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Analysis of cardiac specific transcription factor GATA-4 complex during differentiation of mouse embryonic stem cells into cardiac myocytes

小鼠胚胎干细胞分化为心肌细胞过程中心脏特异性转录因子GATA-4复合物分析

基本信息

批准号：
17590770
负责人：
MORIMOTO Tatsuya
金额：
$ 2.24万
依托单位：
Research Institute for Production Development
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590770/
关键词：
embryonic stem (ES) cells Proteomics Cardiac myocytes differentiation Transcription factor GATA-4

项目摘要

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of HATs, p300 serves as a coactivator of cardiac-specific transcription factors such as a zinc finger protein GATA4. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induces acetylation of GATA4 as well as histones and facilitates their differentiation into cardiac myocytes. Here, we show that cyclin-dependent kinases-9 (CDK9), a component of positive transcription elongation factor b which increases the activitiy of RNA Pol II by hyperphosphorylation, is a novel component of p300/GATA4 complex and is required for myocardial cell differentiation. CDK9 interacted not only with GATA4 but also with p300. A dominant-negative form of CDK9 (DN-CDK9) and … More CDK9 kinase inhibitor, 5,6-dichloro-1-h-ribofuranosyl-benzimidazole (DRB) inhibited p300-induced activation of GATA4-dependent transcription as well as acetylation of GATA4. We examined the effects of DRB and DN-CDK9 on myocardial differentiation by flow cytometry in a mouse ES cell line, in which GFP is expressed under the control of the cardiac-specific Nkx-2.5 promoter. This line of ES cells was cultured in a monolayer on a flat gelatin-coated plate without embryoid body formation. TSA induced the expression of GFP by 8-to 9-fold in these cells. Furthermore, TSA increased the amount of the GATA4/CDK9 complex in mouse ES cells. Administration of DRB repressed TSA-induced expression of GFP controlled by the Nkx-2.5 promoter. Furthermore, we introduced DN-CDK9 into Nkx-2.5/GFP ES cells by lenti-virus-mediated gene transfer. Introduction of the DN-CDK9 gene decreased TSA-induced GFP expression by 50%, while introduction of the null vector had no effect. These findings demonstrate that CDK9, as a novel component of the p300/GATA4 complex, is involved in the differentiation of mouse ES cells into cardiac myocytes. Less

胚胎干细胞（ES）分化为心肌细胞需要激活心脏特异性基因程序。组蛋白乙酰基转移酶（HAT）和组蛋白脱乙酰基酶（HDAC）通过与特定转录因子结合被募集到靶基因来控制基因表达模式。HAT之一，p300作为心脏特异性转录因子如锌指蛋白GATA 4的共激活因子。用一种特异性HDAC抑制剂--曲马斯他丁A（TSA）处理ES细胞，诱导GATA 4以及组蛋白的乙酰化，并促进它们分化为心肌细胞。细胞周期蛋白依赖性激酶9（cyclin-dependent kinases-9，CDK 9）是p300/GATA 4复合物的一个新的组成部分，是心肌细胞分化所必需的，CDK 9是正性转录延伸因子B的一个组成部分，通过过度磷酸化增加RNA Pol II的活性。CDK 9不仅与GATA 4相互作用，而且与p300相互作用。CDK 9的显性阴性形式（DN-CDK 9）和 ...更多信息 CDK 9激酶抑制剂5，6-二氯-1-h-呋喃核糖基-苯并咪唑（DRB）抑制p300诱导的GATA 4依赖性转录激活以及GATA 4的乙酰化。我们通过流式细胞术在小鼠ES细胞系中检测了DRB和DN-CDK 9对心肌分化的影响，其中GFP在心脏特异性Nkx-2.5启动子的控制下表达。该ES细胞系在无胚状体形成的平明胶包被平板上单层培养。TSA在这些细胞中诱导GFP表达8- 9倍。此外，TSA增加小鼠ES细胞中GATA 4/CDK 9复合物的量。施用DRB抑制TSA诱导的由Nkx-2.5启动子控制的GFP表达。此外，我们通过慢病毒介导的基因转移将DN-CDK 9导入Nkx-2.5/GFP ES细胞。DN-CDK 9基因的引入使TSA诱导的GFP表达降低了50%，而空载体的引入没有影响。这些发现表明，CDK 9作为p300/GATA 4复合物的新组分，参与小鼠ES细胞向心肌细胞的分化。少