C/EBPα represses slow myosin heavy chain 2 gene expression in developing avian myotubes

• Published in: Biochimica et biophysica acta Vol. 1860; no. 11; pp. 2355 - 2362
• Main Authors: Cavanaugh, Eric J., DiMario, Joseph X.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2016
• Subjects: Animals; Avian; Avian Proteins - genetics; Avian Proteins - metabolism; CCAAT-Enhancer-Binding Protein-alpha - metabolism; Cells, Cultured; Chick Embryo; Fiber type; Gene Expression Regulation, Developmental; Lineage; Muscle; Muscle Development; Muscle Fibers, Skeletal - cytology; Muscle Fibers, Skeletal - metabolism; Myosin Heavy Chains - genetics; Myosin Heavy Chains - metabolism; Promoter; Transcription; Promoter; Lineage; Muscle; Avian; Transcription; Fiber type
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2016.07.003

The CCAAT/enhancer binding proteins (C/EBP) comprise a family of transcription factors that regulate many cellular processes. Little is known of their function during embryonic and fetal myogenesis. Slow myosin heavy chain 2 (MyHC2) is a marker of the slow avian skeletal muscle fiber type, and slow MyHC2 gene regulation involves molecular pathways that lead to muscle fiber type diversification. The biological effects of C/EBPα and C/EBPβ expression were analyzed by use of a general C/EBP activity reporter and by slow MyHC2 promoter-reporter constructs transfected into specific myogenic cell lineages. The effects of C/EBPα and C/EBPβ expression were also analyzed by immunocytochemical detection of slow MyHC2. C/EBPα interaction with the slow MyHC2 promoter was assessed by electromobility shift assays. C/EBPα and C/EBPβ are present in embryonic fast and fast/slow avian myogenic lineages. Overexpression of C/EBPα cDNA repressed slow MyHC2 promoter activity in embryonic myotubes and in both electrically stimulated fetal myotubes. Deletion analysis of the slow MyHC2 promoter-luciferase reporter demonstrated that the transcriptional repression mediated by C/EBPα occurs within the first 222bp upstream from exon 1 of the slow MyHC2 gene. Electromobility shift assays determined that C/EBPα can bind to a non-canonical C/EBP site within the slow MyHC2 gene, and mutation of this site reduced transcriptional repression of the slow MyHC2 gene. C/EBPα, but not C/EBPβ, represses slow MyHC2 promoter activity via a non-canonical C/EBP binding element. Members of the C/EBP family of transcription factors differentially regulate genes indicative of distinct muscle fiber types. •C/EBPα, but not C/EBPβ represses slow MyHC2 promoter.•C/EBPα mediated promoter repression occurs during embryonic and fetal myogenesis.•C/EBPα binds to a non-canonical C/EBP site in the slow MyHC2 promoter.