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

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/EBP alpha transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBP alpha 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/EBP alpha and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBP alpha binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBP alpha. 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/EBP alpha mediated activation. In primary dermal fibroblasts, C/EBP alpha 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/EBP alpha that are needed to activate tissue-specific genes.