Structural study of the human telomeric chromatin

Telomeres are the special nucleoprotein structures that protect chromosome ends from both recombination and degradation. In most organisms, telomeric DNA consists of short sequences repeated in tandem ending in single stranded G-rich-overhangs. About 80% of telomeric DNA is organized in tightly packed nucleosomes separated by 10-20 bp of linker DNA. Several specific proteins contribute to telomeric structure. The protein hPOT1 binds to single stranded G-rich-overhangs, whereas double stranded telomeric DNA is specifically recognized by hTRF1 and hTRF2. hTRF1, hTRF2, and hPOT1 associate with hRap1, Tin2 and TPP1 to form the complex named shelterin, essential for telomere protection. In addition, several other factors, many of which are involved in DNA repair and recombination, are also recruited to telomeres. At the moment, a satisfactory description of telomere organization is still lacking. The emerging view is that there is an interconversion among different structures along with the cell cycle and development. While the relevance of telomeric proteins has been widely investigated, the role played by nucleosomes and histone post-translational modifications in the protection of chromosome ends represents a research field almost completely unexplored. This thesis addresses a relevant question in telomere biology, that is whether the human telomeric proteins TRF1 and TRF2 interplay with telomeric nucleosomes and whether they affect the organization and the epigenetic status of telomeric chromatin. In particular, the relation of TRF2, the protein essential for telomere protection, with nucleosomes has been studied by overexpressing TRF2, by transient expression of wild-type and dominant-negative genes. Cancer cells (C33A) and immortalized fibroblasts (HT1080) have been transfected with plasmids encoding hTRF2, hTRF2ΔBΔC. The effect of altered concentrations of TRF2 has been analyzed by chromatin immunoprecipitation assay (ChIP). We found that the density of nucleosomes at human telomere depends on TRF2 expression. Moreover, in order to establish if the association of telomeric proteins and histones at telomeres are cell-cycle regulated we performed ChIP experiments after cell synchronization. Cell synchronization has been checked by flow cytometry and ChIP assay has been performed at various times after cell synchronization. We observed that the remodeling effect of TRF2 occurs outside replication. In parallel with ChIP assay, we also evaluated the organization of telomeric chromatin by MNase mapping. Nuclei of control C33A cells and TRF2-overexpressing cells were isolated and then digested with increasing amounts of micrococcal nuclease (MNase). We found that telomeric chromatin shows a higher sensitivity to MNase in TRF2-overexpressing cells. In addition, using Drosophila extracts to assemble nucleosomal arrays in vitro, we showed that the spacing between telomeric nucleosomes is increased by the presence of TRF2. Finally, the epigenetic status of C33A telomeres as a function of TRF2 expression has been characterized by ChIP, using specific antibodies for heterochromatic marks (H3K9me3, H4K20me3), euchromatic marks (H3K4me2), histone variants (H2AX). We observed a decreased density of epigenetic marks in TRF2-overexpressing cells and an enrichment of H2AX at telomeres. All together, these results indicate an impact of TRF2 on nucleosomal organization at mammalian telomeres, further highlighting the importance of TRF2 in telomere protection.