DNA methylation controls unmethylated transcription start sites in the genome in trans

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.

Keywords::

• 5-aza-2′-deoxycytidine
• CRISPR/Cas9
• DNA methylation
• DNMT1
• HNF4A
• Illumina 450k
• RNAPII
• RNAPII-Ser5
• upstream regulators

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2217/3dp-2022-0019

Authors’contribution

Conceptualization, C David and M Szyf; Methodology, C David and M Szyf; Software, R Zhou; Formal Analysis, C David; Investigation, C David, R Stochinsky, A-S Pepin, L Schmeltzer, S Christiansen and S Dymov; Writing – Original Draft, C David and M Szyf; Writing – Review & Editing, C David, M Szyf and S Christiansen.

Financial & competing interests disclosure

The work was supported by a grant from the Canadian Institute of Health Research to M Szyf (MOP-42411). D Cheishvili was supported by a postdoctoral fellowship from the Israel Cancer Research Fund. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

The work was supported by a grant from the Canadian Institute of Health Research to M Szyf (MOP-42411). D Cheishvili was supported by a postdoctoral fellowship from the Israel Cancer Research Fund. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.