DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells

Aims/hypothesis. The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating the expression of the leptin gene, we examined the methylation status and methylation-sensitive transcription factors during 3T3-L1 differentiation. Methods. DNase I footprinting, electrophoretic mobility-shift assays, and a Southwestern analysis were carried out using nuclear extracts from preadipocytes and adipocytes. Promoter activity was measured by luciferase assays. The CpG methylation pattern was determined. Results. Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough trans activation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt -161, all of which were highly methylated ( > 92 %) in preadipocytes. Seven of these sites showed a varying degree of demethylation during differentiation, while the site at nt -54 remained methylated. In electrophoretic mobility-shift assays, DNA fragments from nt -115 to nt -70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kM, protein that binds strongly to the methylated probes. Promoter activity was reduced by methylation of the CpG sites up to nt -115, but not up to nt -70. Conclusion/interpretation. These results suggest that methylation of specific CpG sites between nt -115 and nt - 70 and a methylation-sensitive protein could contribute to leptin gene expression during adipocyte differentiation in 3T3-L1 cells.