title: DNA demethylation during the differentiation of 3T3-L1 cells affects the expression of the mouse GLUT4 gene.
abstract: GLUT4 is the major glucose transporter in adipose tissue and skeletal and cardiac muscles. We examined the mechanisms underlying GLUT4 gene expression in 3T3-L1 cells, which express the gene during their differentiation from preadipocytes to adipocytes. In transient transfections, the activity of a mouse GLUT4 promoter extending to -100 bp in the 5'-flanking region did not differ significantly between 3T3-L1 preadipocytes and adipocytes. Promoter activity up to -590 bp in preadipocytes and adipocytes showed a 70% lower and 228% higher activity, respectively, than promoter activity extending to -100 bp. We also examined methylation status of the GLUT4 promoter. Up to -100 bp, there were five CpG sites at -11, -30, -58, -63, and -75 bp. Two CpG sites at -11 and -30 bp were highly methylated in preadipocytes (60 and 92%, respectively) and highly demethylated in adipocytes (28.6 and 25%, respectively). Conversely, three CpG sites at -58, -63, and -75 bp were highly demethylated in both preadipocytes and adipocytes (<12%). In gel mobility-shift assays, a fragment extending from -40 to -1 bp generated a methylation-sensitive band with nuclear extracts from both preadipocytes and adipocytes when the CpG sites were methylated. Southwestern analysis identified a protein of approximately 55 kDa that bound strongly to the methylated probe. Furthermore, methylation of the CpG sites inhibited promoters extending to -50 or -70 bp. These results suggest that in addition to cell type-specific transcription factor, methylation of specific CpG sites and the methylation-sensitive transcription factor contribute to GLUT4 gene regulation during 3T3-L1 differentiation.
authors: Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Japan.
authors: N
authors: N
authors: Yokomori
authors: M
authors: M
authors: Tawata
authors: T
authors: T
authors: Onaya
mesh_terms: D016475:3T3 Cells / Q000166:cytology* / Q000378:metabolism*; D017667:Adipocytes / Q000502:physiology; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D019175:DNA Methylation; D015870:Gene Expression / Q000502:physiology*; D051275:Glucose Transporter Type 4; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins; D011401:Promoter Regions, Genetic / Q000502:physiology; D013234:Stem Cells / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology
chemical_list: D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse