【作者】 严忠海；

【导师】 曾溢滔； 黄淑帧； 任兆瑞； 张敬之；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 珠蛋白基因簇在个体发育中表现为依次表达,并具有高度的组织特异性和发育阶段特异性。珠蛋白基因的转录受染色质结构、编码基因与其旁侧序列甲基化程度、顺式调节元件及细胞内反式作用因子的调节控制。Locus control regiongs (LCRs,基因座控制区)在功能上被定义为一类发育中的哺乳动物红细胞α珠蛋白和β珠蛋白基因表达所必需的上游调控序列。LCR可提高与其相衔接的基因的表达并具有组织特异型和拷贝数依赖的功能元件,是最重要调节珠蛋白基因表达的一类顺式作用元件。有关珠蛋白基因组织特异性表达的各种研究和理论模型中,都暗示了珠蛋白表观遗传修饰在调控珠蛋白基因的特异性表达中的重要功能。由于缺乏合适的实验模型,以往有关组蛋白修饰与珠蛋白基因表达关心的研究大多在细胞系中进行,因而无法重现珠蛋白基因的组织特异性表达。本论文利用先前构建的由LCR的HS3-2元件和β-珠蛋白基因启动子驱动表达EGFP报告基因的小鼠模型,对DNA的差异性甲基化修饰和内源性的miRNA在珠蛋白基因的特异性表达中的作用进行了研究。首先我们运用定量RT-PCR的技术检测了报告基因在不同组织中的表达水平;在该模型小鼠呈现良好的组织特异性表达的基础上,应用DNA亚硫酸盐修饰的方法,测定了人LCR和β-珠蛋白启动子区域在转基因小鼠各组织中的甲基化水平差异,以揭示DNA的甲基化修饰在组织特异性表达中的作用。这部分工作获得的主要进展是:1.以人珠蛋白基因的LCR和启动子驱动表达EGFP的转基因小鼠在红系组织中高效表达人珠蛋白基因,是在活体水平研究珠蛋白基因表达调控的良好模型;2.运用亚硫酸盐测序的方法,对转基因小鼠不同组织中珠蛋白基因的LCR和启动子区的CpG的甲基化状态进行了检测和比较。结果证实,对应的CpG位点在6种红系组织和非红系组织中的甲基化程度和模式有显著差异。对于区域的甲基化程度,红系组织中较非红系组织中低,而这种较低的甲基化修饰与报告基因EGFP的活跃转录对应;3.运用定量RT-PCR技术,对三种DNA甲基化酶在转基因小鼠各组织内的表达进行了比较分析。结果显示,“起始性”(de novo)的Dnmt3a和Dnmt3b在红系组织和非红系组织中的表达模式有显著差异,而“维持性”(maintenance)的甲基化酶Dnmt1的表达则没有明显的红系特异性。这不仅是DNA甲基化修饰影响珠蛋白基因的表达的直接证据,也说明珠蛋白基因的甲基化修饰是Dnmt3a和Dnmt3b作用的结果;4珠蛋白基因的启动子甲基化存在偏爱性,有若干个甲基化化敏感位点。这些位点是一些转录因子的调节区域。表观遗传修饰可能是作用于转录因子和DNA的结合来调节珠蛋白基因的表达。为了探讨转基因小鼠报告基因EGFP组织特异性表达是否与内源性miRNA的功能相关,本研究对靶向作用于人珠蛋白基因的内源性的miRNA进行了初步研究。应用生物信息学和实验验证的方法,找到了针对珠蛋白基因3’UTR区的25个小鼠内源性miRNA,并对其中5个miRNA进行了较深入的分析。结果显示,miR-34c和miR-379与组织中EGFP mRNA及Dnmt3a和Dnmt3b转录本的水平密切相关。由于转基因小鼠各组织中EGFP的表达水平与该组织中的甲基化状态相关,因此推论组织特异性表达的转基因可能被内源性的miRNA所诱发,进而发生转录本的抑制和甲基化修饰,导致其表达水平呈现红系特异性的特征。更多还原

【Abstract】 Human globin gene cluster shows erythroid tissue-specific and developmental stages-specific expression. The transcription of globin genes is controlled by the chromatin structure, methylation status of the coding sequence and the flanking regions, cis-regulatory elements and trans-factors. Locus control region (LCR) is the most important cis-element in regulation ofβ-globin gene expression. Several models (including looping, tracking, linking, topologic alterations, and modification of proteins associated with chromatin) have been proposed to explain the mechanism of humanβ-globin tissue-specific expression. All the models, directly or indirectly, implicate the methylation pattern of DNA and dynamic chromatin configuration. We previously generated a transgenic mouse strain, in which the spatial and hematopoietic-specific expression of the transgene (enhanced green fluorescent protein, EGFP) is driven by the humanβ-globin promoter and under controlled of LCR elements (HS2-HS3). The present study extends the previous work to further investigate the mechanism of tissue-specific expression of the gene.To explore the potential role of DNA methylation in expression of humanβ-globin, we analyzed the EGFP mRNA expression profiles among tissues of the transgenic mice using real-time RT-PCR. The results showed that the EGFP expression was highly specific in hematopoietic tissues. We then investigated the methylation status of the LCR and promoter regions by using sodium bisulfite genomic sequencing. These researches provided significant clues to elucidate the mechanism of the regulation on tissue-specific expression of genes. The main progresses are as follow:1. Expression patterns of the plasmid HS3-2-βP-EGFP-βE are different among various tissues of the transgenic mice. This hematopoietic-specific transgenic mouse model can be used to investigate the mechanism of regulation of humanβ-globin gene expression in vivo.2). The methylation status of HS3-2 elements in LCR as well asβ-globin promoter with upstream region was analyzed. The results showed that all CpGs of the LCR and promoter region in each hematopoietic tissue revealed a tendency toward a hypomethylation pattern, while there is a hypermethylation pattern in non-hematopoietic tissues. The results also demonstrated that different levels of the expression of EGFP transgene in hematopoietic and non-hematopoietic tissues were tightly correlated with different methylated patterns of theβ-globin LCR and promoter.3. Differential expression patterns of Dnmt3a and Dnmt3b, but not Dnmt1, in hematopietic and non-henatopoietic tissues, that corresponding to the EGFP expression. It provided the evidence that the differ expression of Dnmt3a and Dnmt3b may constitute one of the mechanisms of the methylations status at CpG sites inβ-globin LCR and promoter regions of various tissues.4) Methylation frequencies at some CpG sites ofβ-globin LCR and promoter region were more predominant, and the methylation status was biased along the CpG positions. The nearer the transcription start site of the transgene plasmid, the higher the methylation frequencies were. The transcription initiation complex plays a critical role in methylation and demethylation, possibly be interfering with the methylation modification after DNA replication.We also investigate whether the transgene tissue-specific expression may be related with the functions of the endogenous microRNA. The 3’-UTR region of humanβ-globin gene was searched for complimentarily with the existing human miRNAs from miRBase using RNAhybrid software, and 25 miRNAs were identified to be complementary to 3′-UTR region of humanβ-globin. The analysis of the expression patterns of 5 miRNAs among tissues showed that miR-34c and miR-379 expression profiles were closely correlated with the target EGFP mRNA as well as Dnmt3a and Dnmt3b transcripts in LCR elements andβ-globin promoter. Since the expression of the transgene (EGFP) was tightly linked with the methylation status of the individual tissues, we therefore deduced that the differential expression of the transgene in each tissue may be induced by the endogenous miRNAs, causing the differention of the transcription and methylation modification and then impacting on the gene expression.更多还原