SIRT7基因启动子与其蛋白稳定性的调控机制研究

【作者】 张浪玺；

【导师】 张业； 沈珝琲；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 在酵母中,染色体沉默因子Sir2(silent information regulator-2)是NAD+依赖的组蛋白去乙酰化酶,它参与了基因组稳定和衰老等多种生理过程[1-2]。Sir2在哺乳动物中有七个家族成员(SIRT1-SIRT7)[3]。近年来,大量研究表明Sirtuin家族成员广泛参与凋亡,应激,分化,衰老,基因表达等多种生理过程[4]。SIRT7是这个家族中唯一定位于细胞核仁的蛋白[5]。也是到目前为止,唯一没有被鉴定出酶活性的家族成员。SIRT7是Poll聚合酶的活化子,能够促进rDNA转录。研究表明在乳腺癌和甲状腺癌中SIRT7mRNA高表达,提示SIRT7可能参与癌症的发生过程。因此研究SIRT7基因的表达调控机制对于我们了解SIRT7在肿瘤发生,应激等生理过程中的作用具有重要意义。本文主要研究了SIRT7的启动子和SIRT7蛋白稳定性调控机制,以及SIRT7在细胞中的亚细胞定位,并尝试鉴定了SIRT7的组蛋白与非组蛋白的去乙酰化酶活性。1SIRT7mRNA的表达谱我们通过Northern blotting和RT-PCR在mRNA水平检测了SIRT7在小鼠主要组织和几种人源细胞系中的表达,结果显示SIRT7在小鼠组织中广泛表达,其中在肝脏组织中表达最为丰富。在人胚肾细胞HEK293T, MCF7, Huh7, HepG2, HeLa细胞中SIRT7均有表达,在肺癌细胞H1299中几乎不表达。25’-RACE确定SIRT7转录起始位点提取小鼠肝脏和人急性白血病细胞HL60RNA,利用SIRT7特异引物以及5’-RACE连接子内外引物进行巢式PCR,得到长度为650bp的扩增产物,结果显示SIRT7基因在人和鼠中存在单一的转录起始位点。转录起始位点为C碱基,转录起始位点上游没有典型的TATA box和GC box,也没有明显的CpG岛。3克隆并鉴定SIRT7启动子区我们首次克隆并鉴定了SIRT7启动子区。实验结果表明SIRT7的转录起始位点到上游-2Kb的DNA片段具有启动子活性,并且这种启动子活性具有细胞特异性。通过启动子删切实验表明,-312/-174的DNA片段为SIRT7启动子的关键区域。通过生物信息学的方法确定该区域含有保守的C/EBPα转录因子结合位点。4Doxorubicin能够影响SIRT7蛋白稳定性, SIRT7蛋白稳定性受泛素蛋白酶体途径调控.我们发现在DNA损伤过程中,MCF7细胞内SIRT7蛋白水平快速下降。而SIRT7mRNA水平却不下降。表明SIRT7蛋白调控过程发生于转录后水平。外源表达的SIRT7蛋白和内源的SIRT7蛋白具有较短的半衰期,其稳定性受泛素蛋白酶体的调控。体内泛素化实验表明,SIRT7在细胞内能够发生多聚泛素化。5SIRT7在细胞内的亚细胞定位.免疫荧光显示在HEK293T细胞和MCF7细胞和Huh7细胞中,外源表达的Flag-SIRT7并不定位于核仁,而是分布在细胞核内,并且在核膜内侧富集。在细胞分裂期,Flag-SIRT7不与染色体结合。而全长的人源EGFP-SIRT7却主要定位在核仁,在有丝分裂期,EGFP-SIRT7与染色体紧密结合。结果表明外源表达的SIRT7蛋白在细胞内的定位依赖于表达量和融合标签类型。鉴于商业化SIRT7抗体特异性差,我们首次生产了识别小鼠SIRT7蛋白362-379位氨基酸的多克隆抗体,并证明了SIRT7多克隆抗体的特异性。使用SIRT7多克隆抗体,通过免疫荧光法,发现内源SIRT7在HEK293T, Huh7, MCF7细胞内以点状分布于细胞核,并且分布区域DAPI染色较浅。6尝试鉴定SIRT7的去乙酰化酶的活性我们纯化原核表达的GST融合的SIRT7蛋白,在体外建立组蛋白去乙酰化反应体系。结果表明原核表达的SIRT7没有组蛋白H4K16的去乙酰化的活性。用免疫荧光法发现在MCF7细胞内,外源表达的SIRT7没有H3K9Ac, H3K14Ac, H4K5AC, H4K8AC, H4K12Ac, H4K16Ac和P53的去乙酰化酶活性。更多还原

【Abstract】 The chromatin silencing factor Sir2(silent information regulator-2) catalyses NAD+-dependent histone deacetylation to regulate genomic stability and cellular senescence in budding yeast.Sirtuins in mammals constitute a family of seven genes (SIRT1-SIRT7) recently been proposed to be involved in the control of critical metabolic pathways as well as apoptosis, stress responses, DNA repair, cell cycle, genomic stability and gene expression.SIRT7is the only Sirtuin located in nucleolus, and is the only Sirtuin protein for which a clear enzymatic activity has remained elusive. Previous studies suggest sirt7is an activator of RNA polymerse1transcription. It over expression enhances rDNA transcription. An elevated Sirt7expression has been detected in several human cancers such as breast cancer. The study of SIRT7regulation is valuable for us to understand the exact roles of SIRT7in tumorgensis and metabolic pathways.Here we try to definite the SIRT7gene promoter and investigate the role of ubiquitin-mediated proteasomal degradation in regulation of SIRT7levels. We also examine the sirt7for its cellular localization; protein deacetylase activity.These works will develop our understanding of the role of SIRT7.1SIRT7mRNA ubiquitously expressed.The mRNA level of SIRT7gene was detected by Northern blotting or RT-PCR. Data shows SIRT7mRNA was expressed in all mouse tissues examined, and was most abundant in liver. Human SIRT7mRNA was also expressed in several cell lines such as HEK293T, MCF7, Huh7, HepG2, and HeLa except H1299cells.2The transcription site of SIRT7was determined by5’-RACE assay.Using SIRT7primer and5’RACE outer primer, we amplified a650bp fragment by nest PCR from total mouse live RNA and HL60RNA The data showed that there is only one transcription initiation sites on sirt7gene, and the site is47bp apart from sirt7translation start site ATG. Characterization of the5’-flanking genomic region, which precedes the sirt7transcription initiation site, revealed a TATA-and GC-box less promoter that lacks CpG islands.3Functional identification of the SIRT7gene promoter.A2.1Kb5’-flangking region of human Sirt7was cloned and characterized. Promoter deletion assays suggested the constructed (-312/+30) contained a conserved C/EBPα binding site can efficiently drove luciferase activity.4Anticancer agent doxorubicin can induce the SIRT7degradation and the degradation is mediated by26s proteasome. SIRT7can polyubiquitination of SIRT7in vivo.The SIRT7protein levels but not mRNA levels are decreased by doxorubicin treatment. And the SIRT7is a short-lived protein and the effect on the SIRT7protein degradation is MG132dose and time dependent. SIRT7might be regulated by ubiquitin-mediated proteasomal degradation.5Cellular localization of Sirt7protein.Exogenous expressed Flag-tagged full length human SIRT7locate in nucleus but not in nucleoli in HEK293T and MCF7by immnofluorescence analysis. And it mainly concentrates near nuclear envelope. During M phase, when nucleoli disintegrate, SIRT7did not bind to the condensed mitotic chromatin. In contrast, GFP-SIRT7is mainly located in nucleoli. During M phase, EGFP-SIRT7remained bound to the condensed mitotic chromatin. Antibodies were raised against the amino acids363-379of mSIRT7. And we demonstrate the antibody can specifically recognize the mSIRT7protein. Using anti-SIRT7C-terminal antibodies for immunofluorescence co focal Microscopy, we found SIRT7proteins mainly locate in nucleus of HEK293T, Huh7, MCF7cells. 6The deaceylation activity of SIRT7We Purify GST-fusion SIRT7protein and total histone and set up the vitro deacetylation assay. Data shows SIRT7does not deacetylase H4K16in vitro. SIRT7don’t show deacetylase activity on histone H3K9Ac, H3K14Ac, H4K5AC,H4K8AC, H4K12Ac, H4K16Ac in MCF7cells. SIRT7does not deacetylate p53in MCF7cells.更多还原