Leptin gene expression in human preadipocytes is switched on by maturation-induced demethylation of distinct CpGs in its proximal promoter.

The peptide hormone leptin plays a major role in the regulation of energy intake and expenditure and is predominantly expressed in mature adipocytes but not in preadipocytes. Using bisulfite genomic sequencing, we found that 32 CpGs, distributed within a 317-bp sequence of the proximal leptin promoter, were highly methylated in human preadipocytes (73.4% +/- 9.0%). During maturation toward terminally differentiated adipocytes, this promoter region was extremely demethylated (9.4% +/- 4.4%). CpG methylation-dependent transcriptional activity of the promoter fragment was determined in transfection experiments using a set of 5'-truncated mock-, HhaI-, and SssI-methylated promoter-reporter constructs. Whereas the methylated CpG within the CCAAT/enhancer-binding protein alpha recognition site down-regulated reporter expression, methylated CpGs proximal to the TATA motif and/or in a further upstream region abrogated promoter activity completely. These distinct promoter CpG sequences were found unmethylated in leptin-expressing mature adipocytes. As evidenced by electrophoretic mobility shift assays, nuclear protein complexes were specifically formed on methylated oligonucleotide probes corresponding to the dedicated promoter sequences, indicating that methyl-CpG binding proteins participate in transcriptional repression and regulation of the human leptin gene.