Methylation of conserved CpG sites neighboring the beta retinoic acid response element may mediate retinoic acid receptor beta gene silencing in MCF-7 breast cancer cells.
We investigated the mechanism of retinoic acid receptor (RAR) beta2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous beta2 promoter (-1470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARbeta2. This was true even in the context of replicated chromatin, suggesting a cis-acting rather than a trans-acting defect. Cytosine methylation, a cis-acting DNA modification, has been implicated in RARbeta2 silencing in cancer cells. Upon bisulfite genomic sequencing, we found that 3 CpG sites in the beta2 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 differed in RARbeta2 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 RARbeta2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A beta2 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 RARbeta2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important. Oncogene (2000) 19, 4066 - 4070.
Arapshian
A
A
Department of Medicine, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029-6574, USA.
Kuppumbatti
Y S
YS
Mira-y-Lopez
R
R
D000230:Adenocarcinoma / Q000473:pathology*; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D001943:Breast Neoplasms / Q000473:pathology*; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000502:physiology*; D017422:Sequence Analysis, DNA; D015533:Transcriptional Activation; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D009363:Neoplasm Proteins; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D014212:Tretinoin; D001374:Azacitidine