【作者】 白彦；

【导师】 吴秀山； 刘明耀；

【作者基本信息】 湖南师范大学 ， 遗传学， 2008， 硕士

【摘要】 灵长类特异的KRAB锌指基因约占人类基因组KRAB锌指基因的30%。目前国际上有关灵长类特异锌指基因的功能知之甚少。我们选择锌指基因ZNF480为模型研究灵长类特异锌指蛋白的功能。ZNF480基因在肌肉和心脏组织中高表达,提示该基因可能与肌肉和心脏组织的发育有关。半定量RT-PCR和western blot实验证明,在诱导的C2C12稳定转染细胞的肌分化过程中,ZNF480的mRNA和蛋白质水平持续上调,说明ZNF480与肌分化过程密切相关。为了从形态上检测ZNF480是否调控肌生成的过程,我们通过形态学和肌肉分化特异性抗体MHC对诱导分化的稳定系细胞进行免疫荧光检测。与载体过表达对照组比较,过表达ZNF480促进C2C12细胞形成更多的肌管和多核细胞,并且ZNF480稳转细胞的肌管和多核细胞随着分化过程的进行而逐步增多。Myf-5,myogenin,MyoD是bHLH生肌调节因子,而E47,E12则是普遍表达的bHLH蛋白。在肌分化过程中,MyoD和Myf5表达是决定多能性体节细胞定型为成肌细胞的关键步骤,myogenin在成肌细胞的终极分化中有重要作用。我们检测了ZNF480是否对这些基因进行调控。半定量RT-PCR和western blot实验证明,ZNF480能上调肌肉特异性bHLH转录因子和肌肉特异性基因MCK(肌肉激酶)的mRNA水平,以及MHC和myogenin的蛋白水平,说明ZNF480是通过调控bHLH和MCK促进肌分化。同时,ZNF480过表达的C2C12稳定系分化过程中,p21的蛋白水平明显上调,说明ZNF480通过激活p21使细胞退出细胞周期而进入细胞分化。利用荧光素酶活性实验,我们检测到ZNF480在NIH3T3细胞中并不能促进E12或MyoD对MCK启动子(MCK4800和4RTK)活性的调节,但ZNF480能与E12/MyoD共转时增强MCK启动子的活性,说明ZNF480是通过与E12/MyoD协同调节MCK启动子的活性。为了进一步检测ZNF480是否结合到MCK和myogenin的启动子上对其进行转录调控,我们利用EMSA实验,证明了ZNF480不能直接结合到MCK启动子上,但是能够与E12/MyoD形成蛋白复合体结合到MCK的启动子区域。同时,EMSA实验证明ZNF480能够直接结合到myogenin的启动子区域。这些结果表明ZNF480通过形成蛋白复合体或直接结合的方式来调节MCK和myogenin的启动子活性,从而参与肌肉分化的调控过程。综上所述,灵长类特异人类锌指蛋白ZNF480能够促进肌管发生过程,通过调节肌生成调节因子Myf-5,MyoD和myogenin以及肌肉特异性基因MCK来调控肌生成。更多还原

【Abstract】 Primate-specific KRAB zinc finger proteins account for at least 30% of KRAB zinc finger proteins in the whole human genome. While little related report regards to functional role of primate-specific zinc finger proteins has been published. Therefore, we choose a primate-specific gene ZNF480 as a model to investigate the potential role of primate-specific zinc finger proteins.ZNF480 is highly expressed in muscle and heart tissues, indicating that it may be involved in development of muscle and heart tissues. Half-quantitative RT-PCR and western blot suggest that mRNA and protein levels of ZNF480 are significantly upregulated during stable transfected C2C12 myogenesis, which suggests that ZNF480 may be involved in myogenesis.To investigate whether ZNF480 can regulate myogenesis in morphologic, we examined the information of myotube via morphologic observation and immunofluorescence analysis by muscle-specific antibody MHC. Compared with control group, overexpression of ZNF480 can promote the formation of myotubes and multi-nucleus cells, and the numbers of myotubes and multi-nucleus have been increased following the process of myogenesis.Myf-5, myogenin and MyoD are bHLH transcriptional factors involved in myogenesis. E47 and E12 are bHLH transcriptional factors without tissue-specific. During myogenesis, Myf-5 and MyoD are key genes whose expression will determine whether mesodermal progenitor turn to myoblast, while myogenin plays pivotal role in terminal differentiation of myoblast. Therefore, we examined the possibility whether ZNF480 can regulate these genes. Half-quantitative RT-PCR and western blot show that ZNF480 can upregulate mRNA level of muscle-specific bHLH transcriptional factor and muscle-specific gene MCK, and the protein levels of MHC and myogenin, which in turn documents that ZNF480 promotes myogenesis through regulating bHLH and MCK. Moreover, Western blot suggests that the protein level of p21 is upregulated obviously during cell myogenesis of overexpression of ZNF480. It indicates that ZNF480 cause cell cycle exiting and cell differentiation by upregulating the level of p21 protein.We performed luciferase activity assays which show that over-expression of ZNF480 in NIH3T3 cell can not enhance the activities of E12 or MyoD on MCK promoter, but ZNF480 enhances the activites of MCK promoters (MCK4800 and 4RTK) when cotransfected with E12/MyoD. It suggests that ZNF480 promotes the ability of E12/MyoD to transactivate MCK promoter activity.To probe whether ZNF480 can bind to the promoters of MCK and myogenin and regulate their transcription, we performed EMSA assay. The results show that ZNF480 can not bind to MCK promote region alone but it does through forming protein complex with E12/MyoD. At the same time, EMSA assay showed that ZNF480 can directly bind to the promoter region of myogenin. These results document that ZNF480 regulate transcriptional activity of MCK and myogenin by forming protein complex or binding directly to regulate myogenesis.To sum up, primate-specific human zinc finger protein ZNF480 may enhance myogenesis and play an important role in myogenesis through regulation of myogenic regulation factors Myf-5, MyoD and myogenin and muscle specific gene MCK更多还原