Abstract
Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.
Highlights
Telomeres are an important genome-stabilizing component of linear chromosomes
In order to observe the effect of TRF2 overexpression or depletion on histone density at telomeres, we performed chromatin immunoprecipitation (ChIP) assays 72 h post-infection
In contrast to the study in mouse cells [23], the overexpression of full-length TRF2 did not induce a change in telomere length at the time point selected for cross-linking in our ChIP experiments (Figure S2), showing that the influence of TRF2 on nucleosome organization does not result from telomere shortening
Summary
Telomeres are an important genome-stabilizing component of linear chromosomes. Variations in telomere status critically affect cell senescence, stem cell biology, and the development of many diseases, including cancer [1]. Telomeres are composed of short, tandemly repeated DNA sequences, ending in a G-rich single-stranded 39 tail. They are transcribed in a G-rich RNA named TERRA [4], which is thought to play relevant functions at telomeres. Telosomes are essential for the preservation of chromosome stability; they control telomere length, recombination, and DNA damage checkpoints. Telomere shortening negatively affects histone synthesis, probably via damage signal induction [21]. Overall, it appears that the particular nature of telomeric chromatin plays a role in telomere capping, telomere length regulation, and long-range gene expression
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