of ERalpha and Sp3 increased in the presence of estrogen. Further, we observed increased co-occupancy of Sp3 and serine 5 phosphorylated RNA polymerase II on the TFF1 promoter after estrogen treatment of cells. Taken together, these results provide evidence that Sp3 and ERalpha are involved in the estrogen induced transcription of the TFF1 gene.

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