Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle.

Chromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10-50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1-5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

Protein kinase Msk1 physically and functionally interacts with the KMT2A/MLL1 methyltransferase complex and contributes to the regulation of multiple target genes.

BACKGROUND: The KMT2A/MLL1 lysine methyltransferase complex is an epigenetic regulator of selected developmental genes, in part through the SET domain-catalysed methylation of H3K4. It is essential for normal embryonic development and haematopoiesis and frequently mutated in cancer. The catalytic properties and targeting of KMT2A/MLL1 depend on the proteins with which it complexes and the post-translational protein modifications which some of these proteins put in place, though detailed mechanisms remain unclear. RESULTS: KMT2A/MLL1 (both native and FLAG-tagged) and Msk1 (RPS6KA5) co-immunoprecipitated in various cell types. KMT2A/MLL1 and Msk1 knockdown demonstrated that the great majority of genes whose activity changed on KTM2A/MLL1 knockdown, responded comparably to Msk1 knockdown, as did levels of H3K4 methylation and H3S10 phosphorylation at KTM2A target genes HoxA4, HoxA5. Knockdown experiments also showed that KMT2A/MLL1 is required for the genomic targeting of Msk1, but not vice versa. CONCLUSION: The KMT2A/MLL1 complex is associated with, and functionally dependent upon, the kinase Msk1, part of the MAP kinase signalling pathway. We propose that Msk1-catalysed phosphorylation at H3 serines 10 and 28, supports H3K4 methylation by the KMT2A/MLL1 complex both by making H3 a more attractive substrate for its SET domain, and improving target gene accessibility by prevention of HP1- and Polycomb-mediated chromatin condensation.

Histone deacetylase inhibitors for cancer therapy: An evolutionarily ancient resistance response may explain their limited success.

Histone deacetylase inhibitors (HDACi) are in clinical trials against a variety of cancers. Despite early successes, results against the more common solid tumors have been mixed. How is it that so many cancers, and most normal cells, tolerate the disruption caused by HDACi-induced protein hyperacetylation? And why are a few cancers so sensitive? Here we discuss recent results showing that human cells mount a coordinated transcriptional response to HDACi that mitigates their toxic effects. We present a hypothetical signaling system that could trigger and mediate this response. To account for the existence of such a response, we note that HDACi of various chemical types are made by a variety of organisms to kill or suppress competitors. We suggest that the resistance response in human cells is a necessary evolutionary consequence of exposure to environmental HDACi. We speculate that cancers sensitive to HDACi are those in which the resistance response has been compromised by mutation. Identifying such mutations will allow targeting of HDACi therapy to potentially susceptible cancers. Also see the video abstract here.

Cells adapt to the epigenomic disruption caused by histone deacetylase inhibitors through a coordinated, chromatin-mediated transcriptional response.

BACKGROUND: The genome-wide hyperacetylation of chromatin caused by histone deacetylase inhibitors (HDACi) is surprisingly well tolerated by most eukaryotic cells. The homeostatic mechanisms that underlie this tolerance are unknown. Here we identify the transcriptional and epigenomic changes that constitute the earliest response of human lymphoblastoid cells to two HDACi, valproic acid and suberoylanilide hydroxamic acid (Vorinostat), both in widespread clinical use. RESULTS: Dynamic changes in transcript levels over the first 2 h of exposure to HDACi were assayed on High Density microarrays. There was a consistent response to the two different inhibitors at several concentrations. Strikingly, components of all known lysine acetyltransferase (KAT) complexes were down-regulated, as were genes required for growth and maintenance of the lymphoid phenotype. Up-regulated gene clusters were enriched in regulators of transcription, development and phenotypic change. In untreated cells, HDACi-responsive genes, whether up- or down-regulated, were packaged in highly acetylated chromatin. This was essentially unaffected by HDACi. In contrast, HDACi induced a strong increase in H3K27me3 at transcription start sites, irrespective of their transcriptional response. Inhibition of the H3K27 methylating enzymes, EZH1/2, altered the transcriptional response to HDACi, confirming the functional significance of H3K27 methylation for specific genes. CONCLUSIONS: We propose that the observed transcriptional changes constitute an inbuilt adaptive response to HDACi that promotes cell survival by minimising protein hyperacetylation, slowing growth and re-balancing patterns of gene expression. The transcriptional response to HDACi is mediated by a precisely timed increase in H3K27me3 at transcription start sites. In contrast, histone acetylation, at least at the three lysine residues tested, seems to play no direct role. Instead, it may provide a stable chromatin environment that allows transcriptional change to be induced by other factors, possibly acetylated non-histone proteins.

Immunolabelling of human metaphase chromosomes reveals the same banded distribution of histone H3 isoforms methylated at lysine 4 in primary lymphocytes and cultured cell lines.

BACKGROUND: Using metaphase spreads from human lymphoblastoid cell lines, we previously showed how immunofluorescence microscopy could define the distribution of histone modifications across metaphase chromosomes. We showed that different histone modifications gave consistent and clearly defined immunofluorescent banding patterns. However, it was not clear to what extent these higher level distributions were influenced by long-term growth in culture, or by the specific functional associations of individual histone modifications. RESULTS: Metaphase chromosome spreads from human lymphocytes stimulated to grow in short-term culture, were immunostained with antibodies to histone H3 mono- or tri-methylated at lysine 4 (H3K4me1, H3K4me3). Chromosomes were identified on the basis of morphology and reverse DAPI (rDAPI) banding. Both antisera gave the same distinctive immunofluorescent staining pattern, with unstained heterochromatic regions and a banded distribution along the chromosome arms. Karyotypes were prepared, showing the reproducibility of banding between sister chromatids, homologue pairs and from one metaphase spread to another. At the light microscope level, we detect no difference between the banding patterns along chromosomes from primary lymphocytes and lymphoblastoid cell lines adapted to long-term growth in culture. CONCLUSIONS: The distribution of H3K4me3 is the same across metaphase chromosomes from human primary lymphocytes and LCL, showing that higher level distribution is not altered by immortalization or long-term culture. The two modifications H3K4me1 (enriched in gene enhancer regions) and H3K4me3 (enriched in gene promoter regions) show the same distributions across human metaphase chromosomes, showing that functional differences do not necessarily cause modifications to differ in their higher-level distributions.

Dosage compensation in mammals.

Many organisms show major chromosomal differences between sexes. In mammals, females have two copies of a large, gene-rich chromosome, the X, whereas males have one X and a small, gene-poor Y. The imbalance in expression of several hundred genes is lethal if not dealt with by dosage compensation. The male-female difference is addressed by silencing of genes on one female X early in development. However, both males and females now have only one active X chromosome. This is compensated by twofold up-regulation of genes on the active X. This complex system continues to provide important insights into mechanisms of epigenetic regulation.

Nucleosome signalling; an evolving concept.

The nucleosome core particle is the first stage of DNA packaging in virtually all eukaryotes. It both organises nuclear DNA and protects it from adventitious binding of transcription factors and the consequent deregulation of gene expression. Both properties are essential to allow the genome expansion characteristic of complex eukaryotes. The nucleosome is a flexible structure in vivo, allowing selective relaxation of its intrinsically inhibitory effects in response to external signals. Structural changes are brought about by dedicated remodelling enzymes and by posttranslational modifications of the core histones. Histone modifications occasionally alter nucleosome structure directly, but their more usual roles are to act as receptors on the nucleosome surface that are recognised by specific protein domains. The bound proteins, in turn, affect nucleosome structure and function. This strategy enormously expands the signalling capacity of the nucleosome and its ability to influence both the initiation and elongation stages of transcription. The enzymes responsible for placing and removing histone modifications, and the modification-binding proteins themselves, are ubiquitous, numerous and conserved amongst eukaryotes. Like the nucleosome, they date back to the earliest eukaryotes and may have played integral and essential roles in eukaryotic evolution. The present properties and epigenetic functions of the nucleosome reflect its evolutionary past and the selective pressures to which it has responded and can be better understood in this context. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

The histone deacetylase inhibitor sodium valproate causes limited transcriptional change in mouse embryonic stem cells but selectively overrides Polycomb-mediated Hoxb silencing.

BACKGROUND: Histone deacetylase inhibitors (HDACi) cause histone hyperacetylation and H3K4 hypermethylation in various cell types. They find clinical application as anti-epileptics and chemotherapeutic agents, but the pathways through which they operate remain unclear. Surprisingly, changes in gene expression caused by HDACi are often limited in extent and can be positive or negative. Here we have explored the ability of the clinically important HDACi valproic acid (VPA) to alter histone modification and gene expression, both globally and at specific genes, in mouse embryonic stem (ES) cells. RESULTS: Microarray expression analysis of ES cells exposed to VPA (1 mM, 8 h), showed that only 2.4% of genes showed a significant, >1.5-fold transcriptional change. Of these, 33% were down-regulated. There was no correlation between gene expression and VPA-induced changes in histone acetylation or H3K4 methylation at gene promoters, which were usually minimal. In contrast, all Hoxb genes showed increased levels of H3K9ac after exposure to VPA, but much less change in other modifications showing bulk increases. VPA-induced changes were lost within 24 h of inhibitor removal. VPA significantly increased the low transcription of Hoxb4 and Hoxb7, but not other Hoxb genes. Expression of Hoxb genes increased in ES cells lacking functional Polycomb silencing complexes PRC1 and PRC2. Surprisingly, VPA caused no further increase in Hoxb transcription in these cells, except for Hoxb1, whose expression increased several fold. Retinoic acid (RA) increased transcription of all Hoxb genes in differentiating ES cells within 24 h, but thereafter transcription remained the same, increased progressively or fell progressively in a locus-specific manner. CONCLUSIONS: Hoxb genes in ES cells are unusual in being sensitive to VPA, with effects on both cluster-wide and locus-specific processes. VPA increases H3K9ac at all Hoxb loci but significantly overrides PRC-mediated silencing only at Hoxb4 and Hoxb7. Hoxb1 is the only Hoxb gene that is further up-regulated by VPA in PRC-deficient cells. Our results demonstrate that VPA can exert both cluster-wide and locus-specific effects on Hoxb regulation.

Altered histone modifications in cancer.

In human health and disease the choreographed actions of a wide armory of transcription factors govern the regulated expression of coding and nonprotein coding genes. These actions are central to human health and are evidently aberrant in cancer. Central components of regulated gene expression are a variety of epigenetic mechanisms that include histone modifications. The post-translational modifications of histones are widespread and diverse, and appear to be spatial--temporally regulated in a highly intricate manner. The true functional consequences of these patterns of regulation are still emerging. Correlative evidence supports the idea that these patterns are distorted in malignancy on both a genome-wide and a discrete gene loci level. These patterns of distortion also often reflect the altered expression of the enzymes that control these histone states. Similarly gene expression patterns also appear to reflect a correlation with altered histone modifications at both the candidate loci and genome-wide level. Clarity is emerging in resolving these relationships between histone modification status and gene expression -patterns. For example, altered transcription factor interactions with the key co-activator and co-repressors, which in turn marshal many of the histone-modifying enzymes, may distort regulation of histone modifications at specific gene loci. In turn these aberrant transcriptional processes can trigger other altered epigenetic events such as DNA methylation and underline the aberrant and specific gene expression patterns in cancer. Considered in this manner, altered expression and recruitment of histone-modifying enzymes may underline the distortion to transcriptional responsiveness observed in malignancy. Insight from understanding these processes addresses the challenge of targeted epigenetic therapies in cancer.

Epigenetic distortion to VDR transcriptional regulation in prostate cancer cells.

The current study aimed to examine the gene specific mechanisms by which the actions of the vitamin D receptor (VDR) are distorted in prostate cancer. Transcriptional responses toward the VDR ligand, 1α,25(OH)2D3, were examined in non-malignant prostate epithelial cells (RWPE-1) and compared to the 1α,25(OH)2D3-recalcitrant prostate cancer cells (PC-3). Time resolved transcriptional studies for two VDR target genes revealed selective attenuation and repression of VDR transcriptional responses in PC-3 cells. For example, responses in PC-3 cells revealed suppressed responsiveness of IGFBP3 and G0S2. Furthermore, Chromatin Immunoprecipitation (ChIP) assays revealed that suppressed transcriptional responses in PC-3 cells of IGFBP3 and G0S2 were associated with selective VDR-induced NCOR1 enrichment at VDR-binding regions on target-gene promoter regions. We propose that VDR inappropriately recruits co-repressors in prostate cancer cells. Subsequent direct and indirect mechanisms may induce local DNA methylation and stable transcriptional silencing. Thus a transient epigenetic process mediated by co-repressor binding, namely, the control of H3K9 acetylation, is distorted to favor a more stable epigenetic event, namely DNA methylation. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Recruitment of NCOR1 to VDR target genes is enhanced in prostate cancer cells and associates with altered DNA methylation patterns.

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.

A unified phylogeny-based nomenclature for histone variants.

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

The adjustable nucleosome: an epigenetic signaling module.

This review examines the proposition that the nucleosome, in addition to its role as a DNA packaging device, is a signaling module through which changing environmental and metabolic conditions can influence genomic functions. The role of enzyme-catalyzed post-translational modifications of the core histones is critically assessed, leading to the conclusion that they play varied, often crucial and sometimes causative roles in this signaling process.

Genes are often sheltered from the global histone hyperacetylation induced by HDAC inhibitors.

Histone deacetylase inhibitors (HDACi) are increasingly used as therapeutic agents, but the mechanisms by which they alter cell behaviour remain unclear. Here we use microarray expression analysis to show that only a small proportion of genes (∼9%) have altered transcript levels after treating HL60 cells with different HDACi (valproic acid, Trichostatin A, suberoylanilide hydroxamic acid). Different gene populations respond to each inhibitor, with as many genes down- as up-regulated. Surprisingly, HDACi rarely induced increased histone acetylation at gene promoters, with most genes examined showing minimal change, irrespective of whether genes were up- or down-regulated. Many genes seem to be sheltered from the global histone hyperacetyation induced by HDACi.

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

Epigenetic control of a VDR-governed feed-forward loop that regulates p21(waf1/cip1) expression and function in non-malignant prostate cells.

In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.

Environmental sensing by chromatin: an epigenetic contribution to evolutionary change.

Chromatin structure and function are regulated by families of protein-modifying enzymes that are sensitive to a variety of metabolic and environmental agents. These enzymes, and proteins that read the modifications they maintain, constitute a system by which environmental agents, such as chemical toxins and dietary components, can directly regulate patterns of gene expression. This review describes this environmental sensing system from an evolutionary perspective. It is proposed that persistent environmentally-induced changes in gene expression patterns can cause changes in phenotype that are acted upon by natural selection, and that epigenetic processes can potentially play central roles in evolution.

Immunostaining of modified histones defines high-level features of the human metaphase epigenome.

BACKGROUND: Immunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies. RESULTS: By immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3. CONCLUSIONS: At metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis.