Monomethylation Of Histone H4 Lysine Research Articles

EZH2 Inhibitor Efficacy in Non-Hodgkin’s Lymphoma Does Not Require Suppression of H3K27 Monomethylation

The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkin's lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.

Discovery of a selective, substrate-competitive inhibitor of the lysine methyltransferase SETD8.

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure–activity relationships (SAR) of this series.

Enhancer biology and enhanceropathies

Enhancers are cis-regulatory elements that enable precise spatiotemporal patterns of gene expression during development and are notable for being able to function at large distances from their target genes. Such regulatory elements often bypass intervening genes and typically comprise binding sites for multiple transcription factors that can also be transcribed by RNA polymerase II (Pol II) to produce noncoding enhancer RNAs (eRNAs). Genome-wide analyses have revealed chromatin signatures of enhancers, such as the enrichment for monomethylation of histone H3 lysine 4 (H3K4me1) and the acetylation or methylation of histone H3 lysine 27 (H3K27). Enhancer signatures have been used to describe the transitions of these regulatory elements from inactive to primed and from activated to decommissioned states during development. New mutations of enhancer sequences and of the protein factors regulating enhancer function in human disease continue to be identified, contributing to a growing class of 'enhanceropathies'.

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.

The biological functions of lysine methyltransferase PR-SET7

PR-SET7 (also named SET8 or KMT5a) is a sole lysine methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20me1) in higher eukaryotes. The abundance of PR-SET7 is dynamically mediated by the distinct E3 ubiquitin ligases in different cell cycle phases. PR-SET7 is closely related to the regulation of cell proliferation, and the H4K20me1 catalyzed by PR-SET7 has been implicated in regulating the diverse biological processes, including DNA replication, chromosome condensation and the activation of DNA replication checkpoints. Loss of PR-SET7 results in mas-sive DNA damage, cell cycle arrest and induction of apoptosis. In addition, PR-SET7 involves in regulating the transcrip-tion of several genes, such as ERa, Wnt and p53. PR-SET7 is also essential for individual development and participates in the formation of genomic imprinting. Moreover, PR-SET7 has been reported to promote tumorigenesis and metastasis, sug-gesting that PR-SET7 may be a potential target for cancer treatment. In this review, we focus on analyzing the structure of PR-SET7 and factors influencing histone modification on regulation of PR-SET7, and discuss the mechanisms by which PR-SET7 modulates cell-cycle progression, gene transcription, individual development and tumorigenesis.

Chromatin and higher-order chromosome organization shape the recombination landscape in C. elegans

Meiotic recombination generates crossovers, which physically link homologous chromosomes, and direct their segregation to opposite poles. Despite the benefits, crossovers are restricted both in number as well as spatially. Crossovers in the nematode C. elegans occur most frequently within the distal regions (or “arms”) of the chromosomes, which comprise ~50% of the genome, but ~90% of the genetic map length. Meiotic recombination initiates with the formation of programmed double strand breaks (DSBs) catalyzed by the SPO-11 enzyme. It has been unclear whether the crossover bias in C. elegans is a consequence of DSB distribution, or instead reflects a mechanism that biases the downstream repair outcome of DSBs. In order to address this, we generated a genome-wide map of meiotic DSBs by mapping the distribution of the sole C. elegans RecA-like recombinase, RAD-51, by ChIP-seq. We have found that the RAD-51 binding pattern is strikingly similar to the known recombination pattern in C. elegans. The majority of RAD-51 binding sites occur on chromosome arms. Comparing RAD-51 distribution relative to functional genomic elements revealed that RAD-51 is particularly enriched in intronic DNA while coding sequences are underrepresented in RAD-51 occupied regions. We find that monomethylation of histone H3 lysine 36 (H3K36me1), which has not previously been associated with meiotic recombination, is highly correlated with break site preference. H3K36me1 is particularly enriched over active genes with high intron content, suggesting that the specific chromatin environment associated with these genes favors DSB formation in C. elegans. We have also identified a sequence motif that is highly overrepresented in DSB sites. Although the DSB pattern is highly correlated with crossover frequency, there are interesting exceptions. In particular, the sub-telomeric regions are completely devoid of crossovers but are active DSB sites. We have also identified a DNA sequence motif that appears to be enriched within DSB sites suggesting that sequence specific factors may have a role in DSB specification in C. elegans. In a parallel study, we have developed an Illumina Golden Gate SNP array to rapidly measure genome-wide recombination rates in C. elegans. Using this array we measured meiotic recombination rates in met-2(n4256) mutant animals. MET-2 is one of the two histone methyltransferases responsible for H3K9 methylation, a histone mark associated with heterochromatin. We find that the genetic map is significantly altered in met-2(n4256) mutant animals. In particular, regions that are normally enriched for H3K9 methylation show elevated recombination rates in met-2(n4256) mutants. We are currently investigating whether this change in the genetic map is the result of altered DSB distribution. Taken together, our data provide new insights into the role of chromatin in meiotic recombination. Because meiotic recombination generates genetic diversity, our data implicates chromatin as a key player in the evolution of genomes.

High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine in Mouse Embryonic Stem Cells

We describe the use of a unique DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5-hydroxymethylcytosine (5hmC) at low-occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follow the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the monomethylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

Methyltransferase Inhibitor Adenosine Dialdehyde Suppresses Androgen Receptor Expression and Prostate Cancer Growth

Although most prostate cancers regress after androgen deprivation therapy is given at diagnosis, they eventually regrow in a castration resistant manner, spread systemically and end fatally. Thus, novel therapeutic compounds are needed for prostate cancer. We previously reported that methylation at histone H3 lysine 9 was increased in prostate cancer. In this study we examined the effects of the methyltransferase inhibitor adenosine dialdehyde (Sigma®) on the methylation state of histone H3 lysine 9 and AR gene expression as well as its possible usefulness for prostate cancer. The effect of adenosine dialdehyde on the methylation state of histone H3 lysine 9 and AR gene expression was examined by quantitative real-time polymerase chain reaction and Western blot. We compared methylation at histone H3 lysine 9 at the AR promoter region between androgen dependent and castration resistant prostate cancer by chromatin immunoprecipitation assay. The cytotoxic effect of adenosine dialdehyde on prostate cancer was also evaluated in vitro and in vivo. Adenosine dialdehyde suppressed the monomethylation and dimethylation of histone H3 lysine 9 and inhibited Twist1 as well as androgen receptor expression, which are critical for the survival and growth of androgen dependent, androgen sensitive and castration resistant prostate cancer cells in which monomethylated histone H3 lysine 9 increased at the 5' untranslated region of the AR gene. As a result, adenosine dialdehyde had a cytotoxic effect on androgen dependent, androgen sensitive and castration resistant prostate cancer cells in vitro. Adenosine dialdehyde also suppressed prostate cancer growth in vivo in a mouse xenograft model. Results indicate that the methyltransferase inhibitor adenosine dialdehyde is a promising, novel therapeutic compound for prostate cancer.

Global Mapping of H3K4me1 and H3K4me3 Reveals the Chromatin State-Based Cell Type-Specific Gene Regulation in Human Treg Cells

Regulatory T cells (Treg) contribute to the crucial immunological processes of self-tolerance and immune homeostasis. Genomic mechanisms that regulate cell fate decisions leading to Treg or conventional T cells (Tconv) lineages and those underlying Treg function remain to be fully elucidated, especially at the histone modification level. We generated high-resolution genome-wide distribution maps of monomethylated histone H3 lysine 4 (H3K4me1) and trimethylated H3K4 (H3K4me3) in human CD4+CD25+FOXP3+ Tregs and CD4+CD25+FOXP3− activated (a)Tconv cells by DNA sequencing-by-synthesis. 2115 H3K4me3 regions corresponded to proximal promoters; in Tregs, the genes associated with these regions included the master regulator FOXP3 and the chemokine (C-C motif) receptor 7 (CCR7). 41024 Treg-specific H3K4me1 regions were identified. The majority of the H3K4me1 regions differing between Treg and aTconv cells were located at promoter-distal sites, and in vitro reporter gene assays were used to evaluate and identify novel enhancer activity. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me1 and H3K4me3 patterns in Treg and aTconv cells, which may control cell type-specific gene regulation. This basic principle is likely not restricted to the two closely-related T cell populations, but may apply generally to somatic cell lineages in adult organisms.

Patterns of Histone H3 Lysine 27 Monomethylation and Erythroid Cell Type-specific Gene Expression

Post-translational histone modifications, acting alone or in a context-dependent manner, influence numerous cellular processes via their regulation of gene expression. Monomethylation of histone H3 lysine 27 (K27me1) is a poorly understood histone modification. Some reports describe depletion of K27Me1 at promoters and transcription start sites (TSS), implying its depletion at TSS is necessary for active transcription, while others have associated enrichment of H3K27me1 at TSS with increased levels of mRNA expression. Tissue- and gene-specific patterns of H3K27me1 enrichment and their correlation with gene expression were determined via chromatin immunoprecipitation on chip microarray (ChIP-chip) and human mRNA expression array analyses. Results from erythroid cells were compared with those in neural and muscle cells. H3K27me1 enrichment varied depending on levels of cell-type specific gene expression, with highest enrichment over transcriptionally active genes. Over individual genes, the highest levels of H3K27me1 enrichment were found over the gene bodies of highly expressed genes. In contrast to H3K4me3, which was highly enriched at the TSS of actively transcribing genes, H3K27me1 was selectively depleted at the TSS of actively transcribed genes. There was markedly decreased to no H3K27me1 enrichment in genes with low expression. At some locations, H3K27 monomethylation was also found to be associated with chromatin signatures of gene enhancers.

PR‐Set7‐mediated monomethylation of histone H4 lysine 20 at specific genomic regions induces transcriptional repression

Increasing evidence indicates that the post-translational modifications of the histone proteins play critical roles in all eukaryotic DNA-templated processes. To gain further biological insights into two of these modifications, the mono- and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP-chip experiments were performed to identify the precise genomic regions on human chromosomes 21 and 22 occupied by these two modifications. Detailed analysis revealed that H4K20me1 was preferentially enriched within specific genes; most significantly between the first approximately 5% and 20% of gene bodies. In contrast, H4K20me3 was preferentially targeted to repetitive elements. Among genes enriched in H4K20me3, the modification was typically targeted to a small region approximately 1 kb upstream of transcription start. Our collective findings strongly suggest that H4K20me1 and H4K20me3 are both physically and functionally distinct. We next sought to determine the role of H4K20me1 in transcription since this has been controversial. Following the reduction of PR-Set7/Set8/KMT5a and H4K20me1 in cells by RNAi, all H4K20me1-associated genes analyzed displayed an approximately 2-fold increase in gene expression; H4K20me3-associated genes displayed no changes. Similar results were obtained using a catalytically dead dominant negative PR-Set7 indicating that H4K20me1, itself, is essential for the selective transcriptional repression of H4K20me1-associated genes. Furthermore, we determined that the H4K20me1-associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression in vivo. Our findings reveal the molecular mechanisms of a mammalian transcriptional repressive pathway whereby the DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of H4K20 which, in turn, reduces gene expression by half.

Role of the H4K20me1/H3K9me1 trans‐tail histone code in transcriptional regulation

We previously discovered a novel trans‐tail histone code in which monomethylated histone H3 lysine 9 (H3K9me1) is enriched in genomic regions containing monomethylated histone H4 lysine 20 (H4K20me1). Based on this, we hypothesized that the PR‐Set7 H4K20 monomethyltransferase recruits an H3K9 monomethyltransferase to establish this histone code to regulate the expression of specific genes. A survey of known H3K9 methyltransferases revealed that only PRDM2/Riz1 binds PR‐Set7 in vivo. To further understand the role of this histone code in transcription, gene expression profiling was used to identify a panel of target genes regulated by PR‐Set7. In all cases, the absence of PR‐Set7 and/or H4K20me1 resulted in a >200% increase in their transcription indicating a repressive role for this histone modification. Although both H3K9me1 and H4K20me1 were enriched at these genes, depletion of Riz1 had no effect on H4K20me1 levels and had varying effects on transcription. Collectively, these findings indicate that the repression of a specific set of human genes is conferred by H4K20me1 and suggests that H3K9me1 may be dispensable for gene regulation.

Structural insights into a novel histone demethylase PHF8

Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain (PHD) finger protein 8 (PHF8) are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without alpha-ketoglutarate (alpha-KG) at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 (H3K9me2/1), but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.

Patterns of Monomethylation of Histone H3 Lysine 27 Influence Gene Expression in a Cell-Type Specific Manner.

Abstract 4585Post-translational histone modifications influence expression by creating a chromatin environment which is conducive to or inhibitory of transcription. Modifications such as trimethylation of histone H3 lysine 4 and acetylation of histone H3 lysine 9 are generally associated with euchromatin and gene activation, while modifications such as trimethylation of histone H3 lysine 27 are associated with regions of heterochromatin and/or gene repression. Monomethyl histone H3 lysine 27 (H3K27me1) is a poorly studied post-translational histone modification for which variable associations with mRNA expression have been observed. Initially, H3K27me1 was localized to areas of pericentric heterochromain and was thought to be a marker of gene repression. Later reports described H3K27me1 enrichment throughout the body of actively transcribing genes (Vakoc C et al. MCB 26:9185, 2006; Wang Z, 40:897 Nat Genet, 2008). Some reports describe selective depletion of H3K27Me1 at promoters and transcription start sites (TSS), implying that depletion of H3K27me1 at the TSS is necessary for active transcription, (Vakoc C et al.), while others have associated increased enrichment for H3K27me1 at the promoter with increased levels of mRNA expression (Barski A et al. Cell 129:823, 2007). We hypothesize that the relationship between H3K27me1 occupancy and gene expression varies depending on both the cell-type and the location in the gene (i.e. promoter, transcription start site (TSS) and body of the gene) and that varying H3K27me1 levels in each of these locations is associated with alterations in the level of mRNA expression. To assess the association of H3K27me1 level with mRNA expression, H3K27me1 binding was determined using chromatin immunoprecipitation on microarray analysis (ChIP-chip) and correlated with mRNA levels determined using Illumina human expression arrays. ChIP-chip was performed using an antibody specific for H3K27me1 and the resulting DNA applied the to a custom designed genomic tiling NimbleGen microarray containing over 100 erythroid expressed genes and 10-100kb of flanking DNA for each locus. Probes were tiled with 10-100bp spacing, typically ∼65bp. Regions of repetitive DNA were excluded. ChIP-chip was performed in erythroid (K562) and non-erythroid (SY5Y-neural, RD-muscle) cells and the pattern of H3K27me1 enrichment assessed. mRNA transcript analyses were performed using Illumina human V6-2 expression arrays and quantitative real time RT-PCR. H3K27me1 levels at the promoter (-1000 to +1), in the 200bp surrounding the TSS (-100 to +100), and over the body of the gene were correlated with the level of mRNA expression. Increasing levels of H3K27me1 over the body of the gene lead correlated with increased levels of gene expression (R2=0.6122), while the amount of H3K27me1 at the promoter (-1000 to +1) had no correlation with gene expression (R2=0.2769). In agreement with Vakoc et al., decreased enrichment for H3K27Me1 at the TSS (-100 to +100) correlated with increased levels of mRNA expression. This is in sharp contrast to H3K4Me3, which accumulates at the start site of active genes. H3K27me1 has not been studied in detail in transcriptionally silent genes. Interestingly, genes without H3K27Me1 enrichment had no expression, implying that H3K27me1 is a marker of active transcription. Patterns of H3K27Me1 enrichment were cell-type specific. For example, in erythroid (K562) cells, the beta-globin locus was highly enriched for H3K27me1. This enrichment was not present in non-erythroid cells (RD, SY5Y). Finally, H3K27Me1 may also mark enhancers in a cell type-specific manner. For example, in the well studied HS2 enhancer in the beta-globin LCR, there is significant enrichment for H3K27me1 in K562 cells, but not in SY5Y or RD cells. These data indicate that modulation of chromatin architecture by monomethylation of histone 3 lysine 27 influences the level of gene expression in erythroid and non-erythroid cells. Disclosures:No relevant conflicts of interest to declare.

Catalytic Function of the PR-Set7 Histone H4 Lysine 20 Monomethyltransferase Is Essential for Mitotic Entry and Genomic Stability

Histone-modifying enzymes play a critical role in modulating chromatin dynamics. In this report we demonstrate that one of these enzymes, PR-Set7, and its corresponding histone modification, the monomethylation of histone H4 lysine 20 (H4K20), display a distinct cell cycle profile in mammalian cells: low at G1, increased during late S phase and G2, and maximal from prometaphase to anaphase. The lack of PR-Set7 and monomethylated H4K20 resulted in a number of aberrant phenotypes in several different mammalian cell types. These include the inability of cells to progress past G2, global chromosome condensation failure, aberrant centrosome amplification, and substantial DNA damage. By employing a catalytically dead dominant negative PR-Set7 mutant, we discovered that its mono-methyltransferase activity was required to prevent these phenotypes. Importantly, we demonstrate that all of the aberrant phenotypes associated with the loss of PR-Set7 enzymatic function occur independently of p53. Collectively, our findings demonstrate that PR-Set7 enzymatic activity is essential for mammalian cell cycle progression and for the maintenance of genomic stability, most likely by monomethylating histone H4K20. Our results predict that alterations of this pathway could result in gross chromosomal aberrations and aneuploidy.

Expression of cell fate‐specific genes are regulated by monomethylated Histone H4 Lysine 20

Post translational modifications of N terminal histone tails play an important role in regulating gene expression. In this study, we show that one of these modifications, the monomethylation of Histone H4 Lysine 20 (H4K20) is found within lineage specific genes and that loss of H4K20 monomethylation causes upregulation of these genes. Runx1, a regulator of hematopoeisis, is an example of a gene that is upregulated in the absence of H4K20 monomethylation. Chromatin immunoprecipitation assays (ChIP) confirmed the enrichment of H4K20 monomethylation at Runx1 promoter region but not at an upstream region. The H4K20 monomethyl binding protein, L3MBT, was also enriched at the promoter regions. This is consistent with our previous data and shows that L3MBT binds to H4K20 monomethyation to cause gene repression. Knockdown of H4K20 monomethylation also caused misexpression of Runx1 in HeLa cells. To further investigate the biological relevance of H4K20 monomethylation at Runx1, we dfferentiated the pre‐erythroid cell line, K562, into megakaryocytes, which are Runx1 positive, and found that as Runx1 protein levels increased, global levels of H4K20 monomethylation decreased. Taken together, these data define a novel repression pathway in which H4K20 monomethylation and L3MBT cooperate to repress lineage specific genes such as Runx1.

Aberrant monomethylation of histone H4 lysine 20 activates the DNA damage checkpoint inDrosophila melanogaster

PR-Set7 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (K20) and is essential for cell proliferation. Our results show that in PR-Set7 mutants, the DNA damage checkpoint is activated. This phenotype is manifested by reduction in both the mitotic and the S phase indexes, a delay in the progression through early mitosis, and strong reduction of cyclin B. Furthermore, in a double mutant of PR-Set7 and mei-41 (the fly ATR orthologue), the abnormalities of mitotic progression and the cyclin B protein level were rescued. PR-Set7 also showed a defect in chromosome condensation that was enhanced in the double mutant. We therefore propose that monomethylated H4K20 is involved in the maintenance of proper higher order structure of DNA and is consequently essential for chromosome condensation.

Differentiation-dependent Alterations in Histone Methylation and Chromatin Architecture at the Inducible Chicken Lysozyme Gene

It is now well established that locus-wide chromatin remodeling and dynamic alterations of histone modifications are required for the developmentally regulated activation of tissue-specific genes. However, little is known about the dynamics of these events during cell differentiation and how chromatin of an entire gene locus responds to signal transduction processes. To address this issue we investigated chromatin accessibility, linker histone distribution, and the histone methylation status at the macrophage-specific chicken lysozyme locus and the ubiquitously expressed gas41 locus in multipotent precursor cell lines and BM2 monoblast cells. The latter can be induced to go through macrophage maturation by treatment with phorbol-12-myristate acetate and can be further stimulated with bacterial lipopolysaccharide. We show that expression of the lysozyme gene in undifferentiated monoblasts is low and that a high level of gene expression requires both cell differentiation and lipopolysaccharide stimulation. However, depletion of the linker histone H1 is observed already in lysozyme non-expressing multipotent precursor cells. In undifferentiated monoblasts, the lysozyme regulatory regions are marked by the presence of monomethylated histone H3 lysine 4, which becomes increasingly converted into trimethylated H3 lysine K4 during cell differentiation. We also present evidence for extensive, differentiation-dependent alterations in nuclease accessibility at the lysozyme promoter without alterations of nucleosome and transcription factor occupancy.