Histone H3 Post-translational Modifications Research Articles

The role of H3K4me3 and H3K9/14ac in the induction by dexamethasone of Per1 and Sgk1, two glucococorticoid early response genes that mediate the effects of acute stress in mammals

Glucocorticoids are known to induce or repress the expression of a wide variety of genes with roles in various biological processes such as the circadian clock and the stress response. We studied the changes in the levels of two histone H3 post-translational modifications associated with active chromatin, H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysines 9/14 (H3K9/14ac), that take place in the promoters of two glucocorticoid early response genes, Per1 and Sgk1, during their induction by the synthetic glucocorticoid, dexamethasone. Sgk1 mediates the effects of acute and chronic stress on the prefrontal cortex and other parts of the brain, while Per1 is a core circadian clock gene whose expression is strongly induced by the increased levels of blood-borne glucocorticoids that accompany acute and chronic stress. Here we show that dexamethasone rapidly increases the levels of H3K4me3 and H3K9/14ac in the promoters of both genes. Furthermore, the effect of dexamethasone on these genes, regarding both mRNA levels and the abundance of H3K4me3 and H3K9/14ac in their promoters, can be inhibited by the presence of nicotinamide, a metabolic molecule which has been shown to possess anxiolytic properties.

Anticancer drug mithramycin interacts with core histones: An additional mode of action of the DNA groove binder

Mithramycin (MTR) is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin – histone proteins – as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode). The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR–DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg2+. As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo. Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.

Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.

Chronic valproic acid administration impairs contextual memory and dysregulates hippocampal GSK-3β in rats

Valproic acid (VPA), a long-standing anti-epileptic and anti-manic drug, exerts multiple actions in the nervous system through various molecular mechanisms. Neuroprotective properties have been attributed to VPA in different models of neurodegeneration, but contrasting results on its improvement of learning and memory have been reported in non-pathologic conditions. In the present study, we have tested on a hippocampal-dependent learning test, the contextual fear conditioning, the effect of chronic VPA administration through alimentary supplementation that allows relatively steady concentrations to be reached by a drug otherwise very rapidly eliminated in rodents. Contextual fear memory was significantly impaired in rats chronically treated with VPA for 4weeks. To understand the cellular and molecular correlates of this amnesic effect with particular regard to hippocampus, we addressed three putatively memory-related targets of VPA action in this brain area, obtaining the following main results: i) chronic VPA promoted an increase of post-translational modifications of histone H3 (acetylation and phosphorylation) known to favor gene transcription; ii) adult neurogenesis in the dentate gyrus, which has been controversially reported to be affected by VPA, was unchanged; and iii) GSK-3β, a kinase playing a key role in hippocampal plasticity, as well as in learning and memory, was dysregulated by VPA treatment. These results point at GSK-3β dysregulation in the hippocampus as an important parameter in the amnesic effect of VPA. The VPA amnesic effect in the animal model here reported is also supported by some observations in patients and, therefore, it should be taken into account and monitored in VPA-based therapies.

A Mutational Mimic Analysis of Histone H3 Post-Translational Modifications: Specific Sites Influence the Conformational State of H3/H4, Causing either Positive or Negative Supercoiling of DNA

Histone H3 has specific sites of post-translational modifications that serve as epigenetic signals to cellular machinery to direct various processes. Mutational mimics of these modifications (glutamine for acetylation, methionine and leucine for methylation, and glutamic acid for phosphorylation) were constructed at the relevant sites of the major histone variant, H3.2, and their effects on the conformational equilibrium of the H3/H4 tetramer at physiological ionic strength were determined when bound to or free of DNA. The deposition vehicle used for this analysis was NAP1, nucleosome assembly protein 1. Acetylation mimics in the N-terminus preferentially stabilized the left-handed conformer (DNA negatively supercoiled), and mutations within the globular region preferred the right-handed conformer (DNA positively supercoiled). The methylation mimics in the N-terminus tended to maintain characteristics similar to those of wild-type H3/H4; i.e., the conformational equilibrium maintains similar levels of both left- and right-handed conformers. Phosphorylation mimics facilitated a mixed effect, i.e., when at serines, the left-handed conformer, and at threonines, a mixture of both conformers. When double mutations were present, the conformational equilibrium was shifted dramatically, either leftward or rightward depending on the specific sites. In contrast, these mutations tended not to affect the direction and extent of supercoiling for variants H3.1 and H3.3. Variant H3.3 promoted only the left-handed conformer, and H3.1 tended to maintain both conformers. Additional experiments indicate the importance of a propagation mechanism for ensuring the formation of a particular superhelical state over an extended region of the DNA. The potential relevance of these results to the maintenance of epigenetic information on a gene is discussed.

Differential Cellular and Molecular Effects of Butyrate and Trichostatin A on Vascular Smooth Muscle Cells

The histone deacetylase (HDAC) inhibitors, butyrate and trichostatin A (TSA), are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC) that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi) by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.

Effects of Zinc and DHA on the Epigenetic Regulation of Human Neuronal Cells

Dietary intake of zinc and omega-3 fatty acids (DHA) have health benefits for a number of human diseases. However, the molecular basis of these health benefits remains unclear. Recently, we reported that zinc and DHA affect expression levels of histones H3 and H4 in human neuronal M17 cells. Here, using immunoblotting and densitometric analysis, we aimed to investigate the effect of zinc and DHA on post-translational modifications of histone H3 in M17 cells. In response to increase in zinc concentration, we observed increase in deacetylation, methylation and phosphorylation of H3 and decrease in acetylation. We also investigated the role of zinc in apoptosis, and found that zinc reduced the levels of the anti-apoptotic marker Bcl-2 while increasing the apoptotic marker caspase-3 levels, correlating with cell viability assays. Conversely, DHA treatment resulted in increase in acetylation of H3 and Bcl-2 levels and decrease in deacetylation, methylation, phosphorylation of H3 and caspase-3 levels, suggesting that DHA promotes gene expression and neuroprotection. Our novel findings show the opposing effects of zinc and DHA on the epigenetic regulation of human neuronal cells and highlight the potential benefit of dietary intake of DHA for management of neurodegenerative diseases.

TR-FRET Cellular Assays for Interrogating Posttranslational Modifications of Histone H3

Posttranslational modifications such as phosphorylation, acetylation, and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone-modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. The authors have developed high-throughput LanthaScreen cellular assays for Histone H3 site-specific modifications. These assays use cells expressing green fluorescence protein-tagged Histone H3 transiently delivered via BacMam and terbium-labeled anti-Histone H3 modification-specific antibodies. Robust time-resolved Förster resonance energy transfer signals were detected for H3 lysine-9 acetylation and dimethylation (H3K9me2), serine-10 phosphorylation, K4 di- and trimethylation, and K27 trimethylation. Consistent with previous reports, hypoxic stress increased K4 methylation levels, and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly, with little effects on other modifications. To demonstrate the utility of this assay platform in screening, the K9 acetylation assay was used to profile the Enzo Epigenetics Library. Twelve known HDAC inhibitors were identified as hits and followed up in a dose-response format. In conclusion, this assay platform enables high-throughput cell-based analysis of diverse types of posttranslational modifications of Histone H3.

Mechanisms of Co-Operation of DNMT3A Mutations with JAK2 V617F Through Histone H4 Arginine 3 Provides New Insights in MPN Disease Pathogenesis

Abstract 2823Myeloproliferative neoplasms (MPN), including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary myelofibrosis (PMF), are clonal stem cell disorders characterised by an excess of mature cells of one or more myeloid lineages in the chronic phase, and an associated risk of progression to myelofibrosis and leukemic transformation. Highly specific somatic mutations of JAK2 and MPL are found in the majority of MPN patients, while numerous other somatic mutations have now been identified to be common between MPN and de novo acute myeloid leukemia (AML), including somatic mutations of TET2, ASXL1, CBL, IKZF1, EZH2, IDH1, IDH2 (reviewed in Tefferi, 2010 (Tefferi)), and most recently the DNA methyltransferase, DNMT3A (Abdel-Wahab, et al, Ley, et al, Stegelmann, et al, Walter, et al, Yan et al). We identified somatic heterozygous mutations in DNMT3A affecting amino acids R882 (R882C) and M880 (M880V), detected in peripheral blood mononuclear cells from two JAKV617F-positive PV patients (Rao et al, 2011, accepted for publication).At the level of chromatin and gene regulation, cooperation of DNMT3A mutations with JAK2V617F can occur through direct interaction of DNMT3A with the chromatin modifying protein PRMT5 which is a direct phosphorylation target of JAK2V617F (Liu F et al, 2011). PRMT5 is an arginine methyltransferase which associates with chromatin and methylates the key arginine (R) residue at the N-terminus of histone H4 (R3), which in turn recruits a DNMT3A complex resulting in methylation of adjacent CpG residues and gene-silencing (Zhao Q et al., 2009). To test the functional affects of DNMT3A mutations M880V and R882C, we have performed site- directed mutagenesis to generate GFP-tagged DNMT3A wild-type and mutant retroviral constructs. These DNMT3A clones will be transduced into cord blood CD34+ve cells and GFP-positive cells selected with flow cytometry. For functional analysis, we will examine DNA methyltransferase activity, changes in histone H4 modifications and CpG methylation.In a complementary analysis we have identified a change in a JAK2V617F+ve PV patient in the highly conserved HIST1H4C gene resulting in a substitution of Arg3 for Cys in Histone H4. Although there are 15 histone H4 genes encoding identical proteins, gene expression varies significantly in tissues and in cancer cells (W.F Holmes et. al, J.Biol Chem, 2005). We have used Q-PCR, using long primers to differentiate H4 transcripts and measured relative expression levels. We have confirmed that HIST1H4C is the major contributor of the H4 pool making ≥50% of the total H4 RNA in normal CD34+ve cells, Monocytes, lymphocytes and a variety of haemopoietic cell lines, indicating that the change to this critical residue in this gene may be functionally important. An analysis of a matched buccal sample showed that the HIST1H4C R3C variant in the PV patient was germline. Subsequent sequencing of the entire open reading frame of HIST1H4C in 232 normal individuals did not identify any other amino acid substitutions affecting this gene. Given the interaction of this residue with DNMT3A, we are currently investigating functional effects associated with this histone H4 variant. We have generated HEK293 cells with 4-Hydroxy tamoxifen (4-HT) inducible expression of the H4 R3C variant, and following the addition of 4-HT, we are initially assessing changes in histone H4 post-translational modifications, and markers of DNA damage such as gamma H2AX serine 139 phosphorylation. Disclosures:No relevant conflicts of interest to declare.

Butyrate, an HDAC inhibitor, stimulates interplay between different posttranslational modifications of histone H3 and differently alters G1-specific cell cycle proteins in vascular smooth muscle cells

HDACs and HATs regulate histone acetylation, an epigenetic modification that controls chromatin structure and through it, gene expression. Butyrate, a dietary HDAC inhibitor, inhibits VSMC proliferation, a crucial factor in atherogenesis, and the principle mechanism in arterial and in-stent restenosis. Here, the link between antiproliferation action of butyrate and the portraits of global covalent modifications of histone H3 that it induces are characterized to understand the mechanics of butyrate-arrested VSMC proliferation. Analysis of histone H3 modifications specific to butyrate arrested VSMC proliferation display induction of histone H3-Lysine9 acetylation, inhibition of histone H3-Serine10 phosphorylation, reduction of histone H3-Lysine9 dimethylation and stimulation of histone H3-Lysine4 di-methylation, which is linked to transcriptional activation, cell cycle/mitosis, transcriptional suppression and activation, respectively. Conversely, untreated VSMCs exhibit inhibition of H3-Lysine9 acetylation, induction of H3-Serine10 phosphorylation, stimulation of H3-Lysine9 di-methylation and reduction in H3-Lysine4 di-methylation. Butyrate's cooperative effects on H3-Lysine9 acetylation and H3-Serine10 phosphorylation, and contrasting effects on di-methylation of H3-Lysine9 and H3-Lysine4 suggests that the interplay between these site-specific modifications cause distinct chromatin alterations that allow cyclin D1 and D3 induction, G1-specific cdk4, cdk6 and cdk2 downregulation, and upregulation of cdk inhibitors, p15INK4b and p21Cip1. Regardless of butyrate's effect on D-type cyclins, downregulation of G1-specific cdks and upregulation of cdk inhibitors by butyrate prevents cell cycle progression by failing to inactivate Rb. Overall, through chromatin remodeling, butyrate appears to differentially alter G1-specific cell cycle proteins to ensure proliferation arrest of VSMCs, a crucial cellular component of blood vessel wall.

Glucose-dependent Insulinotropic Polypeptide and Glucagon-like Peptide-1 Modulate β-Cell Chromatin Structure

Chromatin can exert a regulatory effect on gene transcription by modulating the access of transcription factors to target genes. In the present study, we examined whether nuclear actions of the incretin hormones, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, involve modulation of beta-cell chromatin structure. Stimulation of INS-1(832/13) beta-cells or dispersed mouse islets with glucose-dependent insulinotropic polypeptide or glucagon-like peptide-1 resulted in the post-translational modification of core H3 histones, through acetylation and phosphorylation. Both increased histone H3 acetyltransferase and reduced histone deacetylase activities contributed. Subsequent studies demonstrated that incretin-mediated histone H3 modifications involved activation of protein kinase A, p42/44 mitogen-activated protein kinase (MAPK), and p38 MAPK signaling modules, resulting in the activation of mitogen- and stress-activated kinase-1. Additionally, modification of histone H3 increased its association with the transcription factor, phosphorylated cAMP-response element-binding protein (phospho-CREB) and with cAMP-responsive CREB coactivator 2. Incretin-activated CREB-related Bcl-2 transcription was greatly reduced by a histone acetyltransferase inhibitor, demonstrating the functional importance of histone H3 modification. This appears to be the first demonstration of beta-cell chromatin modification in response to the incretins and the studies indicate that their regulatory effects involve coordinated nuclear interactions between specific signaling modules, chromatin-modifying enzymes and transcription factors.

Rosiglitazone synergizes anticancer activity of cisplatin and reduces its nephrotoxicity in 7, 12-dimethyl benz{a}anthracene (DMBA) induced breast cancer rats

BackgroundAntineoplastic drug cisplatin remains the drug of choice for various solid tumours including breast cancer. But dose dependent nephrotoxicity is the major drawback in majority of platinum based chemotherapy regimens. Recent reports have shown that inflammatory pathways are the main offender for cisplatin induced nephrotoxicity. The present study was undertaken to assess the effect of rosiglitazone, a PPARγ agonist and an anti-inflammatory agent, on cisplatin induced nephrotoxicity, and its anticancer activity in DMBA induced breast cancer rats.MethodsMammary tumours were induced in female Sprague-Dawley rats by feeding orally with dimethylbenz [a]anthracene (DMBA) (60 mg/kg). Cisplatin induced nephropathy was assessed by measurements of blood urea nitrogen, albumin and creatinine levels. Posttranslational modifications of histone H3, mitogen-activated protein (MAP) kinase p38 expression and PPAR-γ expression were examined by western blotting.ResultsOur data shows involvement of TNF-α in preventing cisplatin induced nephrotoxicity by rosiglitazone. Rosiglitazone pre-treatment to cisplatin increases the expression of p38, PPAR-γ in mammary tumours and shows maximum tumour reduction. Furthermore, cisplatin induced changes in histone acetylation, phosphorylation and methylation of histone H3 in mammary tumours was ameliorated by pre-treatment of rosiglitazone. Suggesting, PPAR-γ directly or indirectly alters aberrant gene expression in mammary tumours by changing histone modifications.ConclusionTo best of our knowledge this is the first report which shows that pre-treatment of rosiglitazone synergizes the anticancer activity of cisplatin and minimizes cisplatin induced nephrotoxicity in DMBA induced breast cancer.

Insulin induced alteration in post-translational modifications of histone H3 under a hyperglycemic condition in L6 skeletal muscle myoblasts

Chromatin remodelling events, especially histone modifications are proposed to form the mainstay for most of the biological processes. However, the role of these histone modifications in the progression of diabetes is still unknown. Hyperglycemia plays a major role in diabetes and its complications. The present study was undertaken to check the effect of insulin on alterations in post-translational modifications of histone H3 in L6 myoblasts under a hyperglycemic condition. We provide first evidence that insulin under hyperglycemic condition alters multiple histone modifications by enhanced production of reactive oxygen species. Insulin induces dose dependent changes in Lysine 4 and 9 methylation, Ser 10 phosphorylation and acetylation of histone H3. Interestingly, insulin induced generation of reactive oxygen species induces dephosphorylation and deacetylation of histone H3. Preincubation with catalase and DPI prevents these changes in post-translational modifications of histone H3. Furthermore, changes in histone H3 phosphorylation was found to be independent of ERK, p38, RSK2 and MSK1. Moreover, serine/threonine phosphatase inhibitor, okadaic acid attenuates insulin induced dephosphorylation and deacetylation of histone H3, suggesting a role of serine/threonine phosphatases in altering modifications of histone H3. These changes in epigenetic modifications can provide new insights into pathogenesis of diabetes.

Change in post‐translational modifications of histone H3, heat‐shock protein‐27 and MAP kinase p38 expression by curcumin in streptozotocin‐induced type I diabetic nephropathy

Curcumin has been used to treat cancer, diabetes and other pathologies. However, little is known regarding its role in altering post-translational modifications of histone H3. A recent report suggests that acute hyperglycaemia induces a global down-regulation of gene expression in human tissues and epigenetic regulation of gene expression could be a novel mechanism underlying the pathological processes of hyperglycaemia. The present study was undertaken to examine changes in histone modification by curcumin treatment which prevents development of type I diabetic nephropathy. Male Sprague-Dawley rats were rendered diabetic using a single dose of streptozotocin (55 mg kg(-1), i.p.). Diabetic nephropathy was assessed by measurements of blood urea nitrogen, albumin and creatinine levels. Post-translational modifications of histone H3, heat shock protein-27 (HSP-27) and mitogen-activated protein (MAP) kinase p38 expression were examined by western blotting. Treatment of diabetic rats with curcumin significantly decreased blood urea nitrogen and creatinine and increased albumin; variables associated with the development of diabetic nephropathy. There were also increased levels of HSP-27 and MAP kinase (p38) in diabetic kidney. However, curcumin treatment prevented this increase in HSP-27 and p38 expression. Moreover, at nuclear level curcumin prevented the decrease in dephosphorylation and increases acetylation of histone H3. Our results suggested that protection against development of diabetic nephropathy by curcumin treatment involved changes in post-translational modifications of histone H3, expression of HSP-27 and MAP kinase p38 in diabetic kidney.

Mechanism of lymphocyte-specific inactivation of RAG-2 intragenic promoter of NWC: Implications for epigenetic control of RAG locus

NWC, third evolutionarily conserved gene within RAG locus is transcribed at high level in all cells except mature T and B lymphocytes and their RAG negative progenitors. It is so, because in lymphocytes expression of NWC is regulated by RAG-1 promoter, while in other cells it is controlled by RAG-2 intragenic promoter which in T and B lymphocytes is silent. Here we show that lymphocyte-specific inactivation of NWC promoter is caused by CpG island hypermethylation accompanied by site-specific blocking of chromatin accessibility, which in contrast to RAG promoters, is not accompanied by expected posttranslational modifications of histone H3. These results indicate that accessibility of NWC promoter and RAG promoters to trans-acting factors is regulated by different epigenetic mechanisms. The implications of our findings for understanding mechanisms regulating transcription within RAG/ NWC locus in different cells are discussed and the model of epigenetic control of this locus is proposed.

Constitutive heterochromatin during mouse oogenesis: The pattern of histone H3 modifications and localization of HP1α and HP1β proteins

Centromeres are the fragments of DNA that are responsible for proper chromosome segregation. They consist of centromeric chromatin surrounded by blocks of pericentric heterochromatin, playing an important role in centromere function. In somatic cells, the pericentric domains have a specific pattern of epigenetic modifications of core histones and contain specific pericentric proteins. These features are probably more important for the centromere function than the sequence of the centromeric DNA itself. In somatic cells, the HP1alpha and HP1beta proteins are indispensable for constitutive heterochromatin formation and maintenance. We have analyzed the localization of these proteins in the primordial, growing, fully-grown, and maturing mouse oocytes. Additionally, we have analyzed post-translational modifications of histone H3, which can influence HP1alpha and HP1beta association with the heterochromatin. We showed that the regions of constitutive heterochromatin have a distinct pattern of histone H3 acetylation and di-, and trimethylation of its lysine 9. We demonstrated that HP1beta protein was present in pericentric chromatin domains in primordial oocytes, growing (transcriptionally active) oocytes, and in fully-grown oocytes, and was released to the cytoplasm after germinal vesicle breakdown. In contrast, the HP1alpha was never detected in primordial oocytes, was first detected in pericentric heterochromatin in growing oocytes, dissociated from pericentric heterochromatin in fully-grown oocytes, and it was never detected in maturing oocytes. The presence of HP1alpha and HP1beta proteins on the heterochromatin of transcriptionally active oocytes and their absence in transcriptionally silent oocytes suggest that they are necessary for the repression of RNA synthesis in heterochromatin domains of transcribing oocytes.

Crosstalk between site-specific modifications on p53 and histone H3

Previously, we have observed a link between p53 expression and histone H3 post-translational modifications. Here, we ask if specific post-translational modifications of p53 impact upon histone H3 modifications in a selective manner. We have also screened for internal co-operative effects within the repertoire of p53 modifications. Exogenous p53 constructs were expressed in HCT116 p53-/- cells. Four mutant p53 constructs were used, with single 'phosphorylation' mutations at serines 15 and 37 (S15A, S15D, S37A and S37D) and compared with exogenously expressed wild-type p53. The results showed that the replacement of serine 15 with either alanine (S15A) or aspartic acid (S15D) induced phosphorylation at S33P, S37P and S46P. In contrast, phosphorylation mutants p53(S37A) and p53(S37D) were not phosphorylated on S33. S46 phosphorylation appeared specifically enhanced by p53(S37D) relative to p53(S37A). Distal induction of S392 phosphorylation was observed for each of the p53 N-terminal phosphorylation mutants. Analysis of endogenous histone H3 (from the transfected cells) revealed loss of di-methylated K9 following expression of wild type and mutant p53 constructs. Expression of p53 (S15A), (S15D) and (S37A) selectively induced acetylation at K9 and K14. In contrast, wt p53 and p53(S37D) had no effect upon K9 or K14 acetylation. K18 acetylation status was unaffected throughout.

Histone H3 Lysine 4 Dimethylation Signals the Transcriptional Competence of the Adiponectin Promoter in Preadipocytes

Adipogenesis is regulated by a coordinated cascade of sequence-specific transcription factors and coregulators with chromatin-modifying activities that are between them responsible for the establishment of the gene expression pattern of mature adipocytes. Here we examine the histone H3 post-translational modifications occurring at the promoters of key adipogenic genes during adipocyte differentiation. We show that the promoters of apM1, glut4, gpd1, and leptin are enriched in dimethylated histone H3 Lys4 (H3-K4) in 3T3-L1 fibroblasts, where none of these genes are yet expressed. A detailed study of the apM1 locus shows that H3-K4 dimethylation is restricted to the promoter region in undifferentiated cells and associates with RNA polymerase II (pol II) loading. The beginning of apM1 transcription at the early stages of adipogenesis coincides with promoter H3 hyperacetylation and H3-K4 trimethylation. At the coding region, H3 acetylation and dimethylation, as well as pol II binding, are found in cells at later stages of differentiation, when apM1 transcription reaches its maximal peak. This same pattern of histone modifications is detected in mouse primary preadipocytes and adipocytes but not in a related fibroblast cell line that is not committed to an adipocyte fate. Inhibition of H3-K4 methylation by treatment of 3T3-L1 cells with methylthioadenosine results in decreased apM1 gene expression as well as decreased adipogenesis. Taken together, our data indicate that H3-K4 dimethylation and pol II binding to the promoter of key adipogenic genes are distinguishing marks of cells that have undergone determination to a preadipocyte stage.

Ros-Induced Histone Modifications and their Role in Cell Survival and Cell Death

Much is known about the distal DNA damage repair response. In particular, many of the enzymes and auxiliary proteins that participate in DNA repair have been characterized. In addition, knowledge of signaling pathways activated in response to DNA damage is increasing. In contrast, comparatively less is known of DNA damage-sensing molecules or of the specific alterations to chromatin structure recognized by such DNA damage sensors. Thus, precisely how chromatin structure is altered in response to DNA damage and how such alterations regulate DNA repair processes remain important unanswered questions. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after double-strand break formation, extends over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. We have shown that reactive oxygen species (ROS)-induced DNA single-strand breaks induce the incorporation of 32P specifically into histone H3. ADP-Ribosylation of histones may stimulate local chromatin relaxation to facilitate the repair process, and, indeed, histone ribosylation preceded DNA damage-induced histone H3 phosphorylation. However, H3 phosphorylation occurred concomitant with overall chromatin condensation, as revealed by decreased sensitivity of chromatin to digestion by micrococcal nuclease and by DAPI staining of nuclei. Inhibitors of the ERK and p38MAPK pathways and inhibition of poly(ADP-ribose) polymerase all reduced ROS-induced H3 phosphorylation, chromatin condensation, and cell death. Precisely how changes in the post-translational modification of histone H3 regulate the survival response remains unclear. Attempts to determine the precise site of histone H3 phosphorylation, putative histone H3 kinases, and histone H3 interacting proteins are underway.

Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

BackgroundThe soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O2) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber.ResultsEmbryos exposed to brief periods to anoxia (30 minutes) contain prophase blastomeres with chromosomes in close proximity to the nuclear membrane, condensation of interphase chromatin and metaphase blastomeres with reduced spindle microtubules density. Embryos exposed to longer periods of anoxia (1–3 days) display several characteristics including interphase chromatin that is further condensed and in close proximity to the nuclear membrane, reduction in spindle structure perimeter and reduced localization of SAN-1 at the kinetochore. Additionally, we show that the spindle checkpoint protein SAN-1 is required for brief periods of anoxia-induced cell cycle arrest, thus demonstrating that this gene product is vital for early anoxia responses. In this report we suggest that the events that occur as an immediate response to brief periods of anoxia directs cell cycle arrest.ConclusionFrom our results we conclude that the sub-nuclear characteristics of embryos exposed to anoxia depends upon exposure time as assayed using brief (30 minutes), intermediate (6 or 12 hours) or long-term (24 or 72 hours) exposures. Analyzing these changes will lead to an understanding of the mechanisms required for initiation and maintenance of cell cycle arrest in respect to anoxia exposure time as well as order the events that occur to bring about anoxia-induced cell cycle arrest.