Telomeres are the nucleoproteic structures that protect the ends of eukaryotic chromosomes from recombination and degradation. In humans, telomeres consist of several kbp of TTAGGG repeats ending in single-stranded 3’ ends about 150 nt long. Telomeres progressively shorten in replicating cells, as a consequence of end processing and of incomplete DNA replication at chromosome termini, till they reach a critical length that leads to cellular senescence. In germ line cells telomere loss is counteracted by the reverse transcriptase enzyme telomerase, which adds telomeric repeats to the 3’ ends of the chromosomes using as template its RNA moiety. Two hallmarks of cancer can be directly referred to telomeres. First, shortening and/or uncapping of telomeres is one of the main sources leading to the chromosome instability commonly found in cancer. Secondly, in tumors the limitless replicative potential is directly linked to the reactivation of telomerase, or to alternative mechanisms of telomere maintenance. At the moment, a satisfactory description of telomere organization is still lacking. Double-stranded TTAGGG repeats are organized in tightly spaced nucleosomes and are bound by the specific proteins TRF1 and TRF2, essential for chromosome protection. An unresolved question in the organization of mammalian telomeres is whether specific telomeric proteins compete with the histone octamer for binding to telomeric sequences or whether they cooperate to form a telomeric protective structure. By means of in vitro model systems we previously demonstrated that TRF1 is able to recognize nucleosomal binding sites and to alter nucleosome structure. We show that, unlike TRF1, TRF2 is unable to bind to nucleosomal binding sites, suggesting that TRF2 and the histone octamer compete for binding to telomeres. To investigate how TRF2 interplays with telomeric nucleosomes, we overexpressed TRF2 or a dominant negative mutant in C33A cells by transient lentiviral expression. By means of ChIP analysis and MNase digestion assay we found that the density of nucleosomes at telomeres is inversely correlated with TRF2 dosage. These results are in agreement with in vitro assembly of telomeric nucleosomal arrays by using Drosophila embryonic extracts. Remarkably, when added to the Drosophila extracts TRF2 increases nucleosomal repeat length from 155±5 bp to about 195±5 bp, indicating that TRF2 may preferentially position between nucleosomes. The implication of these findings for telomere stability will be discussed.
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|Titolo:||TRF2 negatively regulates nucleosome density and organization at human telomeres|
|Data di pubblicazione:||2009|
|Appartiene alla tipologia:||04a Atto di comunicazione a congresso|