Alpha7 integrin expression is negatively regulated by deltaEF1 during skeletal myogenesis.
Alpha7 integrin levels increase dramatically as myoblasts differentiate to myotubes. A negative regulatory element with putative sites for deltaEF1 is present in the alpha7 proximal promoter region. To define the role of deltaEF1 in regulating alpha7 integrin expression, we overexpressed deltaEF1 in C2C12 myoblasts. This resulted in a major down-regulation of alpha7 protein expression. Promoter assays revealed that C2C12 myoblasts transfected with deltaEF1 showed a decrease in activity of the 2.8-kb alpha7 promoter fragment, indicating regulation of alpha7 integrin at the transcriptional level. We have identified two E-box-like sites for deltaEF1 in the negative regulatory region. Mutation of these sites enhanced alpha7 promoter activity, indicating that these sites function in repression. MYOD, an activator of alpha7 integrin transcription, can compete with deltaEF1 for binding at these sites in gel shift assay. By using chromatin immunoprecipitation, we demonstrated a reciprocal binding of deltaEF1 and MYOD to this regulatory element depending on the stage of differentiation: deltaEF1 is preferentially bound in myoblasts to this region, whereas MYOD is bound in myotubes. The N-terminal region of deltaEF1 is necessary for alpha7 repression, and this region also binds the co-activator p300/CBP. Importantly, we found that the p300/CBP co-activator can overcome repression by deltaEF1, suggesting that deltaEF1 can titrate limiting amounts of this co-activator. These findings suggest that deltaEF1 has a role in suppressing integrin expression in myoblasts by displacing MYOD and competing for p300/CBP co-activator.
Jethanandani
Poonam
P
Department of Cell and Tissue Biology, University of California, San Francisco, California 94143-0640, USA.
Kramer
Randall H
RH
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000378:metabolism / Q000502:physiology*; D039001:Integrin alpha Chains / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology*; D009154:Mutation; D017570:MyoD Protein / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D050880:p300-CBP Transcription Factors / Q000378:metabolism; D000071799:Zinc Finger E-box-Binding Homeobox 1
D015703:Antigens, CD; D017931:DNA Primers; D018398:Homeodomain Proteins; D039001:Integrin alpha Chains; D016023:Integrins; D017570:MyoD Protein; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; C057363:MyoD1 myogenic differentiation protein; C085725:integrin alpha7; C519422:ZEB1 protein, mouse; D000071799:Zinc Finger E-box-Binding Homeobox 1