Cis-elements involved in differential expression of Na(+)-K(+)-ATPase alpha 2 subunit gene in muscle differentiation.
The mRNA of the Na(+)-K(+)-ATPase alpha 2 subunit gene (Atpla2) increases during myogenesis in rat following birth. To investigate the mechanism of the transcriptional regulation underlying this process, we examined the promoter activity of Atpla2 by transient transfection assays in myocyte differentiation, using C2C12 murine skeletal myoblast cell line. The promoter activity was increased about 5-fold in the differentiated cells, comparing with that in the growing cells. By analyses of 5'-deletion mutations of the gene, the responsible elements were localized in the region between -108 and +60. We identified a binding factor that exists only in the nuclear extract from the growing cells but not in the extract from the differentiated cells with a probe DNA fragment from +20 to +50 by gel retardation assays. The element to which the factor binds can mediate the inhibition of alpha 2 gene expression in the growing C2C12 cells but not in the differentiated cells. Finally we revealed protein occupancy in this region in the growing cells by in vivo footprinting. We propose a model that the increased expression of Atpla2 during muscle differentiation is mediated by the disappearance of a negative factor.
Ikeda
K
K
Department of Biology, Jichi Medical School, Tochigi-ken, Japan.
Kawakami
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004794:Enzyme Repression; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D051379:Mice; D008969:Molecular Sequence Data; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000201:enzymology / Q000254:growth & development*; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D004268:DNA-Binding Proteins; D000254:Sodium-Potassium-Exchanging ATPase