Regulation of telomeric repeat-containing RNA expression by telomere length and heterochromatin in human cells

Telomeres are ribonucleoproteic structures that protect the ends of chromosomes from degradation and fusions. In normal somatic cells, telomere length decreases with each cell division, progressively driving cells to senescence. However, cancer cells acquire unlimited replication potential through reactivation of a telomere maintenance mechanism, which can be either telomerase, an enzyme that adds telomeric repeats at the ends of chromosomes, or an alternative mechanism (ALT) based on homologous recombinations between telomeric DNA sequences. Although telomeres have been described as heterochromatic structures, it was recently found that they are transcribed into long noncoding RNAs, dubbed TERRA (TElomeric Repeat-containing RNAs), in a large variety of species. Transcription of human telomeres is initiated at subtelomeric promoters and proceeds all along the telomeric tract. In yeast and drosophila, telomere length is able to modulate the expression level of nearby genes, a phenomenon called telomere position effect and attributable to telomeric heterochromatin spreading. Thus, we wondered if TERRA levels could be regulated by a similar mechanism in human cells. Using pairs of syngenic cell lines displaying distinct telomere lengths, we showed that telomere elongation reduces TERRA expression and that this is mediated by increased density of H3K9me3 and HP1α repressive marks at elongated telomeres. Interestingly, increased H3K9me3 density was restricted to telomeric repeats and did not spread upstream, likely due to the binding of CTCF insulating factor near telomeres. Hence, these data suggested the existence of strong barriers against heterochromatin spreading at human telomeres. In the second part of the PhD thesis work, we focused on the connection between telomeric chromatin properties and the ALT mechanism. Previous studies in mouse suggested that a more open telomeric chromatin may favour ALT by promoting homologous recombinations. To gather more evidences in favour of this hypothesis, we compared telomeric heterochromatin in human fibroblast cell lines with similar genetic background and relying either on ALT or on telomerase and, to increase the number of comparable cell lines, we created cellular hybrids. Agreeing with the hypothesis, we found that ALT telomeres display reduced nucleosome density accompanied by decreased H3K9me3 levels. This more open chromatin of ALT telomeres was associated with increased telomere transcription. Altogether, our results bring new insight on human telomeric heterochromatin and on telomere transcription regulation.