Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding

Abstract

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats – dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.