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

Abstract

In this study, the methylation status of the distal promoter F of estrogen receptor alfa ( ERα) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERα 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 ERα 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 ERα expression. By quantitative RT-PCR analysis we demonstrated that ERα gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERα 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 ERα-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERα-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERα gene promoter F here analyzed may contribute to ERα transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.