Abstract
We investigated the mechanism of retinoic acid receptor (RAR) β2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous β2 promoter (−1470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARβ2. 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 RARβ2 silencing in cancer cells. Upon bisulfite genomic sequencing, we found that 3 CpG sites in the β2 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 RARβ2 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 RARβ2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A β2 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 RARβ2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important.
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Acknowledgements
This work was supported by NCI grant R01 CA54273, The Samuel Waxman Cancer Research Foundation and The Jaffe Family Foundation. We thank the following investigators for reagent gifts: Joyce-Taylor Papadimitriou (MTSV1.7 cells), Reuben Lotan (−1470/+156 RARβ2-LUC construct) and Pierre Chambon (RAR expression vectors).
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Arapshian, A., Kuppumbatti, Y. & Mira-y-Lopez, R. 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. Oncogene 19, 4066–4070 (2000). https://doi.org/10.1038/sj.onc.1203734
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DOI: https://doi.org/10.1038/sj.onc.1203734
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