Cyclin-dependent kinase 9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4

Cyclin-dependent kinase 9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4

Shinji Kaichi1, Koji Hasegawa2, Tomohide Takaya3, Tatsuya Morimoto4, Yoichi Sunagawa4, Teruhisa Kawamura55, Koh Ono3, Hiraku Doi1, Shiro Baba1, Tatsutoshi Nakahata1, Toshio Heike1.

  1. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  2. Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  3. Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  4. Division of Molecular Medicine, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
  5. Career-path Promotion Unit for Young Life Scientists, Kyoto University, Kyoto, Japan.

The 6th China-Japan Cardiovascular Forum (Beijing, China), 2010/10/16 (Poster).

Abstract

During embryogenesis, cell type-specific gene expression plays a pivotal role in the determination of cell fate, including differentiation, proliferation, and apoptosis. In contrast to other cell types, cardiac muscle cells are highly organized and their developmental processes require a number of cell type-specific transcription factors. One of HATs, p300 serves as a coactivator of cardiac-specific transcription factors such as a zinc finger protein GATA4. Recently, we identified cyclin-dependent kinase-9 (Cdk9), a core component of positive elongation factor-b, as a novel GATA4-binding partner by tandem affinity purification and mass spectrometric analyses. Cdk9 also formed a complex with p300 as well as GATA4. p300 was required for the interaction of GATA4 with Cdk9 and for the kinase activity of Cdk9. Conversely, Cdk9 was required for the phosphorylation of p300 and its kinase activity was required for the p300-induced transcriptional activities, DNA-binding, and acetylation of GATA4. The present study examined whether Cdk9 also forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which GFP is expressed under the control of the cardiac specific Nkx2.5 promoter, were induced to differentiate. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP-positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor (ANF). Dominant-negative form of Cdk9, which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole inhibited TSA-induced increase of endogenous Nkx2.5 and ANF mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. TSA did not altered the interaction of GATA4 with Cdk9. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4.