【作者】 徐倩；

【导师】 唐北沙； 宋伟宏；

【作者基本信息】 中南大学 ， 神经病学， 2012， 博士

【摘要】 背景：帕金森病(Parkinson’s disease,PD)是当今仅次于阿尔茨海默病(Alzheimer’s disease,AD)的第二大神经系统退行性疾病。PD的发病机制复杂,尚未完全明确,目前认为可能与老年化、环境因素(比如MPTP.除草剂、杀虫剂、金属等)和遗传因素相关。随着分子遗传学的发展,遗传因素在PD发病机制中的作用越来越受到关注,至少有13个致病基因被克隆,其中α-synucelein、LRRK2、 PRKIN、DJ-1、PINK1以及ATP13A2作为PD的致病基因已经得到了较为一致的肯定。这些基因的功能涉及到了氧化应激、线粒体功能缺陷、泛素蛋白酶体系功能障碍等方面。2001年Hampshire等对一例呈常染色体隐性遗传的合并有痴呆、核上性凝视麻痹的约旦PD家系进行了联锁定位分析,将其致病区间定位于1p36的一个9cM的区域,命名为PARK9。2006年Ramirez等在该家系患者中发现ATP13A2基因的致病突变,且存在家系共分离,从而克隆了PARK9的致病基因。其后,研究者们对不同国家和不同种族来源的PD患者进行了该基因的筛查,不断有新的病例报道。ATP13A2基因(OMIM*610513)定位于人类染色体1p36,包括三个剪切本,其编码的蛋白属于P型ATPase蛋白超家族,该家族主要参与无机离子的跨膜转运。近年来,对于遗传退行性疾病致病基因的转录调控得到越来越多的重视,转录因子可以通过对致病基因的转录调节从而参与疾病的发病机制,比如转录因子GATA能够与SNCA基因启动子区结合,从而使得该基因表达显著上调。目前,转录因子已成为遗传退行性疾病治疗研究的一个新兴的靶点。尽管ATP13A2作为PD的致病基因已经得到了一致的肯定,关于ATP13A2蛋白的转录调控却不明确。为了研究其转录调控的相关分子机制,本研究拟对ATP13A2基因转录起始位点定位并初步分析该基因启动子区活性特点,进而研究其启动子区转录因子结合情况,深入研究感兴趣的转录因子对ATP13A2基因转录的调节。目的：研究ATP13A2基因转录调控的相关分子机制。方法：1.运用RNA连接酶介导的cDNA末端快速扩增法(RNA ligase mediated rapid amplification of cDNA ends, RLM-RACE)确定人类ATP13A2基因的转录起始位点(transcription start site, TSS);2.构建连接有不同长度ATP13A2基因启动子区序列的重组荧光素酶报告基因质粒,采用Dual-luciferase reporter assay system对质粒的启动子活性进行检测；3.运用在线软件结合手工分析,预测ATP13A2基因启动子区潜在的转录因子结合位点；4.采用凝胶电泳迁移率分析(electrophoretic mobility shift assay, EMS A)以及染色质免疫共沉淀分析(chromatin immunopreci-pitation assay, ChIP)对预测的感兴趣的转录因子缺氧诱导因子1α (hypoxia inducible factor1α, HIF-1α)结合位点—缺氧反应元件(hypoxia response element, HRE)进行验证；5.运用HIF-1α蛋白表达质粒pHA-Hif-1α与含ATP13A2启动子的重组荧光素酶报告基因质粒pPK9-A共转染或给予pPK9-A转染的细胞缺氧处理(2%02),观察HIF-1α和缺氧对于ATP13A2基因启动子区活性的影响；6.给予HEK293细胞以及小鼠多巴胺能神经元细胞模型MN9D细胞缺氧处理(2%02),并结合半定量RT-PCR技术,检测两种细胞株内源性ATP13A2mRNA水平,以进一步观察缺氧对ATP13A2转录水平的调控作用。结果：1.对RLM-RACE方法获得的PCR产物测序显示与RNA adapter序列相连的第一位碱基为位于ATP13A2翻译起始位点ATG上游206bp的腺嘌呤A；2.构建了一系列连接有ATP13A2基因启动子区片段的重组pcDNA3质粒,命名为pPK9-A至pPK9-J,其中最长的片段长度为2.0kb(-1954至+84bp,将TSS定位为+1)；对这些质粒的启动子活性分析表明质粒pPK9-A(-1954至+84bp)、pPK9-C(-1538至+84bp)、pPK9-E(-846至+84bp)、pPK9-F(-201至+84bp)、pPK9-G(-78至+84bp)、pPK9-H(-78至+50bp)具有显著的启动子活性；3.通过转录因子结合位点预测软件分析同时结合手工比对,结果显示在人类ATP13A2基因启动子区-1954至+84bp这-区间内包含有9个5’-A/GCGTG-3’序列,并依次命名为HRE1至HRE9；4. EMSA结果显示HRE-1、HRE-3、HRE-4、HRE-8以及HRE-9的寡居核苷酸能够显著的减弱HIF-1α探针与HIF-1α蛋白形成的DNA-蛋白复合物迁移带的浓度；ChIP结果显示HRE-1、HRE-3+4、HRE-8+9的特异性引物能够从HIF-1α特异性抗体免疫共沉淀后分离纯化的DNA样品中成功扩增出目的片段；5.相对于空白质粒共转染的阴性对照,pPK9-A与质粒pHA-Hif-1α共转染显著增加了pPK9-A的启动子活性(2.483±0.241,p<0.05)；相对于正常氧含量培养的阴性对照,pPK9-A转染的HEK293细胞经过缺氧培养(2%02)显著增加了pPK9-A的启动子活性(2.466±0.293,p<0.05)；6.给予HEK293细胞缺氧处理(2%02)Oh、12h、24h及48h后检测细胞内源性ATP13A2的RNA水平,结果在显示缺氧24h及48h情况下,其mRNA水平显著增高(分别为2.356±0.320、2.526±0.203,P值均<0.05)；给予MN9D细胞缺氧处理(2%02)Oh、12h、24h后检测细胞内源性Atp13a2mRNA水平,结果显示缺氧12h及24h情况下,其RNA水平显著增高(分别为1.668±0.148、1.606±0.130,P值均<0.05)。结论：1.人类ATP13A2基因的主要转录起始位点为位于该基因翻译起始位点ATG上游206bp的腺嘌呤A；2.ATP13A2基因-78至+50bp的启动子区域含有ATP13A2基因转录所必须的核心启动子序列；3. EMSA和ChIP分析证实ATP13A2基因启动子区存在功能性的HRE位点,能够与转录因子HIF-1α相结合；4.转录因子HIF-1α能够与ATP13A2基因启动子区的HRE位点相结合,对ATP13A2基因的启动子活性进行调控；5.缺氧可以通过转录因子HIF-1α对ATP13A2基因的转录起到上调作用。更多还原

【Abstract】 Background:Parkinson’s disease (PD) is the second most common neurodeg-enerative disorders with a variable combination of motor and non-motor symptoms. Its characteristic motor symptoms include tremor at rest, rigidity, bradykinesia. Non-motor dysfunctions, such as depression, anxiety, dementia and sexual dysfunction may appear as the disease progresses. The etiology of PD is unclear, however, both genetic and environmental causes contribute to it. Since the first missense mutation in the a-synuclein gene reported in a large PD family of Italian descent in1997, up to date,13genes are reported to be associated with PD. a-synuclein, LRRK2, Parkin, DJ-1, PINK1and ATP13A2have been confirmed to be the pathogenic genes for PD.In2006Alfredo and colleagues identified ATP13A2mutations from a non-consanguineous Chilean family with PD and named it PARK9. Following this report, the mutation screening of ATP13A2was performed on PD patients from different ethnic groups, and several novel mutations in ATP13A2were identified. The human ATP13A2gene (OMIM*610513), locates in chromosome1p36encoding for a protein of1180amino acids with10transmembrane domains which belongs to the P-type ATPase superfamily. However, the protein function of ATP13A2and the pathogenic mechanism of PARK9is still not clear.Recently, abnormal gene expression has been implicated in neurodegenerative disorders. However, the transcriptional regulation of the ATP13A2gene is unknown.Objective:To define the molecular mechanism underlying its transcription and its transcriptional regulation.Methods:1. RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) assay was performed to determine the transcription start site of the human ATP13A2gene.2. To examine the transcriptional regulation of the human ATP13A2, fragments of the5’flanking region of the ATP13A2gene were pulled out by PCR from the human genome DNA and cloned into pG13-basic vector. Luciferase assay was performed to detect the promoter activity of these vectors.3. To further identify the transcription factor binding sites, Computer-based transcription factor binding site analysis was employed and the putative binding sties were confirmed by EMSA and ChIP.4. To determine the effect of hypoxia and HIF-1α on the human ATP13A2promoter activity, HEK293cells were co-transfected by pPK9-A with pHIF-1α or exposed to hypoxic condition after trans-fection with pPK9-A.5. To further investigate the transcrptional regulation of ATP13A2by hypoxia on the mRNA level, HEK293cells and MN9D cells were exposed to hypoxic condition. The endogenous mRNA level of ATP13A2from both cell lines were measured and compared to its own control after hypoxia treatment.Results:1. PCR product from RLM-RACE was sequenced and the sequence result revealed the adenine locating at206bp upstream of the translation start site was the base after RNA adapter;2. A series of luciferase reporter plasmids with different upstream deletions of the ATP13A2gene promoter region were constructed and named according to the original location of insert fragment (TSS was designated as+1); pPK9-A (-1954to+84bp), pPK9-C (-1538to+84bp), pPK9-E (-846to+84bp), pPK9-F (-201to+84bp), pPK9-G(-78to+84bp), pPK9-H(-78to+50bp) present significant promoter activiey;3. Computer-based transcription factor binding site analysis revealed that there are nine putative hypoxia response elements (HRE) in the2.0kb promoter region (-1954to+84) of the human ATP13A2gene;4. EMSA showed that oligos of HRE-1, HRE-3, HRE-4, HRE-8and HRE-9can sharped induced the intensity of the DNA-protein band formed by HIF-1a probe and HIF-1a protein; ChIP assay showed PCR products can be amplified from chromatin samples immune-precipitated by HIF-1a antibody using specific primers of HRE-1, HRE-3+4and HRE-8+9respectively;5. pPK9-A was transfected into HEK293cells with pHIF-1a or empty vector as control, pPK9-A promoter activity was significantly increased compared to control (2.483+0.241, P<0.05); pPK9-A transfected HEK293cells were subjected to hypoxia (2%oxygen) or normoxia (21%oxygen) for24hours and the promoter activity of pPK9-A was significant increased by2.466+0.293(P<0.05) after24hours exposure to low oxygen;6. HEK293cells were incubated in hypoxia chamber with2%oxygen for0,12,24,48hours. Endogenous ATP13A2mRNA levels were measured by qRT-PCR and the results showed the exposure to hypoxic condition for24and48hours resulted in a significant increase of ATP13A2mRNA level,2.356+0.320fold and2.526+0.203fold, respectively; MN9D cells were also inbubated in hypoxia chamber with2%oxygen for0,12, and24hours, and the mRNA level of Atpl3a2after12and24hours hypoxic treatment were increased to1.668±0.148and1.606±0.130, respectively (P<0.05).Conclusions:1. We identified the adenine locating at206bp upstream of the translation start site is the major transcription start site of the ATP-13A2gene;2. We constructed a series of plasmids with different deletions of the5’ flanking region of the ATP13A2gene; the5’flanking region from-78to+50bp contains the minimal promoter sequence necessary for the transcriptional activation of the human ATP13A2gene expression;3. EMS A and ChIP assay confirmed the HRE sites1,3,4,8and9are functional HIF-1a binding sites;4. Overexpression of transcription factor HIF-1a can increase the promoter activity of ATP13A2, and hypoxia can up-regulate the promoter activity of the ATP13A2via HIF-1α;5. Finally, we further confirmed hypoxia can up-regulate the expression of ATP13A2gene at transcription level.更多还原