Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase

Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity, we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds, seven hits were identified. Of those, one inhibitor, BIX-01294 (diazepin-quinazolin-amine derivative), does not compete with the cofactor S-adenosyl-methionine, and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays, transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly, chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin.     

Deacetylation and methylation at histone H3 lysine 9 (H3K9) coordinate chromosome condensation during cell cycle progression

Interphasic chromatin condenses into the chromosomes in order to facilitate the correct segregation of genetic information. It has been previously reported that the phosphorylation and methylation of the N-terminal tail of histone H3 are responsible for chromosome condensation. In this study, we demonstrate that the deacetylation and methylation of histone H3 lysine 9 (H3K9) are required for proper chromosome condensation. We confirmed that H3K9ac levels were reduced, whereas H3K9me3 levels were increased in mitotic cells, via immunofluorescence and Western blot analysis. Nocodazole treatment induced G2/M arrest but co-treatment with TSA, an HDAC inhibitor, delayed cell cycle progression. However, the HMTase inhibitor, AdoX, had no effect on nocodazole-induced G2/M arrest, thereby indicating that sequential modifications of H3K9 are required for proper chromosome condensation. The expression of SUV39H1 and SETDB1, H3K9me3-responsible HMTases, are specifically increased along with H3K9me3 in nocodazole-arrested buoyant cells, which suggests that the increased expression of those proteins is an important step in chromosome condensation. H3K9me3 was highly concentrated in the vertical chromosomal axis during prophase and prometaphase. Collectively, the results of this study indicate that sequential modifications at H3K9 are associated with correct chromosome condensation, and that H3K9me3 may be relevant to the condensation of chromosome length.     

Functional analysis of histone methyltransferase g9a in B and T lymphocytes

Lymphocyte development is controlled by dynamic repression and activation of gene expression. These developmental programs include the ordered, tissue-specific assembly of Ag receptor genes by V(D)J recombination. Changes in gene expression and the targeting of V(D)J recombination are largely controlled by patterns of epigenetic modifications imprinted on histones and DNA, which alter chromatin accessibility to nuclear factors. An important component of this epigenetic code is methylation of histone H3 at lysine 9 (H3K9me), which is catalyzed by histone methyltransferases and generally leads to gene repression. However, the function and genetic targets of H3K9 methyltransferases during lymphocyte development remain unknown. To elucidate the in vivo function of H3K9me, we generated mice lacking G9a, a major H3K9 histone methyltransferase, in lymphocytes. Surprisingly, lymphocyte development is unperturbed in G9a-deficient mice despite a significant loss of H3K9me2 in precursor B cells. G9a deficiency is manifest as modest defects in the proliferative capacity of mature B cells and their differentiation into plasma cells following stimulation with LPS and IL-4. Precursor lymphocytes from the mutant mice retain tissue- and stage-specific control over V(D)J recombination. However, G9a deficiency results in reduced usage of Iglambda L chains and a corresponding inhibition of Iglambda gene assembly in bone marrow precursors. These findings indicate that the H3K9me2 epigenetic mark affects a highly restricted set of processes during lymphocyte development and activation.     

A Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin

G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely interspaced zinc finger (WIZ) associates with the G9a-GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a-GLP with chromatin by depleting WIZ resulted in altered gene expression and protein-protein interactions that were distinguishable from that of small molecule-based inhibition of G9a/GLP, supporting discrete functions of the G9a-GLP-WIZ chromatin complex in addition to H3K9me2 methylation.     

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.     

Histone H3K27 dimethylation landscapes contribute to genome stability and genetic recombination during wheat polyploidization

Bread wheat (Triticum aestivum) is an allohexaploid that was formed via two allopolyploidization events. Growing evidence suggests histone modifications are involved in the response to ‘genomic shock’ and environmental adaptation during polyploid formation and evolution. However, the role of histone modifications, especially histone H3 lysine-27 dimethylation (H3K27me2), in genome evolution remains elusive. Here we analyzed H3K27me2 and H3K27me3 profiles in hexaploid wheat and its tetraploid and diploid relatives. Although H3K27me3 levels were relatively stable among wheat species with different ploidy levels, H3K27me2 intensities increased concurrent with increased ploidy levels, and H3K27me2 peaks were colocalized with massively amplified DTC transposons (CACTA family) in euchromatin, which may silence euchromatic transposons to maintain genome stability during polyploid wheat evolution. Consistently, the distribution of H3K27me2 is mutually exclusive with another repressive histone mark, H3K9me2, that mainly silences transposons in heterochromatic regions. Remarkably, the regions with low H3K27me2 levels (named H3K27me2 valleys) were associated with the formation of DNA double-strand breaks in genomes of wheat, maize (Zea mays) and Arabidopsis. Our results provide a comprehensive view of H3K27me2 and H3K27me3 distributions during wheat evolution, which support roles for H3K27me2 in silencing euchromatic transposons to maintain genome stability and in modifying genetic recombination landscapes. These genomic insights may empower breeding improvement of crops.     

PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state

Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.