【作者】 戴灿；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 遗传学， 2010， 博士

【摘要】 染色体过客复合物(Chromosomal Passenger Complex, CPC)是细胞分裂中的一种重要的动态结构,在精确的时间和空间发挥作用,它在中期染色体排列、姐妹染色体分离、胞质分裂等方面发挥着十分重要的作用。人类细胞分裂周期相关基因8 (cell division cycle associated 8, CDCA8)编码的蛋白称为Borealin/DasraB,与Aurora B、INCENP、和Survivin共同构成CPC,它在定位CPC到着丝粒、纠正动粒结合错误、稳定双极纺锤体等方面发挥重要作用。CDCA8基因的表达分析显示,其在癌细胞和未分化的人类胚胎干细胞(human embryonic stem cells, hESCs)中高表达、在正常细胞中无表达。为了解析CDCA8基因差异表达的调控机制,深入了解该基因的功能,我们从转录水平入手,对CDCA8基因启动子功能及其相关转录因子进行了研究。在前期研究中,我所苗聪秀博士进行了启动子的克隆和体外转录启动能力分析：通过5’-RACE实验发现,CDCA8基因存在3个转录起始位点,分别位于翻译起始位点上游200 bp、194bp、175bp处,其中一个转录本存在选择性剪切；通过在Hela细胞中转染5’末端依次删减的启动子候选片段及活性分析,确定了启动子所在区域；在不同类型细胞中启动子活性的分析实验发现,CDCA8启动子在未分化的hESCs、肿瘤细胞中有高启动活性,而在分化的hESCs和正常细胞中只有低的启动活性或没有活性,提示转录水平的调控可能是CDCA8基因差异表达的重要调控方式。为了检测CDCA8启动子在体内的转录启动能力,本研究首先将“启动子-EGFP"片段显微注射到小鼠受精卵,在体外培养过程中,通过观测报告基因的表达来分析启动子在胚胎早期发育不同阶段的转录活性；然后采用显微注射法,构建“启动子-Luciferase"转基因小鼠模型。通过PCR和Southern Blot鉴定和筛选阳性小鼠。采用小动物活体成像技术、多组织荧光素酶活性测定、组织冰冻切片免疫荧光染色和石蜡切片免疫组化等方法分析转基因小鼠中报告基因在启动子活性下的组织表达情况。为了探寻CDCA8启动子活性差异的可能原因和调控机制,本研究首先应用重亚硫酸盐测序技术(Bisulfite sequencing PCR, BSP)检测hESCs分化前后CDCA8启动子基础活性区甲基化状态的变化情况,在表观遗传调控水平探寻原因。随后在转录因子水平进行了分析：首先对CDCA8启动子区域可能存在的转录因子结合位点进行了生物信息学预测；然后应用Pull-down和蛋白质/DNA芯片技术,比较了hESCs.人恶性多发性畸胎瘤细胞(NTERA-2)和人胚胎成纤维细胞(human embryo fibroblasts cell, hEF)中与启动子结合的转录因子,并用EMSA和Western Blot实验进一步验证；最后通过定点突变、过表达实验分析了转录因子对启动子活性的作用。研究发现,在小鼠胚胎早期发育的2-cell、4-cell和桑葚胚时期,启动子都有转录启动活性,启动了下游报告基因EGFP的表达。转基因小鼠的活体成像和多组织荧光素酶活性检测结果一致显示,CDCA8启动子在睾丸组织中有很强活性。转基因小鼠睾丸组织的冰冻切片免疫荧光染色和石蜡切片免疫组化分析显示,启动子仅在生精小管靠近基底膜的第一层细胞——精原细胞中启动荧光素酶的表达,随着管腔方向细胞分化／精子发生的进行,荧光素酶的表达消失,这与细胞水平的启动子活性一致。BSP结果显示,hESCs分化前后启动子基础活性区甲基化状态未发生明显改变。Pull-down和蛋白质/DNA芯片实验筛选出了在hESCs. NTERA-2中与启动子的结合量较hEF细胞大于2倍以上改变的转录因子。其中NFY的结合差异得到了EMSA和Western Blot的验证,并且定点突变和过表达实验表明,NFY的结合对CDCA8启动子活性起着重要作用,因此它在干细胞、癌细胞和正常细胞中与启动子结合的差异是启动子在这三类细胞中活性差异的重要原因。通过本课题的研究,我们获得了CDCA8启动子的位置、细胞水平和整体水平活性以及参与其调控的转录因子等较详细的研究结果,为进一步研究CDCA8基因的转录调控机制奠定了基础,对深入了解CDCA8基因的功能有着重要意义。更多还原

【Abstract】 Chromosome passenger complex (CPC) is an important dynamic structure during cell division. It plays an important role in ranking of the metaphase chromosomes, separation of the sister chromatid and cytokinesis. Human cell division cycle associated 8 (CDCA8) encodes a protein, termed Borealin/Dasra B, which is one of the four components of CPC. Functional studies revealed that Borealin was required for targeting of the CPC to centromeres, correction of kinetochore attachment errors and stability of the bipolar spindle in human cells.Expression analysis demonstrated that CDCA8 expressed highly in human cancer cells and undifferentiated embryonic stem cells (hESCs), compared with normal cells. In order to resolve the regulatory mechanism of differential expression and in-depth understand the gene function, CDCA8 promoter and its associated transcription factors were studied.In preliminary studies, Dr. Miao Congxiu Cloned and functionally analyzed the CDCA8 promoter in vitro:by 5’-RACE, three transcription start sites of CDCA8 were found at 200 bp,194 bp and 175 bp upstreame of translation start site, one of which is alternative splicing; the location of the promoter was defined by transient transfecting the Hela cells with reporter gene driven by the 5’-Truncated promoter fragments. Transient expression assays in different types of cells revealed that CDCA8 promoter is significantly activated in undifferentiated hESCs and cancer cell lines but repressed in differentiated hESCs and normal primary cells. This suggested the regulation of differential expression of CDCA8 gene was mainly on transcriptional level.In this study, to confirm the promoter activity in vivo, "promoter-EGFP" fragment was microinjected into mouse zygotes and expression of EGFP directed by the promoter was observed in developing embryos; "promoter-Luciferase" transgenic mice were established by microinjection and were identified by PCR and Southern Blot. In vivo imaging, multi-organization Luciferase assay, immunofluorescence and immunohistochemistry were used to test the ability of the promoter to drive gene expression in each issue.In order to explore the regulatory mechanism and possible reasons for the differential promoter activity, here, bisulfite sequencing PCR (BSP) was used firstly to compare the methylation status of basic promoter between undifferentiated and differentiated hESCs. Subsequent analysis was conducted on the level of transcription factors (TFs):First, the potential binding sites for known TFs on CDCA8 promoter were predicted by computer analysis; then the TFs binding to the promoter in hESCs, NTERA-2 and hEF cells were compared using Pull-down and Protein/DNA arrays, and confirmed with EMSA and Western Blot; site-specific mutagenesis and over-expression assay were used to test the effect of TFs on promoter activity.The results are as follows:Expression of EGFP directed by CDCA8 promoter was observed in 2-cell,4-cell and morula of early developing mouse embryos. Both in vivo imaging and multi-organization luciferase assay showed that strong luciferase expression was detected in the testes of adult transgenic mice. Immunohistochemistry and immunofluorescence staining revealed that luciferase expression was restricted to the subpopulation of spermatogonia, adjacent to the basement membrane of seminiferous tubules. The expression was disappeared along with the cell differentiation/spermiogenesis toward the lumen, which agreed with the promoter activity in vitro. BSP showed there was no change in methylation of CpG islands in the basic promoter during differentiation of hESCs. The TFs, whose binding amount on promoter had more than two times changes in hESCs and NTERA-2, compared with hEF, were found by Pull-down and Protein/DNA arrays. Among them, the changes of NFY were further confirmed by EMSA and Western Blot. Moreover, the site-specific mutagenesis and over-expression assay showed the binding of NFY was very important to the promoter activity. Therefore, the changes of binding amount of NFY could considerably responsible for the differential promoter activity in different cell types.Overall, the main characterization of CDCA8 promoter was defined in this study, including promoter location, promoter activity in vitro and in vivo, and TFs involved. These findings are of great significance and establish the basis for further research on transcription regulatory and function of CDCA8 gene.更多还原