Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

Abstract

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.

ACKNOWLEDGMENTS

We are very grateful to Nathalie Bérubé for providing the ATRX shRNA constructs, Michael Dyer for the gift of the IF-GFP-ATRX plasmid, Valérie Gouilleux and François Boussin for the gift of cell lines, Jérome Bourgeais for help with the ChIP experiments, Nicole Ishac for help with the experiments on apoptosis, and Laetitia Corset for technical assistance. We also thank the Département Génomique PPF ASB facility at University François Rabelais of Tours for access to the Storm phosphorimager.

This work was supported by grants from the LIGUE Grand-Ouest contre le Cancer (Comités Charente, Eure-et-Loir, Ille-et-Vilaine, Indre-et-Loire, Morbihan, Sarthe, Vendée, Vienne) and the Fondation de France to M.C. and by grants from the Fonds National de la Recherche Scientifique (Belgium) to H.E. and A.D.