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.

Epigenetic Patterns in a Complete Human Genome

“…While both telomeric and non-telomeric TAR sequences are enriched for CTCF, the non-telomeric TAR sequences are more enriched for activating chromatin marks H3K27ac and H3K4me3, suggesting differences in TERRA activity (Fig 1f). Previous studies have focused on the roles and regulation of TAR in the subtelomere (27)(28)(29), however our results indicate biological functionality of TAR sequences outside the subtelomere as well. Overall, using only publically available short-read data with the new T2T-CHM13 reference and dynamic k-mer assisted mapping, we uncovered novel regulatory features in centromeric, pericentromeric, and subtelomeric repeats and display the usage of this technique for investigating disease related epigenetic dysregulation.…”

Expression and differential regulation of human TERRA at several chromosome ends

“…Impaired de novo methylation during embryogenesis leads to devastating effects in ICF1 patients who are characterized by short telomeres and high TERRA levels. Interestingly a recent study showed that loss of methylation in the subtelomeric CpG islands in HeLa led to an NRF1-dependent up-regulation of TERRA expression strengthening the link between CpG-island containing promoters and TERRA transcription (Le Berre et al 2019). We established that the length of telomeres in DNA methyltransferase deficient DKO cells is shorter at chromosome ends independently of their individual TERRA expression levels.…”

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells

“…1B-D). The intensity of TERRA-FISH foci, however, does not reflect the total levels of UUAGGG repeats in human cells, as shown by slot-blot hybridization of total RNA isolated from HCA2 normal fibroblasts and LB37 lung cancer cells which, because of subtelomeric promoter hypomethylation (Nergadze et al 2009;Diman et al 2016;Sagie et al 2017;Feretzaki et al 2019;Le Berre et al 2019), strongly up-regulate telomeric transcription (Fig. 1E,F).…”