Cyclin-dependent kinase-9 is required for the differentiation of mouse ES cells into cardiac myocytes

Cyclin-dependent kinase-9 is required for the differentiation of mouse ES cells into cardiac myocytes

Shinji Kaichi1, Tomohide Takaya2, Yoichi Sunagawa2, Teruhisa Kawamura2, Koh Ono4, Tatsuya Morimoto2, Toru Kita4, Shiro Baba1, Kyoko Hidaka3, Takayuki Morisaki3, Toshio Heike1, Tatsutoshi Nakahata1, Akira Shimatsu2, Koji Hasegawa2.

  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 Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan.
  4. Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

第72回日本循環器学会学術集会 (福岡), 2008/03/28 (ポスター).

Abstract

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns through association with specific transcription factors. Treatment of embryonic stem (ES) cells with trichostatin A (TSA), an HDAC inhibitor, facilitates their differentiation into cardiac myocytes. We have shown that CDK9 interacts with one of HATs p300 and a cardiac transcription factor GATA4. To determine the role of CDK9 in myocardial differentiation, we examined the effects of CDK9 kinase inhibitor, 5,6-dichloro-1-h-ribofuranosyl-benzimidazole (DRB) and a dominant-negative form of CDK9 (DN-CDK9) on myocardial differentiation. Mouse ES cells and NKX-2.5/GFP ES cells, in which GFP is expressed under the control of the cardiac-specific Nkx-2.5 promoter, were cultured in monolayer on a flat gelatin-coated plate. TSA induced GFP expression by 6.5-fold in Nkx-2.5/GFP ES cells and endogenous Nkx-2.5 mRNA levels by 16-fold in mouse ES cells. Furthermore, TSA induced hyperphosphorylation of RNA Pol II, indicating the activation of the CDK9 kinase activity. Administration of DRB repressed TSA-induced GFP expression in Nkx-2.5/GFP ES cells by 60% and of endogenous Nkx-2.5 mRNA levels in mouse ES cells by 75%. Finally, introduction of DN-CDK9 gene into Nkx-2.5/GFP ES cells by lentivirus-mediated gene transfer significantly decreased TSA-induced GFP expression, while introduction of null vector had no effect. These findings demonstrate that CDK9 is required for the differentiation of mouse ES cells into cardiac myocytes.