WD repeat-containing protein 5, a ubiquitously expressed histone methyltransferase adaptor protein, regulates smooth muscle cell-selective gene activation through interaction with pituitary homeobox 2

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.     

Protein-arginine methyltransferase 1 (PRMT1) methylates Ash2L, a shared component of mammalian histone H3K4 methyltransferase complexes

Multiple enzymes and enzymatic complexes coordinately regulate the addition and removal of post-translational modifications on histone proteins. The oncoprotein Ash2L is a component of the mixed lineage leukemia (MLL) family members 1-4, Setd1A, and Setd1B mammalian histone H3K4 methyltransferase complexes and is essential to maintain global trimethylation of histone H3K4. However, regulation of these complexes at the level of expression and activity remains poorly understood. In this report, we demonstrate that Ash2L is methylated on arginine residues both in vitro and in cells. We found that both protein-arginine methyltransferases 1 and 5 methylate Arg-296 within Ash2L. These findings are the first to demonstrate that post-translational modifications occur on the Ash2L protein and provide a novel example of cross-talk between chromatin-modifying enzyme complexes.     

The JmjN Domain of Jhd2 Is Important for Its Protein Stability,and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.     

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.     

Plant homeodomain (PHD) fingers of CHD4 are histone H3-binding modules with preference for unmodified H3K4 and methylated H3K9

A major challenge in chromatin biology is to understand the mechanisms by which chromatin is remodeled into active or inactive states as required during development and cell differentiation. One complex implicated in these processes is the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains both histone deacetylase and nucleosome remodeling activities and has been implicated in the silencing of subsets of genes involved in various stages of cellular development. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core component of the NuRD complex and contains a nucleosome remodeling ATPase domain along with two chromodomains and two plant homeodomain (PHD) fingers. We have previously demonstrated that the second PHD finger of CHD4 binds peptides corresponding to the N terminus of histone H3 methylated at Lys(9). Here, we determine the solution structure of PHD2 in complex with H3K9me3, revealing the molecular basis of histone recognition, including a cation-π recognition mechanism for methylated Lys(9). Additionally, we demonstrate that the first PHD finger also exhibits binding to the N terminus of H3, and we establish the histone-binding surface of this domain. This is the first instance where histone binding ability has been demonstrated for two separate PHD modules within the one protein. These findings suggest that CHD4 could bind to two H3 N-terminal tails on the same nucleosome or on two separate nucleosomes simultaneously, presenting exciting implications for the mechanism by which CHD4 and the NuRD complex could direct chromatin remodeling.     

Structural and biochemical studies on the chromo-barrel domain of male specific lethal 3 (MSL3) reveal a binding preference for mono- or dimethyllysine 20 on histone H4

We have determined the human male specific lethal 3 (hMSL3) chromo-barrel domain structure by x-ray crystallography to a resolution of 2.5 Å (r = 0.226, R_free = 0.270). hMSL3 contains a canonical methyllysine binding pocket made up of residues Tyr-31, Phe-56, Trp-59, and Trp-63. A six-residue insertion between strands β₁ and β₂ of the hMSL3 chromo-barrel domain directs the side chain of Glu-21 into the methyllysine binding pocket where it hydrogen bonds to the NH group of a bound cyclohexylamino ethanesulfonate buffer molecule, likely mimicking interactions with a histone tail dimethyllysine residue. In vitro binding studies revealed that both the human and Drosophila MSL3 chromo-barrel domains bind preferentially to peptides representing the mono or dimethyl isoform of lysine 20 on the histone H4 N-terminal tail (H4K20Me₁ or H4K20Me₂). Mutation of Tyr-31 to Ala in the hMSL3 methyllysine-binding cage resulted in weaker in vitro binding to H4K20Me₁. The same mutation in the msl3 gene compromised male survival in Drosophila. Combined mutation of Glu-21 and Pro-22 to Ala in hMSL3 resulted in slightly weaker in vitro binding to H4K20Me₁, but the corresponding msl3 mutation had no effect on male survival in Drosophila. We propose MSL3 plays an important role in targeting the male specific lethal complex to chromatin in both humans and flies by binding to H4K20Me₁. Binding studies on the related dMRG15 chromo-barrel domain revealed that MRG15 prefers binding to H4K20Me₃.     

Both combinatorial K4me0-K36me3 marks on sister histone H3s of a nucleosome are required for Dnmt3a-Dnmt3L mediated de novo DNA methylation

A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively.Here,taking advantage of the bivalent histone H3 system in yeast,we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L.The results show that lack of both K4me0 and K36me3 on one sister H3 tail,or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC,revealing a synergy of two sister H3s in DNA methylation regulation.Accordingly,the Dnmt3a or Dnmt3L mutation that disrupts the interaction of Dnmt3aADD domain-H3K4me0,Dnmt3LADD domain-H3K4me0,orDnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation.These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s,and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

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.     

Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.     

组蛋白H3K4甲基转移酶复合物亚基Ash2参与裂殖酵母产孢

在氮源缺乏及信息素存在的条件下,裂殖酵母(Schizosaccharomyces pombe)进行减数分裂并完成产孢。在此过程中,信息素介导的MAPK(Mitogen-activated protein kinases)信号通路调控减数分裂相关基因的表达。Spk1是MAPK通路的核心成员,通过蛋白磷酸化的方式激活转录因子Ste11,从而激活mei2⁺、mam2⁺和map3⁺等减数分裂相关基因的表达。尽管组蛋白H3K4甲基化参与基因转录激活、染色质重塑等诸多生物学过程,但其在裂殖酵母产孢过程中的作用并不清楚。文章通过序列比对,发现裂殖酵母Ash2作为H3K4甲基转移酶复合物COMPASS的亚基具有两个保守的结构域,定位于细胞核内参与H3K4的甲基化修饰。ash2⁺的缺失引起裂殖酵母在氮源缺乏时产孢过程的延迟及产孢率下降。ChIP、定量PCR分析结果显示,ash2⁺的缺失降低了spk1⁺编码区H3K4的二甲基化水平,造成spk1⁺mRNA水平的明显下调。在ash2Δ细胞中,虽然ste11⁺的转录水平没有变化,但Ste11的靶基因mei2⁺、mam2⁺和map3⁺的转录水平明显下降。在裂殖酵母中,组蛋白H3K4甲基转移酶复合物COMPASS的亚基Ash2通过调控二甲基化水平修饰从而调节MAPK信号通路,参与裂殖酵母的有性生殖,为建立表观遗传修饰与减数分裂之间的联系提供了新的线索。     

Dynamic variation of histone H3 trimethyl Lys4 (H3K4me3) and heterochromatin protein 1 (HP1) with employment length in nickel smelting workers

Objective: To investigate the dynamic variation in H3K4me3 and HP1 with employment length in nickel smelting workers.

Methods: Blood samples were collected from 140 nickel smelting workers and 140 age-matched office workers to test for H3K4me3, and HP1 levels.

Results: H3K4me3 was statistically significantly different (p?<?0.05) between the two groups and positively correlated with employment length (r_s?=_?0.267). HP1 was not correlated with employment length (p?=?0.066) but was significantly different between the two groups.

Conclusions: Chronic exposure to nickel can induce oxidative damage, and increase H3K4me3 expression and inhibit HP1 expression.     

Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K‐M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K‐M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice.