TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection

TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

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

Data on the kinetics of in vitro assembled chromatin

AbstractHere, we use LC–MS/MS and SWATH-MS to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos (DREX). This system allows easy manipulation of distinct aspects of chromatin assembly such as post-translational histone modifications, the levels of histone chaperones and the concentration of distinct DNA binding factors. In total, 480 proteins have been quantified as chromatin enriched factors and their binding kinetics have been monitored in the time course of 15min, 1h and 4h of chromatin assembly.The data accompanying the manuscript on this approach, Völker-Albert et al., 2016 “A quantitative proteomic analysis of in vitro assembled chromatin” [1], has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier submission number PRIDE: PXD002537 and PRIDE: PXD003445

Cell-cycle regulation of TRF2-mediated telomeric remodelling.

<p>(<b>A</b>) Scheme of the synchronization experiment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034386#s3" target="_blank">Materials and Methods</a> for details). (<b>B</b>) ChIP of C33A cells overexpressing TRF2^FL and control C33A cells using the indicated antibodies and hybridized with Telo and Alu probes. From the left: slot-blots in asynchronous control cells; at the G1/S boundary; 5 hours after release from thymidine block; 12 hours after release; cells maintained in thymidine block for 12 additional hours (12 h+Th). (<b>C</b>) Quantifications of the data in (<b>B</b>) expressed as Telo/Alu hybridization signals. Error bars are s.d. of three independent experiments. Asterisks, p<0.05 based on unpaired Student's t-test.</p

Nucleosome density at human telomeres depends on TRF2 expression.

<p>(<b>A</b>) ChIP of C33A cells overexpressing TRF2^FL or TRF2^ΔBΔM and control C33A cells using the indicated antibodies. Slot-blots were hybridized with a labelled Telo repeat probe and an Alu probe. (<b>B</b>) Quantification of the data in (<b>A</b>) expressed as probe/input hybridization signals. Error bars are s.d. of three independent experiments. Asterisks, p<0.05 based on unpaired Student's t-test.</p

Models for TRF2-induced remodeling of telomeric chromatin.

<p>See the text for details.</p

Altered nucleosome spacing at C33A telomeres.

<p>(<b>A</b>) Digestion of chromatin from C33A cells infected with an empty vector and C33A cells overexpressing TRF2^FL with increasing amounts of MNase. From the left: MNase digests separated on 1.5% agarose gel detected by hybridization with Telo probe; detection of telomeric nucleosomes after hybridization with Alu probe. (<b>B</b>) Ratio of the overall hybridization signal of the telomeric probe with respect to the Alu probe. Ratio values for control C33A cells have been normalized to 100. Error bars are s.d. of three independent experiments. Asterisks, p<0.05 based on unpaired Student's t-test.</p