CpG methylation of half-CRE sequences creates C/EBPalpha binding sites that activate some tissue-specific genes

Proc Natl Acad Sci U S A. 2010 Nov 23;107(47):20311-6. doi: 10.1073/pnas.1008688107. Epub 2010 Nov 8.

Abstract

DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene silencing. Genomic analyses have identified low CpG promoters that are both methylated and transcriptionally active, but the mechanism underlying the activation of these methylated promoters remains unclear. Here we show that CpG methylation of the CRE sequence (TGACGTCA) enhances the DNA binding of the C/EBPα transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBPα activates the CRE sequence only when it is methylated. The biological significance of this observation was seen in differentiating primary keratinocyte cultures from newborn mice where certain methylated promoters are both bound by C/EBPα and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBPα binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBPα. Transfection studies in cell cultures using methylated tissue-specific proximal promoters identified half-CRE (CGTCA) and half-C/EBP (CGCAA) sequences that need to be methylated for C/EBPα mediated activation. In primary dermal fibroblasts, C/EBPα activates a different set of methylated tissue-specific promoters upon differentiation into adipocytes. These data identify a new function for methyl CpGs: producing DNA binding sites at half-CRE and half-C/EBP sequences for C/EBPα that are needed to activate tissue-specific genes.

MeSH terms

  • Animals
  • Binding Sites / genetics
  • Blotting, Western
  • CCAAT-Enhancer-Binding Protein-alpha / metabolism*
  • Cell Differentiation / physiology
  • Chromatin Immunoprecipitation
  • Chromatography, High Pressure Liquid
  • CpG Islands / physiology*
  • Cytosine / metabolism
  • DNA Methylation*
  • DNA Primers / genetics
  • Electrophoretic Mobility Shift Assay
  • Enhancer Elements, Genetic / genetics*
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression Profiling
  • Gene Expression Regulation / genetics*
  • Immunohistochemistry
  • Immunoprecipitation
  • Keratinocytes / cytology
  • Mice
  • Mice, Transgenic
  • Oligonucleotide Array Sequence Analysis
  • Promoter Regions, Genetic / genetics*

Substances

  • CCAAT-Enhancer-Binding Protein-alpha
  • DNA Primers
  • Cytosine