A transcript arising from the CEBPA locus binds DNMT1 and blocks methylation of the CEBPA gene.
Major finding: A transcript arising from the CEBPA locus binds DNMT1 and blocks methylation of the CEBPA gene.
Concept: DNMT1–RNA interactions occur genome wide and are correlated with decreased gene locus methylation.
Impact: This regulatory mechanism could potentially be exploited for gene-specific DNA demethylation.
Noncoding RNAs (ncRNA) have been implicated in the regulation of gene expression through interactions with chromatin remodelers, transcription factors, and microRNAs, but the relationship between ncRNAs and DNA methylation is unknown. Di Ruscio and colleagues evaluated transcription upstream and downstream of the methylation-sensitive gene CEBPA and found that expression of a long transcript encompassing the entire mRNA sequence, which they termed extracoding CEBPA (ecCEBPA), was correlated with CEBPA mRNA expression in all cell lines tested. ecCEBPA was not polyadenylated and was enriched in the nucleus, suggesting that it might have a noncoding function. ecCEBPA knockdown decreased CEBPA expression in association with increased CEBPA promoter methylation, whereas ectopic ecCEBPA expression increased CEBPA mRNA levels and selectively induced demethylation throughout the CEBPA gene locus. ecCEBPA expression also increased during S phase, when DNA methyltransferase 1 (DNMT1) activity peaks, suggesting that ecCEBPA might promote CEBPA expression by interfering with DNMT1-dependent methylation of the CEBPA locus. Consistent with this model, ecCEBPA physically interacted with DNMT1, transcription interfered with the ability of DNMT1 to methylate nearby hemimethylated DNA in vitro, and DNMT1 enzymatic activity was suppressed in the presence of ribonucleotides. In general, DNMT1 was found to have higher affinity for stem-loop structured RNAs than DNA, raising the possibility that DNMT1 may interact with RNAs other than ecCEBPA. Indeed, DNMT1 coimmunoprecipitated with gene loci-derived RNA transcripts throughout the genome, the vast majority of which were nonpolyadenylated like ecCEBPA. DNMT1–RNA interactions were negatively correlated with gene locus methylation, and DNMT1-bound RNAs were more likely to correspond to hypomethylated and expressed genes. Together, these results suggest that ncRNAs can selectively regulate DNA methylation, providing a potential framework for therapeutic manipulation of methylation at the single-gene level.