| The absence of estrogen receptor-α (ERα) is perhaps the most distinctive pathological feature of breast cancers arising in women who inherit a mutation in the breast cancer susceptibility gene 1 (BRCA1). Two hypotheses, not necessarily mutually exclusive, exist in the literature that describe mechanisms of ERα transcriptional repression in breast cancer. One hypothesis suggests that methylation of cytosine-guanine dinucleotides (CpGs) primarily mediates repression, while the other maintains that transcriptional repression is mediated by certain positive and negative promoter elements. To determine if methylation of CpGs within the ERα promoter was associated with BRCA1-linked breast cancers, we evaluated methylation within exon 1. CpG methylation was evaluated by three independent methods, one of which was direct sequencing of bisulfite-treated genomic DNA. Results from direct sequencing showed an overall average methylation of 25% among non BRCA1-linked cancers and 40% among BRCA1-linked cancers (p = 0.003). The most notable difference was at 5 particular CpGs, each of which exhibited a greater than 2-fold increase in the BRCA1-linked group (p < 0.03 for each CpG). To determine if wild type BRCA1 could induce activity of the ERα promoter, we performed a series of transient transfections with ERα promoter segments hooked to a luciferase reporter. Following cotransfection with a BRCA1 expression plasmid, we observed that luciferase activity was significantly increased in both MCF10A and IMEC cells (p < 0.005 and 0.0005 respectively). Specifically, the full length ERα promoter construct showed approximately 50-fold (MCF10A) and 30-fold (IMEC) increases in luciferase activity following BRCA1 transfection. In contrast, MCF10A and IMEC cells transfected with an empty expression plasmid (i.e. no BRCA1) showed a 9-fold and 3-fold increase respectively. We localized the ERα P1 segment responsible for transactivation by BRCA1 to a 109 bp region containing an AP2γ homologous site. Gel shifts indicated that two nuclear complexes were upregulated following transfection with BRCA1, one of which was specific to the 109 bp ERα promoter segment. The work described here indicates that CpG methylation, as well as the activity of certain transcriptional regulatory elements, both represent important mechanisms by which the ERα gene is rendered inactive in breast cancers associated with BRCA1 mutations. |
