Intragenic DNA methylation and its role in transcription regulation

Abstract

DNA methylation is usually correlated with the suppression of transcription when located within promoter regions. However, the absence of methylation at promoters is not an indicator of active transcription, as many inactive genes have unmethylated promoters. The role of DNA methylation outside promoters is less understood, prompting our interest in analyzing the role of DNA methylation from a genome-wide perspective.;We paradoxically found that euchromatic regions of the genome, in contrast to heterochromatin, have higher levels of DNA methylation, especially in the bodies of actively transcribed genes. Therefore, it appears that a major driver of DNA methylation being targeted to euchromatin is transcriptional activity. We pursued these findings focusing on understanding the biological role of transcription-targeted cytosine methylation (TTCM), by employing two distinct approaches of demethylating the genome.;As a first approach, a Kdm5b mouse ES cell knockdown was used, as prior findings indicated the enrichment of H3K4me3 within gene bodies in Kdm5b knockdown cells, a histone mark negatively associated with DNA methylation. Interestingly, the Kdm5b knockdown experiments did not replicate the prior published findings, with no acquisition of new H3K4me3 peaks. As expected, there was no indication of DNA methylation changes in these cells either.;Secondly, we focused on the use of the DNMT1 inhibitor 5-aza-2'-deoxycytidine (5-azaCdR). Treatment of HEK 293T cells decreased DNA methylation levels in a dose-dependent manner, which persisted as an "imprinted" memory following drug withdrawal despite recovery of growth characteristics of the treated cells indistinguishable from control cells. We identified that 5-aza-CdR treatment is mainly targeting actively transcribed, euchromatic regions of the genome. Strikingly, despite the dramatic changes in DNA methylation, we observed limited changes in protein-coding and IncRNA transcriptional levels. However, we did find evidence supporting the activation of intragenic promoters. The few induced genes had interesting features, being enriched for cellular pathways altered in cancer, suggesting that 5-aza-CdR treatment could be predisposing non-tumorigenic cells to promote cancer formation.;These new insights establish a foundation for understanding the therapeutic and toxic effects of DNA demethylating drugs in clinical use, establishing the importance of understanding global DNA methylation patterns beyond promoter regions.