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题名	人类小头症基因MCPH1的进化与功能研究
作者	石磊
学位类别	博士
答辩日期	2012-05
授予单位	中国科学院研究生院
授予地点	北京
导师	宿兵
关键词	灵长类 MCPH1 基因表达调控的进化 转基因小鼠 转基因猕猴
其他题名	Evolutionary and functional studies of microcephalin gene MCPH1 in primates
学位专业	遗传学
中文摘要	人类区别于其他非人灵长类最显著的特征就是显著增加的大脑容量和高度发达的认知能力。但是一直以来，人们对于人类在进化过程中中枢神经系统的这些显著变化的遗传机制仍然缺乏认识。研究这一问题的一个有效手段就是找出那些由于突变会导致人类大脑发育异常的基因。这方面最典型的一个例子就是对人类原发性小头症发病机制的遗传学解析。MCPH1基因是最早被鉴定出来的小头症基因。通过比较灵长类代表物种的MCPH1序列，人们发现MCPH1基因受到强烈的自然选择的作用，且这种选择作用在现代人群中仍然可以看到。但是，这些基于序列比较的进化分析，特别是针对于编码区的分析不能给我们带来对基因功能进化的明确解释。有鉴于此，在本论文中我们对MCPH1基因进行了很详细的功能研究。 我们不仅在细胞水平分析了MCPH1对下游基因的调控作用以及物种特异突变对这种调控作用的影响，更进一步我们制作了MCPH1转基因小鼠模型，包括人的MCPH1转基因小鼠和猕猴的MCPH1转基因小鼠；同时我们还制作了人的MCPH1转基因猕猴模型。对这些转基因动物模型的研究将为了解MCPH1在灵长类进化和 人类起源中对大脑发育影响的分子机制提供有价值的信息。 我们着重开展了四个方面的研究。主要研究结果如下： 第一，我们的实验证明，人的MCPH1基因属于转录抑制子，它能够抑制端粒酶相关基因hTERT的启动子活性。通过各种突变实验，我们发现MCPH1抑制hTERT的活性主要依赖于MCPH1的BRCT结构域；进一步通过EMSA实验，我们发现MCPH1蛋白能够结合hTERT启动子。对hTERT的启动子序列进行逐段敲除并进行EMSA实验，我们发现了MCPH1结合的元件序列。我们还利用TRAP实验证实MCPH1能够抑制端粒酶的活性以及hTERT基因的表达。 第二，我们克隆了人的MCPH1启动子，通过对MCPH1启动子分析，我们发现E2F1是MCPH1的调控基因，而且E2F1可以上调MCPH1的启动子活性。 通过序列分析我们发现这种上调的机制是由于MCPH1核心启动子含有一段GC串联区域。通过对GC串联区域进行突变，我们发现这一段区域是E2F1的结合区域。进一步通过EMSA实验，我们发现E2F1蛋白能够结合这段区域，证实了了我们之前的结论。另外，通过比较脊椎动物中MCPH1上游2Kb序列，我们发现这段GC序列是灵长类特有的,这提示E2F1对MCPH1的调控是最近才起源的。我们由此提出了MCPH1在灵长类的进化中获得了E2F1的结合序列，这与MCPH1蛋白序列的快速进化是平行发生的。 第三，我们制作了人的MCPH1转基因小鼠和猕猴的MCPH1转基因小鼠。这些转了人和猕猴MCPH1基因拷贝的小鼠生长和发育基本正常，但在脑的重量和睾丸重量方面与野生型小鼠有显著差异。我们的初步分析结果表明，MCPH1的过表达能够影响小鼠的脑发育。进一步的表型分析表明，人的MCPH1转基因小鼠和猕猴的MCPH1转基因小鼠在脑重方面没有显著差异，但它们对下游基因的表达调控上有差异。 第四，我们制作了人的MCPH1转基因猕猴模型，这也是首例从进化研究角度建立的非人灵长类的转基因模型。我们前期的试验结果表明，我们成功制作了人的MCPH1转基因猕猴模型。初步的核磁共振（MRI）脑影像学分析发现，转基因猕猴的脑容量比野生型的更大，它们在大脑白质部分和灰质部分的体积上也有差异。 综上所述，我们的研究结果表明，MCPH1基因通过和E2F1相互作用调控一系列参与细胞周期控制、细胞增殖分化和细胞凋亡的基因，从而参与灵长类大脑发育的调控。转基因动物的初步研究显示，过表达MCPH1会影响大脑和睾丸的发育，人和非人灵长类的MCPH1在功能可能已经产生分化。
英文摘要	Human evolution is characterized by a dramatic increase in brain size, but the underling molecular mechanism that caused this expansion is unknown. It had been difficult to address this question using genetic tools because the dramatically enlarged brain is one of the human-specific traits. Fortunately, the dissections of a rare brain developmental disorder, i.e. the human primary microcephaly (MCPH) syndrome have discovered a set of genes regulating brain development. MCPH1 is the first identified microcephalin gene. Evolutionary studies of MCPH1 have suggested a rapid evolution of the protein coding sequences in primates, which is associated with the brain enlargement during primate evolution and human origin, especially in the lineage leading to humans. It has been reportedthat MCPH1 is highly polymorphic in human populations with a signature of on-going positive selection. However, sequence comprison analysis of gene coding regions could not give us functional hints. Hence, in this study, we aim to conduct a systematic dissection of the function of MCPH1, including not only the in vitro assays of revealing the role of MCPH1 for down-stream gene regulation, but also the construction of transgenic animal models (transgenic mouse and rhesus macaque models). The have performed the following four studies of MCPH1 and the major findings are: Firstly, we identified MCPH1 as a transcriptional repressor. We examined the role of MCPH1 in regulating the hTERT promoter in vitro. Co-transfection of the hTERT promoter with MCPH1 in Hela cells could inhibit the hTERT promoter activity. The EMSA assay demonstrated that MCPH1 could bind to the proximal hTERT promoter. Over expression of MCPH1 could repress telomerase activity, and the repression was abolished by knocking down the MCPH1 expression using siRNA in U2OS cells. We propose that MCPH1 functions as a transcriptional repressor of hTERT in vitro. Secondly, we cloned the human MCPH1 promoter and we identified a novel E2F1 binding motif located in the proximal promoter region of MCPH1. The experiments using electrophoretic mobility shift and promoter assays (EMSA) showed that E2F1 could stimulate MCPH1 transcription by direct binding to the E2F1 motif. Over-expression of E2F1 led to the up-regulation of MCPH1 transcription, and knocking down the endogenous E2F1 resulted in the inhibition of the MCPH1 promoter activity. Surprisingly, sequence comparison of vertebrate species suggested that the identified E2F1 binding motif is primate specific, consistent with the previous observation of rapid evolution of MCPH1 protein sequence in primates. We propose that during primate evolution, MCPH1 has acquired a novel E2F1 binding motif in its promoter which may act as a parallel mechanism acting together with the rapid protein sequence changes in primates, and eventually contributed to brain enlargement during primate evolution and human origin. Thirdly, we have constructed the human MCPH1 transgenic mouse and the macaque MCPH1 transgenic mouse. These mice are generally healthy, and they are different from wild type mice on brain weight and testis weight. Phenotype analysis indicates that the human MCPH1 transgenic mouse is not different from the macaque MCPH1 transgenic mouse in view of brain weight. The primary differences are reflected by the regulatory effect of MCPH1 on the expression of its downstream target genes. Finally, We have constructed the human MCPH1 transgenic monkey to test wether human MCPH1 could affect macaque’s brain size. Our preliminary functional experiments indicate that we successfully constructed the human MCPH1 transgenic macaque. The MRI data suggests that the total brain volume of the transgenic macaque is larger than that of the wild-type macaque, so are the white matter volume and the grey matter volume. In summary, we have shown that through interaction with E2F1, MCPH1 can regulate a series of genes involved in cell cycle control, cell proliferation and differentiation, and apoptosis, and eventually contribute to brain development in primates. The preliminary data from our transgenic animal studies suggest that over expression of MCPH1 can influence the development of brain and testis, and there might be functional divergence of MCPH1 between humans and nonhuman primates.
语种	中文
公开日期	2012-06-07
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6968]
专题	昆明动物研究所_比较基因组学
推荐引用方式 GB/T 7714	石磊. 人类小头症基因MCPH1的进化与功能研究[D]. 北京. 中国科学院研究生院. 2012.

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