Methylation analysis of the promoter F of estrogen receptor alpha gene: effects on the level of transcription on human osteoblastic cells.

In this study, the methylation status of the distal promoter F of estrogen receptor alfa (ERalpha) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERalpha gene transcription in bone tissue. The methylation status of promoter F was here evaluated, for the first time, by direct sequencing of bisulfite-treated genomic DNA, at 10 CpG specific sites localized in a region of about 800 bp. An heterogeneous methylation pattern was observed. The most notable difference was found at four particular CpGs, distant from the exon F transcription start site, showing a methylation status that correlates with the expression level, being ERalpha mRNA transcription reduced in a partially methylated cells but preserved in demethylated cells. The other CpG sites, localized around the transcription start site, were always demethylated except for MG-63 cells showing the lowest level of ERalpha expression. By quantitative RT-PCR analysis we demonstrated that ERalpha gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERalpha mRNA is represented by the isoform F. The same 10 CpG sites were investigated in non-osseous cell lines and were found fully methylated in ERalpha-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERalpha-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERalpha gene promoter F here analyzed may contribute to ERalpha transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.