We investigated the mechanism of retinoic acid receptor (RAR) b2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous b2 promoter (71470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARb2. This was true even in the context of replicated chromatin, suggesting a cisacting rather than a trans-acting defect. Cytosine methylation, a cis-acting DNA modi®cation, has been implicated in RARb2 silencing in cancer cells. Upon bisul®te genomic sequencing, we found that 3 CpG sites in the b2 RARE region were variably methylated in MCF-7 cells but were not methylated in MTSV1.7 cells or in 2 MDA-MB-231 subclones that di ered in RARb2 expression (high in clone A2, low in clone A4). However, the 5'-UTR region was hypermethylated in clone A4 relative to clone A2 cells. Following 5-azacytidine treatment, RA and trichostatin A markedly induced RARb2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A b2 RARE reporter construct in which the methylation-susceptible cytosines in the sense strand were replaced by thymine displayed marked loss of activity in a replicated chromatin-dependent manner. We conclude that cytosine methylation contributes to RARb2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important.

Background: The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer.