David Cheishvili1, Steffan Christiansen1,2, Rebecca Stochinsky1, Anne-Sophie Pepin1, Daniel M Sapozhnikov1, Rudy Zhou1, Lauren Schmeltzer1, Sergey Dymov1, Moshe Szyf1,3,4. 1. Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada. 2. Department of Biomedicine, Aarhus University, Aarhus, Denmark. 3. Department of Pharmacology & Therapeutics, Sackler Program for Epigenetics & Developmental Psychobiology, McGill University Medical School, Montreal, Quebec, Canada. 4. Department of Pharmacology & Therapeutics, Canadian Institute for Advanced Research, Montreal, Quebec, Canada.
Abstract
AIM: DNA methylation downregulates transcription. However, a large number of genes, which are unmethylated in the promoter region, are inactive. We tested the hypothesis that these genes are regulated by DNA methylation of upstream regulators. METHODS: We inhibited DNMT1 with 5-aza-2'-deoxycytidine or depleted it with shRNA to map the transcription initiation positions controlled by DNMT1 using ChIPseq with RNApolIIser5 antibody. Ingenuity pathway analysis identified potential methylated upstream regulators. Their functional role in controlling unmethylated promoters was determined by CRISPR/Cas9 gene editing. RESULTS: We show that a large group of unmethylated promoters is regulated by DNMT1 through DNA methylation dependent silencing of upstream regulators such as transcription factor HNF4A. CONCLUSION: The landscape of genes regulated by DNA methylation is more wide-ranging than genes downregulated by methylation of their own cis-regulatory sequences; regulation of unmethylated promoters is dependent on the methylation state of upstream trans regulators.
AIM: DNA methylation downregulates transcription. However, a large number of genes, which are unmethylated in the promoter region, are inactive. We tested the hypothesis that these genes are regulated by DNA methylation of upstream regulators. METHODS: We inhibited DNMT1 with 5-aza-2'-deoxycytidine or depleted it with shRNA to map the transcription initiation positions controlled by DNMT1 using ChIPseq with RNApolIIser5 antibody. Ingenuity pathway analysis identified potential methylated upstream regulators. Their functional role in controlling unmethylated promoters was determined by CRISPR/Cas9 gene editing. RESULTS: We show that a large group of unmethylated promoters is regulated by DNMT1 through DNA methylation dependent silencing of upstream regulators such as transcription factor HNF4A. CONCLUSION: The landscape of genes regulated by DNA methylation is more wide-ranging than genes downregulated by methylation of their own cis-regulatory sequences; regulation of unmethylated promoters is dependent on the methylation state of upstream trans regulators.