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

胚胎干细胞向心肌细胞分化需要心脏特异性基因程序的激活。组蛋白乙酰转移酶(HATS)和组蛋白脱乙酰基酶(HDACs)通过与特定转录因子结合，被招募到靶基因，从而控制基因表达模式。作为HATS的一种，p300是心脏特异转录因子的共激活因子，如锌指蛋白GATA4。用HDAC特异性抑制剂曲古抑素A(TSA)处理ES细胞，可诱导GATA4和组蛋白的乙酰化，促进其分化为心肌细胞。在这里，我们发现细胞周期蛋白依赖的蛋白-9(CDK9)是p300/GATA4复合体的一个新的成分，是心肌细胞分化所必需的。CDK9是正转录延伸因子b的一个成分，通过过度磷酸化增加RNA Pol II的活性。CDK9不仅与GATA4相互作用，还与p300相互作用。一种显性-阴性形式的CDK9(DN-CDK9)和…更多的CDK9激酶抑制剂5，6-二氯-1-h-呋喃核糖基苯并咪唑(DRB)可抑制p300诱导的GATA4依赖的转录和乙酰化。我们用流式细胞术检测了DRB和dN-CDK9对小鼠ES细胞系心肌分化的影响，在该细胞系中，GFP在心脏特异的Nkx-2.5启动子控制下表达。该ES细胞系在平板明胶涂层上单层培养，没有形成类胚体。TSA诱导GFP在这些细胞中的表达增加8~9倍。此外，TSA还能增加小鼠ES细胞中GATA4/CDK9复合体的数量。DRB可抑制TSA诱导的Nkx-2.5启动子调控的GFP表达。此外，我们还通过Lenti病毒介导的基因转移将DN-CDK9导入Nkx-2.5/GFP ES细胞。导入dN-CDK9基因可使TSA诱导的GFP表达减少50%，而导入空载体则没有影响。这些结果表明，CDK9作为p300/GATA4复合体的一个新的成分，参与了小鼠ES细胞向心肌细胞的分化。较少

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