Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS

COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.     

Molecular regulation of H3K4 trimethylation by Wdr82, a component of human Set1/COMPASS

In yeast, the macromolecular complex Set1/COMPASS is capable of methylating H3K4, a posttranslational modification associated with actively transcribed genes. There is only one Set1 in yeast; yet in mammalian cells there are multiple H3K4 methylases, including Set1A/B, forming human COMPASS complexes, and MLL1-4, forming human COMPASS-like complexes. We have shown that Wdr82, which associates with chromatin in a histone H2B ubiquitination-dependent manner, is a specific component of Set1 complexes but not that of MLL1-4 complexes. RNA interference-mediated knockdown of Wdr82 results in a reduction in the H3K4 trimethylation levels, although these cells still possess active MLL complexes. Comprehensive in vitro enzymatic studies with Set1 and MLL complexes demonstrated that the Set1 complex is a more robust H3K4 trimethylase in vitro than the MLL complexes. Given our in vivo and in vitro observations, it appears that the human Set1 complex plays a more widespread role in H3K4 trimethylation than do the MLL complexes in mammalian cells.     

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.     

Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes

MLL complexes are homologs of yeast COMPASS capable of methylating histone H3 Lys4 (H3K4). ASH2L, RbBP5 and WDR5 are conserved subunits of MLL complexes with homology to the Cps40/Cps60, Cps50 and Cps30 subunits of COMPASS, respectively. We report that ASH2L differentially regulates MLL's catalysis of H3K4 trimethylation similarly to Cps40 and Cps60. Furthermore, WDR5 is required to maintain MLL complex integrity, including the stability of ASH2L within the complex. These findings offer insight into the molecular role of ASH2L, and by extension that of WDR5, in proper H3K4 trimethylation.     

The COMPASS family of H3K4 methylases in Drosophila

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.     

Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.     

OsASHL1 and OsASHL2, two members of the COMPASS-like complex,control floral transition and plant development in rice

COMPASS or COMPASS-like is a highly conserved polyprotein complex in eukaryotes that is often involved in methylation of histone H3 lysine 4 (H3K4). However, the biological function of this complex in rice (Oryza sativa) is unclear. Here, we report the identifiction of their functions in growth and development. The osashl1 osashl2 double mutant shows a dwarf and late-flowering phenotype. Lower expression of Ehd1, OsVIL4, and OsMADS51 in the osashl1 osashl2 double mutant background accompanies a delayed vegetative growth phase and photoperiod-sensitive phase compared with that in wild type. Notably, there is less H3K4 mono-, di- and tri-methylation genome-wide in the double mutant, in particular less H3K4 tri-methylation at OsVIL4. Consistent with this result, knockout of OsVIL4 gives rise to a late-flowering phenotype similar to that of the osashl1 osashl2 double mutant, suggesting that OsVIL4 is a target of the COMPASS-like complex. In addition, the expression of key genes in brassinosteroid and gibberellic acid metabolism is altered in the osashl1 osashl2 double mutant, suggesting that the COMPASS-like complex regulates plant growth and development by modulating the levels of these two phytohormones. In summary, we demonstrate that OsASHL1 and OsASHL2 are important for floral transition and plant development.     

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co‐factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre‐implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1‐ to the 4‐cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1‐cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4‐cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4‐cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst. Mol. Reprod. Dev. 77: 540–549, 2010. © 2010 Wiley‐Liss, Inc.     

Ash2l-1调控小鼠卵黄囊早期造血

作为甲基转移酶MLL/SET1复合体的核心成分之一,ASH2L能够促进组蛋白H3K4me3修饰的形成,并在小鼠早期胚胎发育过程中行使重要功能.在小鼠中,由于启动子的选择性使用,Ash2l会转录成两种不同长度的转录本并形成两种蛋白质亚型:ASH2L-1和ASH2L-2.目前有关该基因在小鼠胚胎发育中的作用机制及不同亚型的功能还不清楚.本文利用CRISPR/Cas9技术特异敲除Ash2l-1并研究该亚型的生理学功能.研究结果发现,当Ash2l-1缺失时,小鼠胚胎在E9.5~E10.5时发生致死.特别是Ash2l-1-/-E9.5胚胎的卵黄囊血管和早期造血发育存在明显缺陷.转录组测序结果显示,Ash2l-1的缺失影响红细胞发育和成熟、血管发生和形成相关基因的表达.H3K4me3的CUT&RUN结果显示,在一些表达下调关键基因的启动子区,H3K4me3修饰水平出现下降.以上结果表明,Ash2l-1在小鼠卵黄囊的早期造血和血管形成过程中是必不可少的,它可能是通过调控关键基因启动子区的H3K4me3修饰水平而控制这些基因的表达,从而在相关过程中行使功能.     

Automethylation Activities within the Mixed Lineage Leukemia-1 (MLL1) Core Complex Reveal Evidence Supporting a “Two-active Site” Model for Multiple Histone H3 Lysine 4 Methylation

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex.     

The Arginine Methyltransferase PRMT6 Cooperates with Polycomb Proteins in Regulating HOXA Gene Expression

Protein arginine methyltransferase 6 (PRMT6) catalyses asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a), which has been shown to impede the deposition of histone H3 lysine 4 trimethylation (H3K4me3) by blocking the binding and activity of the MLL1 complex. Importantly, the genomic occurrence of H3R2me2a has been found to coincide with histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone mark generated by the Polycomb repressive complex 2 (PRC2). Therefore, we investigate here a putative crosstalk between PRMT6- and PRC-mediated repression in a cellular model of neuronal differentiation. We show that PRMT6 and subunits of PRC2 as well as PRC1 are bound to the same regulatory regions of rostral HOXA genes and that they control the differentiation-associated activation of these genes. Furthermore, we find that PRMT6 interacts with subunits of PRC1 and PRC2 and that depletion of PRMT6 results in diminished PRC1/PRC2 and H3K27me3 occupancy and in increased H3K4me3 levels at these target genes. Taken together, our data uncover a novel, additional mechanism of how PRMT6 contributes to gene repression by cooperating with Polycomb proteins.