The histone methyltransferase SET8 is required for S-phase progression

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.     

一个新的HMT家族成员SET07的理化性质及其功能

近年来发现组蛋白的甲基化可以改变染色体的状态,进而调节基因的转录、细胞周期、个体发育以及肿瘤发生.对本实验室新近克隆的组蛋白甲基转移酶基因家族新成员SET07理化性质,是否具有甲基转移酶的功能进行初步研究.构建pGEX4T2P2RP2质粒,并对其进行表达和纯化;利用硝纤膜及原核表达产物免疫昆明鼠制备抗SET07多克隆抗体;采用Western印迹对其蛋白水平的表达进行观察;利用HMT(组蛋白甲基转移酶)实验观察表达纯化的SET07蛋白是否具有甲基转移酶的功能.经大肠杆菌表达及纯化获得了较纯的GSTSET07片段,制备获得的多克隆抗体效价高于1∶1000;Western印迹发现SET07蛋白存在于胞核及胞浆.其中在胞核中以49kD存在,在胞浆中SET07以4种形式存在;HMT实验证明,表达纯化的SET07蛋白具有组蛋白甲基转移酶的功能.上述结果提示,SET07可能在合成后经过加工修饰以复合体的形式在胞核内发挥甲基转移酶作用,为进一步研究SET07基因的作用机制、SET07与个体发育、肿瘤发生之间的关系奠定基础.     

Incompatible effects of p53 and HDAC inhibition on p21 expression and cell cycle progression

Nutlin-3 selectively activates p53 by inhibiting the interaction of this tumor suppressor with its negative regulator murine double minute 2 (mdm2), while trichostatin A (TSA) is one of the most potent histone deacetylase (HDAC) inhibitors currently available. As both Nutlin-3 and TSA increase the levels of the cell cycle inhibitor p21(cip1/waf1) in cells, we investigated whether a combination of these compounds would further augment p21 levels. Contrary to expectations, we found that short-term exposure to Nutlin-3 and TSA in combination did not have an additive effect on p21 expression. Instead, we observed that activation of p53 prevented the ability of TSA to increase p21 levels. Furthermore, TSA inhibited Nutlin-3-induced expression of p53-dependent mRNAs including P21. This negative effect of TSA on Nutlin-3 was significantly less pronounced in the case of hdm2, another p53 downstream target. Aside from suggesting a model to explain these incompatible effects of Nutlin-3 and TSA, we discuss the implications of our findings in cancer therapy and cell reprogramming.     

p53-p21通路抑制组蛋白甲基转移酶NSD2的表达

NSD2(nuclear receptor-binding SET domain 2)是一种在黑色素瘤等多种肿瘤细胞中高表达的组蛋白甲基转移酶,其在Wolf-Hirschhorn综合症(wolf-Hirschhorn syndrome,WHS)和多发性骨髓瘤(multiple myeloma,MM)疾病中表达异常的原因已经得到了较好的阐明。而NSD2在其它肿瘤中的表达为何失调还未阐明。本研究选用p53野生型的恶性黑色素瘤细胞系92-1作为细胞模型,采用DNA损伤试剂依托泊苷处理和RNA干扰技术,通过定量PCR和蛋白质免疫印迹的方法首次证实了p53-p21通路对NSD2具有抑制作用。     

Structure of human SMYD2 protein reveals the basis of p53 tumor suppressor methylation

SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel β7 and β8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.     

Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron‐enriched genes

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron‐specific ChIP‐seq and RNA‐seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron‐specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B‐dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.