Islet-1介导组蛋白乙酰化修饰对心脏发育相关转录因子的调控作用

发布时间：2018-06-27 10:56

  本文选题：Islet-1 + 组蛋白乙酰化　； 参考：《重庆医科大学》2012年硕士论文

【摘要】：目的 组蛋白乙酰化为常见的表观遗传方式。本课题组前期研究发现，心脏发育相关转录因子受到组蛋白乙酰化的调控，而组蛋白乙酰化状态则通过组蛋白乙酰化酶（histone acetyltransferases，HATs）和组蛋白去乙酰化酶（histone deacetylase，HDACs）调节。然而，组蛋白乙酰化并没有特异性，在心脏发育过程中是否存在辅助因子，招募并协助HATs识别心脏发育相关转录因子以介导其启动子区组蛋白的乙酰化？Islet-1为心脏发育早期的重要转录因子，属于LIM-Homeodomain家族，其结构符合该辅助因子的需求。本研究拟通过慢病毒转染干扰Islet-1的表达，探讨Islet-1是否作为此种辅助因子介导组蛋白乙酰化参与心脏发育相关转录因子的调控。 材料与方法 培养心肌祖细胞，，以转染Islet-1RNAi慢病毒作为Islet-1抑制组，转染未携带任何基因的空载体慢病毒作为空载体对照组，未转染慢病毒的细胞作为空白对照组。通过流式细胞计数检测转染效率，运用荧光定量聚合酶链反应（quantitative polymerase chain reaction,Q-PCR）检测心脏发育相关转录因子mRNA的表达水平。通过Western blotting检测心肌祖细胞内组蛋白H3乙酰化水平。通过ChIP抗乙酰化H3抗体和抗p300抗体，运用染色质免疫共沉淀（Chromatin immunoprecitation,ChIP）及Q-PCR技术检测心脏发育相关转录因子启动子区乙酰化水平及与HATs亚型p300结合情况。 结果 1.流式细胞计数显示空载体组及Islet-1抑制组的转染效率均大于30%。Q-PCR结果显示Islet-1抑制组中心脏发育相关转录因子Islet-1、Mef2c、Tbx5的mRNA相对表达量与空白对照组和空载体对照组相比有明显降低（P 0.05）。而Gata4的mRNA相对表达量各组间差异不具有统计学意义（P0.05）。 2. Western blotting结果显示空白对照组、空载体对照组及Islet-1抑制组之间组蛋白H3乙酰化水平没有明显变化差异不具有统计学意义（P0.05）。ChIP PCR结果提示Islet-1抑制组中通过乙酰化H3抗体和p300抗体募集下来的Mef2c的启动子DNA含量较空白对照组和空载体对照组有降低，并且差异具有统计学意义（P 0.05），而Gata4、Tbx5的启动子DNA含量各组间则没有明显统计学差异（P 0.05）。 结论 以上数据表明，抑制Islet-1可能降低p300与Mef2c启动子区的结合，从而下调Mef2c启动子区乙酰化水平，进而影响Mef2c的表达。但Gata4、Tbx5可能并非通过该途径而受到组蛋白乙酰化的调控，其调控方式仍需进一步研究。
[Abstract]:Objective histone acetylation is a common epigenetic pattern. Our previous study found that cardiac development-related transcription factors are regulated by histone acetylation, and the histone acetylation state is regulated by histone acetyltransferes (HATs) and histone deacetylase (histone deacetylase). However, histone acetylation is not specific. The acetylated Islet-1 (Islet-1) involved in the identification of cardiac developmental related transcription factors by HATs is an important transcription factor in the early stage of cardiac development. It belongs to the LIM-Homeodomain family and its structure meets the needs of this cofactor. In this study, lentivirus transfection interfered with the expression of Islet-1 to investigate whether Islet-1 is involved in the regulation of transcription factors associated with cardiac development as an auxiliary factor mediating histone acetylation. Materials and methods Myocardial progenitor cells were cultured. Islet-1RNAi lentivirus was transfected as Islet-1 inhibition group, empty vector lentivirus without any gene was transfected as control group, and cells without lentivirus were used as blank control group. The transfection efficiency was detected by flow cytometry and the expression level of cardiac development-related transcription factor mRNA was detected by fluorescence quantitative polymerase chain reaction (quantitative polymerase chain reaction- Q-PCR). The level of histone H 3 acetylation in myocardial progenitor cells was detected by Western blotting. The acetylation level of the promoter region of cardiac development-associated transcription factor and its binding to Hats subtype p300 were detected by chromatin immunoprecipitation (chip) and Q-PCR using ChIP anti-acetylated H3 and anti-p300 antibodies. Result 1. Flow cytometry showed that the transfection efficiency of empty vector group and Islet-1 inhibition group was higher than that of 30. Q-PCR group. The results showed that the relative expression of Islet-1Mef2ctbx5 mRNA in Islet-1 inhibition group was significantly lower than that in blank control group and empty vector control group (P 0.05). However, the relative expression of Gata4 mRNA was not significantly different among the groups (P0.05). The results of Western blotting showed that the blank control group, There was no significant difference in the level of histone H3 acetylation between empty vector control group and Islet-1 inhibition group (P0.05). The PCR results showed that Mef2c was initiated by acetylated H3 antibody and p300 antibody in Islet-1 inhibition group. The DNA content of the subunit was lower than that of the blank control group and the empty vector control group. The difference was statistically significant (P 0.05), but there was no significant difference in the promoter DNA content of Gata4 (Tbx5) among the three groups (P 0.05). Conclusion the above data suggest that inhibition of Islet-1 may decrease the binding of p300 to Mef2c promoter, thus down-regulate the acetylation level of Mef2c promoter, and then affect the expression of Mef2c. However, Gata4Tbx5 may not be regulated by histone acetylation through this pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.4

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