组蛋白巴豆酰化修饰对小鼠胚胎干细胞干性调控的研究
发布时间：2019-01-26 12:29
【摘要】：巴豆酰化修饰是在巴豆酰基转移酶作用下将巴豆酰基从巴豆酰-辅酶A转移到赖氨酸残基上的一种新型翻译后修饰。其它实验室及我们实验室的研究表明,乙酰基转移酶CBP/p300可以催化组蛋白巴豆酰化,组蛋白去乙酰化酶HDAC1、HDAC2、HDAC3、HDAC8以及SIRT1也具有组蛋白去巴豆酰化酶活性。我们用小鼠的胚胎干细胞CGR8为材料,体外诱导分化形成拟胚体,检测组蛋白乙酰化和巴豆酰化水平发现胚胎干细胞分化后组蛋白乙酰化修饰和巴豆酰化修饰水平明显下降。为了研究巴豆酰化修饰在胚胎干细胞中的作用,我们的策略是利用实验室前期筛选得到的具有去巴豆酰化但丧失了去乙酰化酶活性的HD AC1突变体(HDAC1-VRPP),在胚胎干细胞中选择性降低组蛋白巴豆酰化修饰。因此我们在小鼠胚胎干细胞CGR8中构建了可诱导表达野生型HDAC1和突变型HDAC1-VRPP的稳系。我们通过蛋白印迹方法和RT-PCR方法分别检测了Dox诱导野生型HDAC1和突变型HDAC1-VRPP表达不同时间对胚胎干性因子Oct4、Sox2和Nanog蛋白水平的影响和不同胚层的标记基因在转录水平的变化,发现诱导野生型和突变型的HDAC1都能导致胚胎干性因子蛋白水平的下降和分化标志基因表达不同程度的上调,表明选择性去巴豆酰化修饰诱导胚胎干细胞分化。通过显微镜观察,我们也证实了 Dox诱导表达HDAC1-WT及HDAC1-VRP P九天后胚胎干细胞的克隆团形成明显变小。以上结果表明胚胎干细胞具有相比分化细胞而言更高水平的组蛋白巴豆酰化修饰,并且发现高水平的组蛋白巴豆酰化修饰对维持胚胎干细胞的干性具有重要作用。
[Abstract]:Crotonyl modification is a new post-translational modification that transfers crotonyl from crotonyl-coenzyme A to lysine residues under the action of crotonyl transferase. Studies in other laboratories and in our laboratory have shown that acetyltransferase CBP/p300 can catalyze crotonylation of histone, histone deacetylase HDAC1,HDAC2,HDAC3,HDAC8 and SIRT1 also have histone debutamylase activity. Mouse embryonic stem cells (CGR8) were used as materials to induce differentiation into embryoid bodies in vitro. Histone acetylation and crotonylation levels were detected and the levels of histone acetylation and crotonylation modification were significantly decreased after differentiation of embryonic stem cells. In order to study the role of crotonyl modification in embryonic stem cells, our strategy is to use the HD AC1 mutants (HDAC1-VRPP), which have desuccinylation but have lost deacetylase activity, which were obtained from pre-laboratory screening. Selective reduction of histone crotonylation modification in embryonic stem cells. Therefore, we constructed a stable line in mouse embryonic stem cell CGR8 that could induce the expression of wild type HDAC1 and mutant HDAC1-VRPP. The expression of wild type HDAC1 and mutant HDAC1-VRPP induced by Dox were detected by Western blot and RT-PCR, respectively. The effects of Sox2 and Nanog protein levels and the changes of marker genes in different embryo layers at transcription level showed that both wild-type and mutant HDAC1 could induce the decrease of embryonic dry-factor protein level and the up-regulation of differentiation marker gene expression. The results showed that selective decrotonylation could induce the differentiation of embryonic stem cells. By microscope observation, we also confirmed that the colony formation of embryonic stem cells induced by Dox induced expression of HDAC1-WT and HDAC1-VRP P was significantly smaller after 9 days. These results suggest that embryonic stem cells have higher levels of histone crotonylation than differentiated cells and that high levels of histone crotonylation play an important role in maintaining the dryness of embryonic stem cells.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q75
本文编号：2415482
[Abstract]:Crotonyl modification is a new post-translational modification that transfers crotonyl from crotonyl-coenzyme A to lysine residues under the action of crotonyl transferase. Studies in other laboratories and in our laboratory have shown that acetyltransferase CBP/p300 can catalyze crotonylation of histone, histone deacetylase HDAC1,HDAC2,HDAC3,HDAC8 and SIRT1 also have histone debutamylase activity. Mouse embryonic stem cells (CGR8) were used as materials to induce differentiation into embryoid bodies in vitro. Histone acetylation and crotonylation levels were detected and the levels of histone acetylation and crotonylation modification were significantly decreased after differentiation of embryonic stem cells. In order to study the role of crotonyl modification in embryonic stem cells, our strategy is to use the HD AC1 mutants (HDAC1-VRPP), which have desuccinylation but have lost deacetylase activity, which were obtained from pre-laboratory screening. Selective reduction of histone crotonylation modification in embryonic stem cells. Therefore, we constructed a stable line in mouse embryonic stem cell CGR8 that could induce the expression of wild type HDAC1 and mutant HDAC1-VRPP. The expression of wild type HDAC1 and mutant HDAC1-VRPP induced by Dox were detected by Western blot and RT-PCR, respectively. The effects of Sox2 and Nanog protein levels and the changes of marker genes in different embryo layers at transcription level showed that both wild-type and mutant HDAC1 could induce the decrease of embryonic dry-factor protein level and the up-regulation of differentiation marker gene expression. The results showed that selective decrotonylation could induce the differentiation of embryonic stem cells. By microscope observation, we also confirmed that the colony formation of embryonic stem cells induced by Dox induced expression of HDAC1-WT and HDAC1-VRP P was significantly smaller after 9 days. These results suggest that embryonic stem cells have higher levels of histone crotonylation than differentiated cells and that high levels of histone crotonylation play an important role in maintaining the dryness of embryonic stem cells.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q75
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1 高申楠;组蛋白巴豆酰化修饰对小鼠胚胎干细胞干性调控的研究[D];华东师范大学;2017年
，本文编号：2415482
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