Structural and epigenetic dysfunctions of telomeric chromatin in cancer cells

Telomeres are nucleoprotein structures that protect chromosome termini in eukaryotes. Functional telomeres need the establishment of a protective chromatin structure based on the interplay between the specific complex named shelterin and a tight nucleosomal organization. In somatic cells, progressive telomere reduction brings to the destabilization of the telomere capping structure and to the activation of a DNA damage response (DDR) signalling. Then, cells enter into replicative senescence, which constitute a protective barrier against unlimited proliferation. A crucial step in cancer development is the acquirement of a telomere maintenance mechanism that gives the neoplastic cell unlimited replicative potential, one of the main hallmarks of cancer. Despite the crucial role that telomeres play in cancer development, little is known about the epigenetic alterations of telomeric chromatin that affect telomere protection and are associated with tumorigenesis. Here, we explore two different aspects of this wide issue that involves telomeric chromatin and its dysfunctions in cancer development. The first aspect involves the interaction between SIRT6 and TRF2 in heterochromatin stability and cancer. Based on previous evidences reporting the telomeric protein TRF2 as a novel substrate of SIRT6, and that an inverse correlation between SIRT6 and TRF2 protein expression levels was present in a cohort of CRC patients (Rizzo et al., 2017), we decided to investigate the dynamic and the effects of this interaction. We performed several in vitro binding assays which showed that SIRT6 has the capability of stabilize the interaction of TRF2 with the telomeric nucleosome in vitro. Additionally, chromatin extractions after silencing SIRT6 showed that this stabilization is recapitulated also in HCT cancer cell line. Then, we performed chromatin immunoprecipitation of TRF2 coupled with sequencing (ChIP-seq) in HCT-116 upon SIRT6 silencing. We found that TRF2 delocalize both from telomeres and from pericentromeres. Furthermore, we also found TRF2 bound on several gene promoters involved in cancer development. Collectively these data unveil new interesting elements about the interplay of SIRT6 and TRF2 with heterochromatin in colon cancer cells, which could be relevant for a deeper understanding of the mechanisms of tumour onset and potentially brings to the development of new anti-cancer therapies. The second aspect regards the role played by the histone variant H3.3 at telomeres. Among the histone variants, H3.3 is the most common non-centromeric variant of histone H3. Despite being enriched in transcriptionally active regions, it has also been found at pericentromeres and telomeres. Some dominant mutations in the H3F3A/B genes have been described in several paediatric cancers. Interestingly, a positive correlation with mutations in chaperones responsible for H3.3 deposition at telomeres and pericentromeres, and an association with ALT phenotype was also reported. In light of these evidences, unravelling H3.3 function(s) at telomeres could be pivotal for the purposes of both basic and applied research. Telomeric sequences, together with the limitations of a classical methodologies used for studies on telomeres, complicated the analyses of the telomeric nucleosome spacing, and in general of the repeated long sequences. Therefore, we directed the second part of this project towards the development of a novel approach to precisely map H3.3-containing nucleosomes at human telomeres. We generated transgenic cell lines expressing a H3.3Q85C mutated gene. This particular mutation made chromatin sensitive to phenantroline, which cuts the DNA generating short DNA fragments after the addition of copper and hydrogen peroxide which catalyse the formation of short-lived hydroxyl radicals. Then, to overcome the difficulties generated by the uniformly repeated telomeric sequence, we developed a strategy to map nucleosome positions using subtelomeric sequences as starting point. To characterize the fragments of different length emerging from MNase or chemical cleavage, we took advantage of Oxford Nanopores, a technique that can handle very long fragments and does not require any amplification step sequencing. We performed pilot sequencing runs of MNase digested DNA to test the feasibility of nanopores to map telomeric nucleosome positions and spacing. The analysis of the read lengths showed clearly that nucleosome spacing at telomeres is shorter than in the rest of chromatin, but the number of reads obtained is too small to allow nucleosome mapping of single telomeres. For this reason, we developed a protocol to enrich telomeric DNA, by using biotinylated telomeric probes and streptavidin magnetic beads to capture telomeric sequences. With the application of these tools, we aim – in the near future - to obtain a detailed map of telomeric (and genome wide) H3.3 containing nucleosomes and to develop a reliable method to study the H3.3 mutations found in ALT and paediatric cancers