Overrunning cellular replicative capacities, through telomere maintenance or oncogene activation, alters epigenetic profiles

(eng) Telomeres are specialized structures that cap the ends of eukaryotic chromosomes and, together with the subtelomeres, constitute the heterochromatin of chromosomes ends. In most normal somatic cells, telomere length decreases with cell divisions and this progressive shortening eventually results in cell cycle arrest. In cancer cells, telomere loss is prevented by the activation of a telomere maintenance mechanism (TMM) dependent on either telomerase (TEL+ cells) or alternative mechanism(s) (ALT cells). Although it has been established that ALT cells rely on homologous recombination between telomeric sequences to extend telomeres, mechanisms of ALT activation still require elucidation. Our initial goal was to identify genes involved in ALT pathway(s) and/or genes directly or indirectly regulated by telomerase. To this end, we compared gene expression profiles of TEL+ and ALT SV40-immortalized fibroblasts by representational difference analysis (RDA). This approach did not allow us to identify genes implicated in ALT, suggesting that ALT activation may reside at another level, like post-translational or epigenetic modifications. Recent studies from mice suggested that subtelomeric DNA hypomethylation might contribute to the ALT process by facilitating telomeric sister chromatid exchanges (T-SCE). Hence, we wished to investigate the possible link between epigenetic modifications of chromosome ends and ALT process in human cells. We found that ALT/T-SCEhigh tumor cells display low methylation levels at D4Z4 and DNF92 subtelomeric DNA sequences. Surprisingly however, the same sequences retained high methylation level in ALT/T-SCEhigh SV40-immortalized fibroblasts, suggesting that subtelomeric DNA hypomethylation is not required for T-SCE in human ALT cells. We also found that hypomethylation of subtelomeric sequences in ALT tumor cells was correlated with genome-wide hypomethylation. To understand why genome-wide DNA hypomethylation is prevalent in ALT tumor cells and, in a broader perspective, to investigate the mechanisms of DNA demethylation during the tumorigenic process, we relied on an in vitro model of tumorigenesis. Following replicative senescence or oncogene activation, we detected a two-fold reduction in meCpG content of Sat2 DNA, a pericentromeric region of chromosome 1 displaying strong hypomethylation in tumors and reported to be associated with chromosomal translocations. Our preliminary FISH experiments suggested that hypomethylated Sat2 may indeed be associated with chromosomal translocations in RasV12 oncogeneexpressing fibroblasts. In particular, we detected translocations of Sat2 to chromosome ends, a feature also observed in ALT tumor cells, raising the interesting hypothesis that Sat2 sequences may contribute to ALT telomere maintenance. We propose that this extensive Sat2 DNA demethylation may coincide with an overrunning of replication capacities under low DNMT1 level conditions and/or with delocalization of DNMT1 to DNA damage foci induced by excessive replication. In both cases, DNMT1 maintenance activity at genomewide level would be impaired. This hypothesis remains to be further tested and transposed to the understanding of ALT activation during tumorigenesis.