Basic helix-loop-helix transcription factor epicardin/capsulin/Pod-1 suppresses differentiation by negative regulation of transcription.
Epicardin/capsulin/Pod-1, expressed in skeletal myoblasts within brachial arches and in the condensing mesenchyme, is a member of the basic helix-loop-helix (bHLH) transcription factor family that is involved in various cell differentiation processes. In this study, we examined the functional properties of epicardin/capsulin/Pod-1 in differentiation. The yeast and mammalian two-hybrid systems showed physical associations between epicardin/capsulin/Pod-1 and E2A, both of which were present in the nuclei. The bHLH domains mediated this association. Ectopic expression of epicardin/capsulin/Pod-1 inhibited E2A-dependent activation of the exogenous and endogenous expression of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) gene, and the muscle creatine kinase gene that encodes the predominant creatine kinase isoform expressed in mammalian skeletal muscle. Transfection with epicardin/capsulin/Pod-1 small interfering RNA abolished the epicardin/capsulin/Pod-1-mediated suppression of E12-dependent activation of the p21 promoter. Chromatin immunoprecipitation assay showed that epicardin/capsulin/Pod-1 was physically associated with the muscle creatine kinase promoter in vivo. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of epicardin/capsulin/Pod-1. These inhibitory functions of epicardin/capsulin/Pod-1 closely resemble those of the bHLH inhibitor Twist protein. These results indicate that epicardin/capsulin/Pod-1 functions as a negative regulator of differentiation of myoblasts through transcription in at least two distinct steps, cell growth arrest and lineage-specific differentiation.
Funato
Noriko
N
Human Gene Sciences Center and Maxillofacial Orthognathics Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Ohyama
Kimie
K
Kuroda
Takayuki
T
Nakamura
Masataka
M
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000166:cytology; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D051792:Basic Helix-Loop-Helix Proteins; D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008156:Luciferases; D034741:RNA, Small Interfering; D014157:Transcription Factors; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; C491825:TCF21 protein, human; C112923:Tcf21 protein, mouse