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 Sertoli cells. To date, the mechanism(s) responsible for Sertoli cell-specific expression of the FSHR gene are unknown. In this study, DNA methylation at specific sites in the promoter are shown to lead to changes in the DNA-protein interactions at those sites and, subsequently, to transcriptional repression of the gene. The extent of methylation of cytosine residues within the core promoter region of genomic DNA isolated from cells/tissues that expressed, or did not express, the FSHR gene was analyzed by the sodium bisulfite conversion technique. All seven cytosine residues in CpG dinucleotides within the core promoter region were found to be unmethylated in primary cultured 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 interactions in three primary regulatory regions of the promoter were examined by electrophoretic mobility shift assays (EMSA) with synthetic oligonucleotides containing selectively methylated cytosine residues. Methylation of a CpG sequence within a consensus E box element (CACCTG, -124/-119) decreased the binding affinity of USF1/2 transcription factors for this element. Methylation of the CpG sequence in the Inr region (CCGG, -85/-82) allowed the formation of an additional DNA-protein complex. Methylation at both cytosine residues in the E2F element ((m)CG(m)CG) generated a new methylcytosine-specific 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 transcription of the FSHR gene. Taken together, these results suggest that cytosine methylation is a major factor in the repression of the expression of the FSHR gene.