The Telomere Binding Protein TRF2 Induces Chromatin Compaction

Abstract

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

Nucleosome organization and chromatin dynamics in telomeres

“…When nucleosome arrays were assembled on telomeric repeats in the presence of TRF2 in vitro, the distance between nucleosomes increased and became less regular (52). In vitro analytical agarose gel electrophoresis and AFM studies revealed that TRF2, as well as the TRF2 DNA binding domain, promotes the folding of nucleosomal arrays into more compact structures (53,54), thus generating results that seem to be inconsistent with the in vivo studies mentioned earlier.…”

“…The effects of TRF2 on nucleosome organization have also been extensively studied in vivo (50)(51)(52) and in vitro (52)(53)(54). A recent chromatin immunoprecipitation and MNase mapping assay demonstrated that TRF2 overexpression reduces the nucleosome density and increases the internucleosomal distance at telomeres in vivo (52).…”

Functional characterization of the TERRA transcriptome at damaged telomeres

“…TRF2 and TRF1 both consist of four domains: a C-terminal MYB domain required for binding to telomeres, a flexible hinge domain involved in protein–protein interactions, a TRFH domain required for homodimerization 40 and chromatin compaction 41,42 , and a divergent amino (N)-terminal region rich in basic amino acids in TRF2 and acidic residues in TRF1. To identify the molecular properties of TRF2 involved in regulating TERRA transcription, we swapped, inspired by a previous study 41 , several domains between TRF2 and TRF1 (refs 43,44) in cDNA constructs (Fig.…”

“…To identify the molecular properties of TRF2 involved in regulating TERRA transcription, we swapped, inspired by a previous study 41 , several domains between TRF2 and TRF1 (refs 43,44) in cDNA constructs (Fig. 5a) and expressed them in HeLa cells (Fig.…”

“…Overall, these data suggest that TRF2 represses TERRA transcription through its TRFH domain. This domain may elicit this function through its capacity in structural compaction of chromatin 41,42 , making telomeres less accessible to RNAPII. Interestingly, it has recently been shown that the TRFH domain of TRF2 is also required to prevent the activation of the ATM pathway and the initial steps in the telomeric DDR 44 .…”