On your histone mark,SET, methylate!

Lysine methylation of histones and non-histone proteins has emerged in recent years as a posttranslational modification with wide-ranging cellular implications beyond epigenetic regulation. The molecular interactions between lysine methyltransferases and their substrates appear to be regulated by posttranslational modifications surrounding the lysine methyl acceptor. Two very interesting examples of this cross-talk between methyl-lysine sites are found in the SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain-containing lysine methyltransferases SET7 and SETDB1, whereby the histone H3 trimethylated on lysine 4 (H3K4^me3) modification prevents methylation by SETDB1 on H3 lysine 9 (H3K9) and the histone H3 trimethylated on lysine 9 (H3K9^me3) modification prevents methylation by SET7 on H3K4. A similar cross-talk between posttranslational modifications regulates the functions of non-histone proteins such as the tumor suppressor p53 and the DNA methyltransferase DNMT1. Herein, in cis effects of acetylation, phosphorylation, as well as arginine and lysine methylation on lysine methylation events will be discussed.     

组蛋白甲基转移酶G9a在表观遗传调控中的作用

组蛋白赖氨酸甲基化在表观遗传调控中起着关键作用。组蛋白甲基转移酶G9a(又称作常染色质组蛋白赖氨酸N-甲基转移酶2(euchromatic histone-lysine N-methyltransferase 2,EHMT2))含经典的SET结构域,是常染色质主要的甲基转移酶之一,可以甲基化组蛋白H3K9、H3K27和H1bK26等。此外,G9a也可以直接甲基化一些非组蛋白,并与DNA甲基化密切相关。G9a功能紊乱可以导致胚胎发育异常、免疫系统及神经系统发育障碍、甚至癌症的发生发展。     

组蛋白赖氨酸甲基化在表观遗传调控中的作用

组蛋白赖氨酸的甲基化在表观遗传调控中起着关键作用。组蛋白H3的K4、K9、K27、K36、K79和H4的K20均可被甲基化。组蛋白H3第9位赖氨酸的甲基化与基因的失活相关连; 组蛋白H3第4位赖氨酸和第36位赖氨酸的甲基化与基因的激活相关连; 组蛋白H3第27位赖氨酸的甲基化与同源盒基因沉默、X染色体失活、基因印记等基因沉默现象有关; 组蛋白H3第79位赖氨酸的甲基化与防止基因失活和DNA修复有关。与此同时, 组蛋白的去甲基化也受到更为广泛的关注。 关键词: 组蛋白赖氨酸甲基转移酶; 组蛋白赖氨酸甲基化; 组蛋白去甲基化     

Epigenetic regulation: methylation of histone and non-histone proteins

Histone methylation is believed to play important roles in epigenetic memory in various biological processes. However, questions like whether the methylation marks themselves are faithfully transmitted into daughter cells and through what mechanisms are currently under active investigation. Previously, methylation was considered to be irreversible, but the recent discovery of histone lysine demethylases revealed a dynamic nature of histone methylation regulation on four of the main sites of methylation on histone H3 and H4 tails (H3K4, H3K9, H3K27 and H3K36). Even so, it is still unclear whether demethylases specific for the remaining two sites, H3K79 and H4K20, exist. Furthermore, besides histone proteins, the lysine methylation and demethylation also occur on non-histone proteins, which are probably subjected to similar regulation as histones. This review discusses recent progresses in protein lysine methylation regulation focusing on the above topics, while referring readers to a number of recent reviews for the biochemistry and biology of these enzymes     

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.     

SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.     

植物组蛋白赖氨酸甲基化建立过程及其遗传性研究进展

表观遗传学主要包括DNA甲基化、组蛋白修饰和非编码RNA,组蛋白甲基化作为组蛋白修饰中的一种重要修饰,在植物体的发育和环境适应中发挥着重要作用。组蛋白甲基化主要发生在赖氨酸残基上,同时根据不同的赖氨酸位点和每个赖氨酸位点甲基化程度的不同,形成了不同的赖氨酸甲基化修饰。根据对基因的不同功能,通常将组蛋白赖氨酸甲基化修饰分为2大类:(1)能够促进基因表达的,如H3K4me3和H3K36me3;(2)能够抑制基因表达的,如H3K9me2和H3K27me3。不同的组蛋白赖氨酸甲基化去甲基化过程需要相应的阅读(reader)、书写(writer)和擦除(eraser)3种蛋白。同时,组蛋白赖氨酸甲基化的遗传性质目前还不是很清楚。综述了植物中组蛋白赖氨酸甲基化建立与去除过程,以及对组蛋白赖氨酸甲基化可遗传性的探讨。     

The histone methyltransferase SDG8 regulates shoot branching in Arabidopsis

Histone lysine methylation is an evolutionally conserved modification involved in determining chromatin states associated with gene activation or repression. Here we report that the Arabidopsis SET domain group 8 (SDG8) protein is a histone H3 methyltransferase involved in regulating shoot branching. Knockout mutations of the SDG8 gene markedly reduce the global levels of histone H3 trimethylation at lysines 9 and 36 as well as dimethylation at lysine 36. The sdg8 mutants produce more shoot branches than wild-type plants. The expression of SPS/BUS (supershoot/bushy), a repressor of shoot branching, is decreased in sdg8 mutants, while UGT74E2 (UDP-glycosyltransferase 74E2), a gene associated with increased shoot branching, is up-regulated in sdg8 mutants. The altered expression of SPS/BUS and UGT74E2 correlates with changed histone H3 methylation at these loci. These results suggest that SDG8 regulates shoot branching via controlling the methylation states of its target genes.     

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.     

PLMT家族成员SET7/9的非组蛋白甲基化作用

SET7/9是蛋白赖氨酸甲基化转移酶（protein lysine methyltransferases,PLMTs或PKMTs）家族成员,具有SET结构域。现已发现SET7/9是一种赖氨酸单甲基化转移酶,除了能使组蛋白H3第四位赖氨酸（lysine4 of histone 3,H3K4）单甲基化外,更重要的能使一些转录因子、肿瘤抑制因子、膜相关受体等非组蛋白单甲基化,其甲基化作用主要与蛋白稳定和转录活化有关。该效应受赖氨酸特异性去甲基酶1（lysine specifcdemethylase,LSD1）的抑制。SET7/9与LSD1两者效应的平衡对维持体内活性蛋白质含量、调节基因表达具有重要意义。     

Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

The Legionella pneumophila Methyltransferase RomA Methylates Also Non-histone Proteins during Infection

RomA is a SET-domain containing protein lysine methyltransferase encoded by the Gram-negative bacterium Legionella pneumophila. It is exported into human host cells during infection and has been previously shown to methylate histone H3 at lysine 14 [Rolando et al. (2013), Cell Host Microbe, 13, 395–405]. Here, we investigated the substrate specificity of RomA on peptide arrays showing that it mainly recognizes a G-K-X-(PA) sequence embedded in a basic amino acid sequence context. Based on the specificity profile, we searched for possible additional RomA substrates in the human proteome and identified 34 novel peptide substrates. For nine of these, the corresponding full-length protein or protein domains could be cloned and purified. Using radioactive and antibody-based methylation assays, we showed that seven of them are methylated by RomA, four of them strongly, one moderately, and two weakly. Mutagenesis confirmed for the seven methylated proteins that methylation occurs at target lysine residues fitting to the specificity profile. Methylation of one novel substrate (AROS) was investigated in HEK293 cells overexpressing RomA and during infection with L. pneumophila. Methylation could be detected in both conditions, confirming that RomA methylates non-histone proteins in human cells. Our data show that the bacterial methyltransferase RomA methylates also human non-histone proteins suggesting a multifaceted role in the infection process.     

Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.     

Histone lysine methyltransferases and demethylases in Plasmodium falciparum

Dynamic histone lysine methylation, regulated by methyltransferases and demethylases, plays fundamental roles in chromatin structure and gene expression in a wide range of eukaryotic organisms. A large number of SET-domain-containing proteins make up the histone lysine methyltransferase (HKMT) family, which catalyses the methylation of different lysine residues with relatively high substrate specificities. Another large family of Jumonji C (JmjC)-domain-containing histone lysine demethylases (JHDMs) reverses histone lysine methylation with both lysine site and methyl-state specificities. Through bioinformatic analysis, at least nine SET-domain-containing genes were found in the malaria parasite Plasmodium falciparum and its sibling species. Phylogenetic analysis separated these putative HKMTs into five subfamilies with different putative substrate specificities. Consistent with the phylogenetic subdivision, methyl marks were found on K4, K9 and K36 of histone H3 and K20 of histone H4 by site-specific methyl-lysine antibodies. In addition, most SET-domain genes and histone methyl-lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. Furthermore, the malaria parasite and other apicomplexan genomes also encode JmjC-domain-containing proteins that may serve as histone lysine demethylases. Whereas prokaryotic expression of putative active domains of four P. falciparum SET proteins did not yield detectable HKMT activity towards recombinant P. falciparum histones, two protein domains expressed in vitro in a eukaryotic system showed HKMT activities towards H3 and H4, respectively. With the discovery of these Plasmodium SET- and JmjC-domain genes in the malaria parasite genomes, future efforts will be directed towards elucidation of their substrate specificities and functions in various cellular processes of the parasites.     

A Non-Active-Site SET Domain Surface Crucial for the Interaction of MLL1 and the RbBP5/Ash2L Heterodimer within MLL Family Core Complexes

The mixed lineage leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1–MLL4 and SETd1a,b. Dimethylation of H3K4 requires a sub-complex including WRAD (WDR5, RbBP5, Ash2L, and DPY-30), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active-site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5/Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or KIS, are required for formation of a second active site within SET1 family core complexes.