Steric structure–activity relationship of cyproheptadine derivatives as inhibitors of histone methyltransferase Set7/9

Set7/9 is a histone lysine methyltransferase, but it is also thought to be involved in a wide variety of pathophysiological functions. We previously identified cyproheptadine, which has a characteristic butterfly-like molecular conformation with bent tricyclic dibenzosuberene and chair-form N-methylpiperidine moieties, as a Set7/9 inhibitor. In this work, we synthesized several derivatives in order to examine the steric structure–inhibitory activity relationship. We found that even a small change of molecular shape due to reduction or replacement of the 10,11-olefinic bond of the tricyclic ring generally resulted in a drastic decrease of the inhibitory activity. Our results should be useful not only for development of more potent and selective inhibitors, but also for the construction of novel inhibitor scaffolds.     

Development of novel bisubstrate-type inhibitors of histone methyltransferase SET7/9

Histone modification, for example, by histone deacetylase (HDAC) and histone lysine methyltransferase (HMT), plays an important role in regulating gene expression. To obtain novel inhibitors as tools for investigating the physiological function of members of the HMT family, we designed and synthesized novel inhibitors, which are amine analogues of adenosylmethionine (AdoMet; the cofactor utilized in the methylation reaction) bearing various alkylamino groups coupled via an ethylene linker. The inhibitory activities of these compounds towards SET7/9, an HMT, were evaluated. It was found that introduction of an alkylamino group increased the inhibitory activity.     

Crystal structure and functional analysis of the histone methyltransferase SET7/9

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.     

Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly

Epigenetic gene silencing in eukaryotes is regulated in part by lysine methylation of the core histone proteins. While histone lysine methylation is known to control gene expression through the recruitment of modification-specific effector proteins, it remains unknown whether nonhistone chromatin proteins are targets for similar modification-recognition systems. Here we show that the histone H3 methyltransferase G9a contains a conserved methylation motif with marked sequence similarity to H3 itself. As with methylation of H3 lysine 9, autocatalytic G9a methylation is necessary and sufficient to mediate in vivo interaction with the epigenetic regulator heterochromatin protein 1 (HP1), and this methyl-dependent interaction can be reversed by adjacent G9a phosphorylation. NMR analysis indicates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3K9, demonstrating that the chromodomain functions as a generalized methyl-lysine binding module. These data reveal histone-like modification cassettes - or "histone mimics" - as a distinct class of nonhistone methylation targets and directly extend the principles of the histone code to the regulation of nonhistone proteins.     

Linc-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b

Long noncoding RNAs (lncRNAs) are involved in the regulation of skeletal muscle development. In the present study, differentially expressed lncRNAs were identified from RNA-seq data derived from myoblasts and myotubes. We conducted studies to elucidate the function and molecular mechanism of action of Linc-smad7 during skeletal muscle development. Our findings show that Linc-smad7 is upregulated during the early phase of myoblasts differentiation. In in vitro studies, we showed that overexpression of Linc-smad7 promoted the arrest of myoblasts in G1 phase, inhibited DNA replication, and induced myoblast differentiation. Our in vivo studies suggest that Linc-smad7 stimulates skeletal muscle regeneration in cardiotoxin-induced muscle injury. Mechanistically, Linc-smad7 overexpression increased smad7 and IGF2 protein levels. On the contrary, overexpression of miR-125b reduced smad7 and IGF2 protein levels. Results of RNA immunoprecipitation analysis and biotin-labeled miR-125b capture suggest that Linc-smad7 could act as a competing endogenous RNA (ceRNA) for miRNA-125b. Taken together, our findings suggest that the novel noncoding regulator Linc-smad7 regulates skeletal muscle development.     

The methyltransferase Set7/9 (Setd7) is dispensable for the p53-mediated DNA damage response in vivo

p53 is the central regulator of cell fate following genotoxic stress and oncogene activation. Its activity is controlled by several posttranslational modifications. Originally defined as a critical layer of p53 regulation in human cell lines, p53 lysine methylation by Set7/9 (also called Setd7) was proposed to fulfill a similar function in?vivo in the mouse, promoting p53 acetylation, stabilization, and activation upon DNA damage (Kurash et?al., 2008). We tested the physiological relevance of this circuit in an independent Set7/9 knockout mouse strain. Deletion of Set7/9 had no effect on p53-dependent cell-cycle arrest or apoptosis following sublethal or lethal DNA damage induced by radiation or genotoxic agents. Set7/9 was also dispensable for p53 acetylation following irradiation. c-myc oncogene-induced apoptosis was also independent of Set7/9, and analysis of p53 target genes showed that Set7/9 is not required for the p53-dependent gene expression program. Our data indicate that Set7/9 is dispensable for p53 function in the mouse.     

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。