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

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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