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PubMed2004

Histone modifications defining active genes persist after transcriptional and mitotic inactivation.

We examined various histone modifications across the promoter and the coding regions of constitutively active hepatic genes in G0/G1-enriched, mitotically arrested and alpha-amanitin-blocked cells. Gene activation correlated with localized histone hyperacetylation, H3-K4 tri- or dimethylation and H3-K79 dimethylation and localized nucleosome remodeling at the promoter and the 5' portion of the coding regions. Nucleosomes at more downstream locations were monomethylated at H3-K4. CBP, PCAF, Brg-1, SNF2H and FACT were recruited to the coding regions in a gene-specific manner, in a similarly restricted promoter-proximal pattern. Elongator, however, associated with the more downstream regions. While all factors were dissociated from the chromatin after transcriptional inactivation by alpha-amanitin, the histone modifications remained stable. In mitotic cells, histone modifications on parental nucleosomes were preserved and were regenerated in a transcription-dependent manner at the newly deposited nucleosomes, as the cells entered the next G1 phase. The findings suggest that histone modifications may function as molecular memory bookmarks for previously active locations of the genome, thus contributing to the maintenance of active chromatin states through cell division.

Acetyltransferases↗
PubMed2006

The genomic landscape of histone modifications in human T cells.

To understand the molecular basis that supports the dynamic gene expression programs unique to T cells, we investigated the genomic landscape of activating histone modifications, including histone H3 K9/K14 diacetylation (H3K9acK14ac), H3 K4 trimethylation (H3K4me3), and the repressive histone modification H3 K27 trimethylation (H3K27me3) in primary human T cells. We show that H3K9acK14ac and H3K4me3 are associated with active genes required for T cell function and development, whereas H3K27me3 is associated with silent genes that are involved in development in other cell types. Unexpectedly, we find that 3,330 gene promoters are associated with all of these histone modifications. The gene expression levels are correlated with both the absolute and relative levels of the activating H3K4me3 and the repressive H3K27me3 modifications. Our data reveal that rapidly inducible genes are associated with the H3 acetylation and H3K4me3 modifications, suggesting they assume a chromatin structure poised for activation. In addition, we identified a subpopulation of chromatin regions that are associated with high levels of H3K4me3 and H3K27me3 but low levels of H3K9acK14ac. Therefore, these regions have a distinctive chromatin modification pattern and thus may represent a distinct class of chromatin domains.

Acetylation↗
PubMed2025

Proteins driving liquid-liquid phase separation and histone modifications cooperatively associate with chromatin looping and transcriptional regulation.

BACKGROUND: Although liquid-liquid phase separation (LLPS) proteins are known to participate in genome organization and transcriptional regulation through the formation of biomolecular condensates, their functional interplay with other regulatory proteins and histone modifications in chromatin loop formation remains poorly characterized. By combining Hi-C chromatin interaction data with ChIP-seq profiles of 12, 27, and 24 LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively, we identified chromatin loops associated with LLPS proteins and systematically analysed patterns of cooperative protein binding and histone modification enrichment within these loop-associated peaks. RESULTS: We identified 162, 313, and 431 chromatin loops associated with LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively. These loops were relatively small in size and predominantly anchored at enhancer regions. Examination of cooperative binding of proteins within loop-associated peaks revealed that transcriptional repressor IKZF1, HDAC1, and SAP130 most frequently co-localized with LLPS proteins in GM12878, K562, and HepG2 cells, respectively. Further analysis of histone modification enrichment patterns revealed that active histone modifications, such as H3K4me2, H3K4me3, H3K9ac, and H3K27ac, co-localized at loop-associated peaks, with H3K4me1 exhibiting additional specific co-localization with these four histone modifications at enhancer-localized loop-associated peaks. Notably, bivalent chromatin domains where H3K27me3 co-localized with active histone modifications were identified at promoter-localized loop-associated peaks in HepG2 cells, and elevated H3K27me3 occupancy at these peaks was associated with transcriptional repression of target genes. Moreover, quantitative RNA-seq analysis revealed that the expression of target genes associated with enhancer-promoter loops was correlated with both the binding of LLPS proteins and the enrichment patterns of histone modifications within their ChIP-seq peaks at loop anchors. CONCLUSIONS: Our study suggests that LLPS proteins may cooperate with transcriptional repressors to facilitate chromatin looping. Furthermore, local enrichment of histone modifications at loop-associated peaks provides additional regulatory control over chromatin architecture and gene transcription.

Humans↗
PubMed2007

Developmentally regulated histone modifications in Drosophila follicle cells: initiation of gene amplification is associated with histone H3 and H4 hyperacetylation and H1 phosphorylation.

We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication.

Acetylation↗
PubMed2006

Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels.

In addition to DNA sequence information, site-specific histone modifications are another important determinant of gene expression in a eukaryotic organism. We selected four modification sites in common histones that are known to significantly impact chromatin function and generated monoclonal or polyclonal antibodies that recognize each of those site-specific modifications. We used these antibodies to demonstrate that the site-specific histone modification levels remain relatively constant in different organs of the same organism. We also compared the levels of selected histone modifications among several representative organisms and found that site-specific modifications are highly variable among different organisms, providing new insight into the evolutionary divergence of specific histone modifications.

Acetylation↗
PubMed2006

Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos.

Histone modifications are thought to play important roles in various cellular functions. In this article, the distribution patterns of acetylation on histone H4, methylation on histone H3 lysine 9, and phosphorylation on histone H3 serine 10 were examined in in vivo and in vitro fertilization (IVF) preimplantation mouse embryos by using indirect immunofluorescence and scanning confocal microscopy. We desired to know whether the IVF, which has been widely used as a routine assisted reproductive technology in animal and human, was safe at the epigenetic level. As results, we found that there was no difference in these histone modification patterns in in vivo and IVF mouse embryos from zygote to blastocyst stage. Moreover, these histone modifications had different distributions at all examined stages, but they were consistent with the mouse embryo developmental stages.

Acetylation↗
PubMed2002

Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component.

Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

Fluorescent Antibody Technique↗
PubMed2006

Mapping histone modifications by nucleosome immunoprecipitation.

Studies of histone modification patterns and their role in gene regulation have led to the proposal that there is a "histone code." We have developed a method for nucleosome immunoprecipitation that can precisely identify the specific nucleosomes that carry a posttranslational modification of interest. The process involves the isolation and micrococcal nuclease digestion of minichromosomes to generate nucleosome core particles. These are then used in immunoprecipitation reactions with an antibody directed against the histone modification of interest. Subsequently, nucleosome core particle DNA is purified and end labeled. The original locations of the nucleosomes in the immunoprecipitate can be determined at low resolution (using a modified Southern blot hybridization procedure) or at maximal resolution (using the monomer extension method). Using the latter method, the positions of specific nucleosomes that carry the posttranslational modification of interest can be identified precisely. This method is sensitive, provides maximal resolution, and is inexpensive. The approach described here may serve as a paradigm for the study of histone-modifying patterns.

Animals↗
PubMed2006

Histone modifications in kainate-induced status epilepticus.

To understand the molecular actions of status epilepticus at the chromatin level, we studied the effects of kainate-induced status epilepticus on two different histone modifications at amino terminal tails: histone H3 phosphorylation at serine 10 and histone H4 acetylation. In addition to induction of c-fos and c-jun immediate early genes (IEGs) expression in mouse hippocampus, we also found the upregulation of acetylation and phosphorylation of histones, coupled with status epilepticus after kainate administration. c-fos and c-jun mRNA were sequentially induced in response to kainate, in different hippocampal subpopulations, starting from the dentate gyrus (DG) and spreading to the cornus ammonis regions. Immunohistochemical analysis showed that the spatio-temporal distribution of histone H4 hyperacetylation after kainate treatment was well correlated with the expression of c-fos and c-jun genes. Additionally, there was a transient appearance of phosphorylated histone H3 specifically in the DG region. CREB-binding protein or CBP, a well-known transcriptional co-activator with histone acetyltransferase (HAT) activity, was also induced by kainate and its expression pattern well correlated with histone H4 hyperacetylation in the hippocampus. Chromatin immunoprecipitation analysis showed that both histone modifications were associated with c-fos gene promoter after kainate stimulation, but only histone acetylation with c-jun gene. Pretreatment with curcumin, which has a HAT inhibitory activity specific for CBP/p300, attenuated histone modifications, IEGs expression and also the severity of status epilepticus after kainate treatment. Our findings suggest the involvement of histone modifications induced by kainate not only in IEGs expression but also in the development of epilepsy.

Animals↗
PubMed2004

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin.

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.

Acetylation↗
PubMed2004

Histone modifications and DNA double-strand break repair.

The roles of different histone modifications have been explored extensively in a number of nuclear processes, particularly in transcriptional regulation. Only recently has the role of histone modification in signaling or facilitating DNA repair begun to be elucidated. DNA broken along both strands in the same region, a double-strand break, is damaged in the most severe way possible and can be the most difficult type of damage to repair accurately. To successfully repair the double-strand break, the cell must gain access to the damaged ends of the DNA and recruit repair factors, and in the case of homologous recombination repair, the cell must also find, colocalize, and gain access to a suitable homologous sequence. In the repair of a double-strand break, the cell must also choose between homologous and non-homologous pathways of repair. Here, we will briefly review the mechanisms of double-strand-break repair, and discuss the known roles of histone modifications in signaling and repairing double-strand breaks.

Acetylation↗
PubMed2004

Temporal and spatial changes in transcription factor binding and histone modifications at the steroidogenic acute regulatory protein (stAR) locus associated with stAR transcription.

We investigated the binding of transcription factors and histone modifications associated with expression of the steroidogenic acute regulatory protein (StAR) gene in cultured MA-10 Leydig cells and in granulosa cells isolated from mouse periovulatory follicles before and after in vivo human chorionic gonadotropin administration. Quantitative chromatin immunoprecipitation assays were employed to prove association of specific transcription factors (GATA-4, steroidogenic factor 1/adrenal-4 binding protein, CCAAT/enhancer-binding protein beta, cAMP response element binding protein/cAMP response element modulator) and a coactivator (cAMP response element binding protein-binding protein) with the promoter, to define patterns of binding to test hypotheses regarding interactions among these factors, and to correlate changes in histone modification at the StAR locus with transcription. Although each of the transcription factors bound to the StAR proximal promoter, we observed cell-specific binding patterns for individual factors. From these findings we infer that associations among some of the factors can be more complex than can be explained by simple models of stable protein-protein interactions. Histone modifications were also found to exhibit cell-specific, temporal and spatial differences across the StAR locus. In MA-10 cells, these modifications included increased acetylation of histone H3, increased dimethylation of lysine 4 on histone H3 in exonic/intronic sequences (a modification that marks transcriptionally permissive chromatin), and reduced dimethylation of lysine 9 on histone H3 (a modification linked with gene silencing). In mouse granulosa cells, we observed no change in histone H3 or H4 acetylation, but a rapid loss of the dimethyl K9 histone H3 mark. Our findings demonstrate that increased StAR transcription can occur in the context of different patterns of transcription factor binding and histone modification.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor.

We have used chromatin immunoprecipitation (ChIP) assay to follow transcription factor loading and monitor changes in covalent histone modifications associated with the prostate-specific antigen and kallikrein (KLK2) genes in response to androgen and antiandrogen in LNCaP cells. The dynamics of testosterone (T)-induced loading of androgen receptor (AR) onto the proximal promoters of the genes differed significantly from that onto the distal enhancers. Significantly more holo-AR was loaded onto the enhancers than the promoters, but the receptor's residence time was more transient on the enhancers. Even though holo-AR recruited some RNA polymerase II (Pol II) onto the enhancers, the principal Pol II transcription complex was assembled on the promoters. The pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the prostate-specific antigen promoter, but not that of the enhancer, whereas the partial antagonists cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading also onto the enhancer. In contrast to the CDX-occupied receptor, both CPA- and RU486-bound AR recruited Pol II and coactivators p300 and glucocorticoid receptor-interacting protein 1 (GRIP1) onto the promoter and enhancer. However, CPA and RU486 also brought about a simultaneous recruitment of the nuclear receptor corepressor (NCOR) onto the promoter as efficiently as CDX. There were dynamic changes in covalent modifications of histone H3: acetylation of lysine 9 and 14, methylation of arginine 17, phosphorylation of serine 10 as well as di- and tri-methylation at lysine 4 of the H3 N-terminal tail were enhanced in response to T, but not after CDX treatment. Collectively, these results indicate that transcriptional activation by AR is accompanied by a cascade of distinct covalent histone modifications and that the pure antiandrogen CDX and the partial antagonists CPA and RU486 exhibit clear differences in their ability to promote recruitment of histone-acetylating and histone-deacetylating complexes in human prostate cancer cells.

Acetylation↗
PubMed2003

Covalent histone modifications underlie the developmental regulation of insulin gene transcription in pancreatic beta cells.

Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3-K4). To determine whether covalent histone modifications play a role in the beta cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from beta cell lines, non-beta cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in beta cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in beta cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of beta cells is hypermethylated at H3-K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of beta cells and non-beta cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the beta cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.

Acetylation↗
PubMed2005

Changes in histone modifications during in vitro maturation of porcine oocytes.

Nuclear core histone modifications influence chromosome structures and functions. Recently, the involvement of histone acetylations in the cell memory of gene expression has been suggested in mouse oocyte maturation. At present, there is little available data on histone modifications in mammalian oocyte maturation. In the present study, we examined changes in the acetylation of histone H3 lysines 9 (H3K9) and 14 (H3K14), and histone H4 lysines 5 (H4K5), 8 (H4K8) and 12 (H4K12), and trimethylation of H3K9 during in vitro maturation of porcine oocytes. Immunocytochemical analyses revealed that the all of the lysines examined were highly acetylated in the germinal vesicle stage, and this level of acetylation was maintained until the first prometaphase. In the first metaphase, the lysines near the N-terminal end, H3K9 and H4K5, were completely deacetylated. The acetylation of the lysines far from the N-terminal end, H3K14, H4K8, and H4K12, was markedly decreased but still present. The acetylations were increased transiently at the first anaphase and telophase, and then decreased again at the second metaphase to the same level as the first metaphase. Since effective concentrations of trichostatin A (TSA) to inhibit the deacetylation were different in various lysine residues, multiple histone deacetylases (HDACs) were suggested to function during meiotic maturation. The trimethylation of H3K9 was maintained in a high level throughout maturation. These results suggest that the histone acetylation during porcine oocyte maturation is precisely controlled by the cell cycle.

Acetylation↗
PubMed2004

Dynamic alterations of specific histone modifications during early murine development.

In order to investigate whether covalent histone modifications may be involved in early embryonic reprogramming events, changes in global levels of a series of histone tail modifications were studied during oocyte maturation and pre-implantation mouse development using indirect immunofluorescence and scanning confocal microscopy. Results showed that histone modifications could be classified into two strikingly distinct categories. The first contains stable 'epigenetic' marks such as histone H3 lysine 9 methylation [Me(Lys9)H3], histone H3 lysine 4 methylation [Me(Lys4)H3] and histone H4/H2A serine 1 phosphorylation [Ph(Ser1)H4/H2A]. The second group contains dynamic and reversible marks and includes hyperacetylated histone H4, histone H3 arginine 17 methylation [Me(Arg17)H3] and histone H4 arginine 3 methylation [Me(Arg3)H4]). Our results also showed that removal of these marks in eggs and early embryos occurs during metaphase suggesting that the enzymes responsible for the loss of these modifications are probably cytoplasmic in nature. Finally, we provide data demonstrating that treatment of cellular histones with peptidylarginine deiminase (PAD) results in loss of staining for the histone H4 arginine 3 methyl mark, suggesting that PADs can reverse histone arginine methyl modifications.

Acetylation↗
PubMed2005

Histone modifications in Rett syndrome lymphocytes: a preliminary evaluation.

Most cases of Rett syndrome (RTT) are associated with mutations in the coding region of the transcriptional regulator MeCP2. This gene appears to repress gene expression through chromatin conformational changes secondary to histone modifications, mainly histone deacetylation of core histones H3 and H4. There is limited and contradictory information about histone modifications in RTT tissues. The present study intended to provide a preliminary characterization of histone acetylation (AcH3, AcH4) and methylation (MeH3) in RTT, with emphasis on non-selected peripheral cells and molecular-neurologic correlations. We compared 17 females with RTT, 11 of them with MeCP2 mutations, with 10 gender-matched controls in terms of lymphocyte lysate immunoblotting-based levels. We found that immunoreactivities for MeCP2 and AcH3/AcH4 are variable in both control and RTT subjects. Despite this variability, RTT subjects with nonsense mutations showed the expected reduction in C-terminal MeCP2 immunoreactivity. Regardless of MeCP2 levels, both subjects with (RTTPos) and without (RTTNeg) mutations had decreased levels of AcH3. The latter reductions were mainly driven by decreases in levels of H3 acetylated at lysine residue 14 (AcH3K14) and independent of parallel, but milder, decreases in immunoreactivity for MeH3 lysine residues (MeH3K4/MeH3K9). Within our study sample, reductions in AcH3 were correlated with severity of head growth deceleration in the RTTPos group. This contrasted with the lack of significant association between location of MeCP2 mutation and severity of the RTT neurologic phenotype. We concluded that there were distinctive profiles of histone acetylation/methylation in RTT peripheral cells, which reflect pathogenetic mechanisms common to subjects with clinical features of this disorder, regardless of mutation status, and that these patterns may be relevant to neurologic dysfunction in RTT.

Acetylation↗