Transcriptional Activation of TINF2, a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

The expression of the telomere-associated protein TIN2 has been shown to be essential for early embryonic development in mice and for development of a variety of human malignancies. Recently, germ-line mutations in TINF2, which encodes for the TIN2 protein, have been identified in a number of patients with bone-marrow failure syndromes. Yet, the molecular mechanisms that regulate TINF2 expression are largely unknown. To elucidate the mechanisms involved in human TINF2 regulation, we cloned a 2.7 kb genomic DNA fragment containing the putative promoter region and, through deletion analysis, identified a 406 bp region that functions as a minimal promoter. This promoter proximal region is predicted to contain several putative Sp1 and NF-κB binding sites based on bioinformatic analysis. Direct binding of the Sp1 and NF-κB transcription factors to the TIN2 promoter sequence was demonstrated by electrophoretic mobility shift assay (EMSA) and/or chromatin immunoprecipitation (ChIP) assays. Transfection of a plasmid carrying the Sp1 transcription factor into Sp-deficient SL2 cells strongly activated TIN2 promoter-driven luciferase reporter expression. Similarly, the NF-κB molecules p50 and p65 were found to strongly activate luciferase expression in NF-κB knockout MEFs. Mutating the predicted transcription factor binding sites effectively reduced TIN2 promoter activity. Various known chemical inhibitors of Sp1 and NF-κB could also strongly inhibit TIN2 transcriptional activity. Collectively, our results demonstrate the important roles that Sp1 and NF-κB play in regulating the expression of the human telomere-binding protein TIN2, which can shed important light on its possible role in causing various forms of human diseases and cancers

The Telomere Binding Protein TRF2 Induces Chromatin Compaction

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

The Protein Network Surrounding the Human Telomere Repeat Binding Factors TRF1, TRF2, and POT1

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres

Telomeric localization of TRF2, a novel human telobox protein

International audienceno abstrac

Telomeric localization of TRF2, a novel human telobox protein

Natural chromosomal ends are stabilized by proteins that bind duplex telomeric DNA repeats. In human cells, the TTAGGG Repeat Factor 1 (TRF1) was identified by two independent studies, one screening for factors that bind duplex telomeric DNA and the other screening for proteins containing a particular Myb motif called the telobox, which is required for telomeric repeat recognition (Fig. 1a; refs 3-5). A second human open reading frame, orf2, contains a telobox sequence and encodes a polypeptide that specifically recognizes mammalian telomeric repeat DNA in vitro. We show that two proteins of 65 and 69 kD, expressed in HeLa cells, contain the orf2 telobox sequence. These proteins are collectively termed TRF2. Affinity-purified antibodies specific for anti-TRF2 label the telomeres of intact human chromosomes, strengthening the correlation between occurrence of telobox and telomere-repeat recognition in vivo

In vitro binding of nucleolin to double-stranded telomeric DNA

We have purified a 100 kDa protein, resolved in a Southwestern binding screen of total nuclear proteins from Hela cells with double-stranded human telomeric probe. A polyclonal antiserum raised by this protein recognizes purified nucleolin and stains nucleoli in growing Hela cells. We demonstrate that a truncated form of human nucleolin and a purified deletion derivative of mouse nucleolin bind in vitro to duplex telomeric DNA. This study suggests a new link between telomeres and the nucleolus

In vitro binding of nucleolin to double-stranded telomeric DNA.

International audienc