Cancer-linked satellite 2 DNA hypomethylation does not regulate Sat2 non-coding RNA expression and is initiated by heat shock pathway activation

Abstract

Epigenetic dysfunctions, including DNA methylation alterations, play major roles in cancer initiation and progression. Although it is well established that gene promoter demethylation activates transcription, it remains unclear whether hypomethylation of repetitive heterochromatin similarly affects expression of non-coding RNA from these loci. Understanding how repetitive non-coding RNAs are transcriptionally regulated is important given that their established upregulation by the heat shock (HS) pathway suggests important functions in cellular response to stress, possibly by promoting heterochromatin reconstruction. We found that, although pericentromeric satellite 2 (Sat2) DNA hypomethylation is detected in a majority of cancer cell lines of various origins, DNA methylation loss does not constitutively hyperactivate Sat2 expression, and also does not facilitate Sat2 transcriptional induction upon heat shock. In melanoma tumor samples, our analysis revealed that the HS response, frequently upregulated in tumors, is probably the main determinant of Sat2 RNA expression in vivo. Next, we tested whether HS pathway hyperactivation may drive Sat2 demethylation. Strikingly, we found that both hyperthermia and hyperactivated RasV12 oncogene, another potent inducer of the HS pathway, reduced Sat2 methylation levels by up to 27% in human fibroblasts recovering from stress. Demethylation occurred locally on Sat2 repeats, resulting in a demethylation signature that was also detected in cancer cell lines with moderate genome-wide hypomethylation. We therefore propose that upregulation of Sat2 transcription in response to HS pathway hyperactivation during tumorigenesis may promote localized demethylation of the locus. This, in turn, may contribute to tumorigenesis, as demethylation of Sat2 was previously reported to favor chromosomal rearrangements.

Keywords: :

• DNA hypomethylation
• heat shock response
• non-coding RNA
• pericentromeric satellite DNA
• Ras oncogene

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by a Mandat d’Impulsion Scientifique from the Fonds National de la Recherche Scientifique (MIS/FNRS) and by the Special Funds for Research (FSR) from the Catholic University of Louvain. GT was supported by the Télévie/FNRS and the de Duve Institute. AD, NA, SL and AL were supported by the FNRS. We are grateful to M Swinarska, F Brasseur and E De Plaen (Ludwig Institute for Cancer Research, Brussels), E Sokal (UCL, Brussels), C Heirman (VUB, Brussels), F Fuks, F Journe and H Id Boukfer (ULB, Brussels), J Piette (ULg, Liège), J Lingner (ISREC, Lausanne, Switzerland), A Londoño-Vallejo (Institut Curie, Paris, France), C Hill (Cancer Research UK, London) and F d’Adda di Fagagna (IFOM foundation, Milan, Italy) for sharing reagents, plasmids, melanoma tumor RNA samples and cell lines. We thank all members of the GEPI group for helpful discussions and the de Duve Institute for constant support.