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. 2000 Aug 17;19(35):4066-70. doi: 10.1038/sj.onc.1203734.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma / pathology*
  • Azacitidine / pharmacology
  • Base Sequence
  • Breast Neoplasms / pathology*
  • Clone Cells / drug effects
  • Clone Cells / metabolism
  • CpG Islands*
  • DNA Methylation*
  • Female
  • Gene Expression Regulation, Neoplastic*
  • Gene Silencing / physiology*
  • Genes, Reporter
  • Humans
  • Molecular Sequence Data
  • Neoplasm Proteins / physiology*
  • Promoter Regions, Genetic
  • Receptors, Retinoic Acid / physiology*
  • Sequence Analysis, DNA
  • Transcriptional Activation
  • Transfection
  • Tretinoin / pharmacology*
  • Tumor Cells, Cultured / drug effects
  • Tumor Cells, Cultured / metabolism

Substances

  • Neoplasm Proteins
  • Receptors, Retinoic Acid
  • retinoic acid receptor beta
  • Tretinoin
  • Azacitidine