表观遗传学关键蛋白UHRF1稳定性调节的研究

【作者】 陈浩；

【导师】 施扬； 石雨江；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 表观遗传学是主要是在基因组的核苷酸序列之外,其额外所携带的信息并可以遗传给下一代的一门遗传学分支学科。许多传统遗传所无法解释的现象均属于表观遗传学范畴,主要包括基因组印记(genomic impriting),母体效应(maternal effects)等。其中,DNA甲基化和组蛋白甲基化是表观遗传学的两个主要研究领域,DNA甲基化主要是指发生在CpG的胞嘧啶5位碳原子上添加—个甲基修饰形式,这种修饰是通过DNA甲基转移酶DNMT3A/B以及辅助因子DNMT3L催化生成新的甲基化修饰(de novo methylation),而DNMT1则在DNA复制过程中,以高度保真的形式将母链上的DNA甲基化传递给新生成的子链,维持原有的DNA甲基化状态。进一步的研究表明,DNA甲基化的遗传过程需要另外一重要重要蛋白UHRF1的协助方可完成。UHRF1的SRA结构能够特异性识别并结合半甲基化的DNA链(hemi-methylated DNA),并招募DNMT1至DNA上,催化新生的子链至CpG甲基化,从而确保了DNA甲基化的稳定遗传。UHRF1包含UBL,TUDOR,PHD,SRA,RING等多个重要结构功能域,这些结构域赋予它多种生物学功能。前期研究发现,UHRF1不仅可以维持DNA甲基化稳定遗传,还可能通过DNA甲基化或者组蛋白的修饰定位于常染色体区域,参与基因的转录调控。早期研究暗示UHRF1蛋白与DNA损伤应答以及细胞通过G1/S相关调控密切相关。然而,UHRF1蛋白参与这些过程的具体机制尚不清楚,并且,UHRF1蛋白本身功能如何被调控也不明了。为寻找UHRF1功能调节蛋白,我们首先使用亲和纯化及质谱鉴定,进行分析后发现去泛素化酶USP7与UHRF1稳定结合,并且这种相互作用随着细胞周期的进行呈现动态变化,使UHRF1蛋白稳定性随之相应波动。进一步的分析显示,其具体的分子机制为UHRF1蛋白652位丝氨酸在M期特异磷酸化,阻遏UHRF1和USP7的相互作用。UHRF1在被USP7去泛素化之前,必然首先能够被泛素化修饰。我们综合使用生物信息学及分子生物学手段,对UHRF1蛋白序列进行分析后发现,在UHRF1的N端区域存在有E3连接酶SCFβ-TrCP的识别序歹(DSG motif),该区域对于UHRF1蛋白降解是必不可缺的。SCFβ-TrCP底物的DSG motif首先必须要进行磷酸化之后,方可被SCF复合物识别并随后的泛素化反应,我们经过筛选后发现CK1δ能够在体外及体外催化UHRF1蛋白108位丝氨酸磷酸化,进一步的体外泛素化反应则证实这种磷酸化是UHRF1泛素化的必要条件,并且DNA损伤能够诱导UHRF1该位点磷酸化,导致UHRF1在DNA损伤应答中的加速降解。因此,我们的工作阐明了UHRF1蛋白稳定性正负调控的详细机制,尤为关键的是,磷酸化信号对于UHRF1稳定性的调节的揭示,对于理解细胞内外环境刺激如何通过信号传导通路影响表观遗传学的建立及转换(establishment&switch)具有重要意义。更多还原

【Abstract】 Epigenetics refers to the study of heritable changes transmitted by mechanisms other than changes in the DNA sequence. Many phenomenoa, which couldn’t be explained by traditional geneticsincludinggenomic imprinting and maternal effects, are subjectsof investigation in the fields of epigenetics. The researches towards DNA methylation and histone modifications are two of most important subfields in epigenetics. DNA methylation is a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides (cytosine of CpG dinucleotides in mammal), which is mediated by DNA methyltransferases DNMT3A/B and an accessory factor DNMT3L, while DNMT1mainly plays a role in maintaining DNA methylation during DNA replication.Recent studies demonstrate that UHRF1(Ubiquitin-like, with PHD and RING finger domains1) is critical for the maintenance of DNA methylation patterns in the heritance of DNA methylation profiles. The SRA domain of UHRFl selectively binds hemimethylated CpG and recruits DNMT1to methylate the newly synthesized DNA strand, which ensures the faithful propagation of DNA methylation patterns. The epigenetic regulator UHRFl is composed of multiple functional domains, including the UBL, Tudor, PHD, SRA, and RING domains, which are responsible for the recognition of histone and DNA methylation to regulate gene expression in euchromatic region.Previous studies demonstrate that UHRF1is involved in the DNA damage response and the G1/S transition during cell cycle. However, it remains unclear about the function of UHRF1or the regulation of UHRF1in these processes. To address these questions, we purified UHRF1complex via TAP (Tandem Affinity Purification) and identified that the deubiquitylase USP7(HAUSP) interacts with UHRF1. Their interaction is dynamic during cell cycle, along with oscillation of UHRF1levels. Further experiments indicate that UHRF1Ser652is specifically phosphorylated in M phase of the cell cycle, which disrupts the interaction between UHRFl and USP7and subsequently destablizes UHRF1.As UHRFl is expected to be ubiquitylated before being deubiquitylated by USP7. We analyzed UHRF1protein sequence with bioinformatic and biochemical methods and found that a DSG motif in N-terminus of UHRF1is critical for UHRF1degradation by SCFβ-TxCP. It’s well documented that SCFβ-TrCP complex is targeted to its substrates by recognition of phosphorylated DSG, and we uncovered that CK18phosphorylates UHRF1in vitro and in vivo. Importantly, the phosphorylation is necessary for the ubiquitylation by SCF complex, which is triggered by DNA damage and induces subsequent UHRFl degradation.Taken together, we have identified asophisticated mechanism of positive and negative regulation of UHRF1protein level in vivo. More importantly, the demonstration of phosphorylation regulation of UHRF1stability sheds light on understanding how extracellular and intracellular stimuli influence the establishment and switch of epigenetic patterns through signaling pathways.更多还原