Site-specific methylation of the promoter alters deoxyribonucleic acid-protein interactions and prevents follicle-stimulating hormone receptor gene transcription

In the male gonad, the FSH receptor (FSHR) gene is expressed only in Sertol i cells. To date, the mechanism(s) responsible for Sertoli cell-specific ex pression of the FSHR gene are unknown. In this study, DNA methylation at sp ecific sites in the promoter are shown to lead to changes in the DNA-protei n interactions at those sites and, subsequently, to transcriptional repress ion of the gene. The extent of methylation of cytosine residues within the core promoter region of genomic DNA isolated from cells/tissues that expres sed, or did not express, the FSHR gene was analyzed by the sodium bisulfite conversion technique. All seven cytosine residues in CpG dinucleotides wit hin the core promoter region were found to be unmethylated in primary cultu red rat Sertoli cells that were actively expressing FSHR mRNA. In contrast, in tissues not expressing FSHR the same region of the gene was methylated at each of the CpG dinucleotides examined. In addition, DNA-protein interac tions in three primary regulatory regions of the promoter were examined by electrophoretic mobility shift assays (EMSA) with synthetic oligonucleotide s containing selectively methylated cytosine residues. Methylation of a CpG sequence within a consensus E box element (CACCTG, -124/-119) decreased th e binding affinity of USF1/2 transcription factors for this element. Methyl ation of the CpG sequence in the Inr region (CCGG, -85/-82) allowed the for mation of an additional DNA-protein complex. Methylation at both cytosine r esidues in the E2F element ((m)CG(m)CG) generated a new methylcytosine-spec ific DNA-protein complex. The core FSHR promoter region of a mouse Sertoli cell line (MSC-1) that does not express FSHR was shown to be methylated at four CpG dinucleotides. The demethylation of these four sites by treatment of the MSC-1 cells with 5-aza-2'-deoxycytidine (5-azaCdR) activated the tra nscription of the FSHR gene. Taken together, these results suggest that cyt osine methylation is a major factor in the repression of the expression of the FSHR gene.