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ZNF524 directly binds telomeres and supports their integrity via TRF2/RAP1

ABSTRACT: Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex consists of six proteins (TRF1, TRF2, RAP1, POT1, TPP1 and TIN2) and blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. While shelterin does not work autonomously, additional direct telomere binding proteins have been described to function in a supplementary role. We here describe ZNF524, a zinc finger protein that directly binds to telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting the other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, we identified ZNF524 as a direct telomere binding protein and propose that ZNF524 is involved in the maintenance of telomere integrity by promoting TRF2/RAP1 subcomplex binding.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Bone Tissue

DISEASE(S): Osteosarcoma

SUBMITTER: Dennis Kappei

LAB HEAD: Dennis Kappei

PROVIDER: PXD024630 | Pride | 2023-10-21

REPOSITORIES: Pride

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ZNF524 directly binds telomeres and supports their integrity via TRF2/RAP1

Project description:Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex consists of six proteins (TRF1, TRF2, RAP1, POT1, TPP1 and TIN2) and blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. While shelterin does not work autonomously, additional direct telomere binding proteins have been described to function in a supplementary role. We here describe ZNF524, a zinc finger protein that directly binds to telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting the other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, we identified ZNF524 as a direct telomere binding protein and propose that ZNF524 is involved in the maintenance of telomere integrity by promoting TRF2/RAP1 subcomplex binding.

2023-10-22 | PXD031416 | Pride

ZNF524 directly interacts with telomeric DNA and supports telomere integrity

Project description:Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

2023-10-21 | PXD043084 | Pride

ZNF524 directly binds telomeres and supports their integrity via TRF2/RAP1

Project description:Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres in vivo, as shown among other approaches by ChIP-seq analysis, and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting the other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

2023-10-23 | GSE196661 | GEO

TEBP-1 and TEBP-2 are double-stranded telomere binders required for fertility

Project description:Telomeres are the ends of linear chromosomes and consist of repetitive double- and single-stranded DNA sequences. Telomeres are bound by dedicated protein complexes, such as shelterin in mammals. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp 2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp 1;tebp-2 double mutant animals are synthetic sterile, with germlines showing signs of severe mitotic and meiotic arrest. These results define the first telomere-binding complex of C. elegans, including TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility.

2021-03-09 | PXD019241 | Pride

ZBTB10 binds the telomeric variant repeat TTGGGG and interacts with TRF2

Project description:Telomeres are the ends of linear chromosomes and together with the shelterin complex present an essential feature for genome integrity. Vertebrate telomeres usually consist of hexameric TTAGGG repeats, however, in cells that use the alternative lengthening of telomeres (ALT) mechanism, variant repeat sequences are interspersed throughout telomeres. Previously, it was shown that NR2C/F transcription factors bind to TCAGGG variant repeats and contribute to telomere maintenance in ALT cells. While specific binders to other variant repeat sequences have been lacking to date, we here identify ZBTB10 as the first TTGGGG-binding protein and demonstrate direct binding via the two zinc fingers with affinity in the nanomolar range. Concomitantly, ZBTB10 co-localizes with a subset of telomeres in ALT-positive U2OS cells and interacts with TRF2/RAP1 via the N-terminal region of TRF2. Our data establishes ZBTB10 as a novel variant repeat-specific binding protein at ALT telomeres.

2018-12-17 | PXD010672 | Pride

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA repair at damaged telomeres

Project description:Protein phosphatase magnesium-dependent 1 delta (PPM1D) is involved in termination of the cell cycle checkpoint by counteracting activity of the tumour suppressor protein p53. By dephosphorylating various proteins at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified interaction between PPM1D and the components of the shelterin complex that protects telomeric DNA. Interaction between PPM1D and TRF2 was confirmed by immunoprecipitation and proximity ligation assay. Confocal microscopy revealed colocalisation of the endogenous PPM1D with TRF2 at telomeres. Further, we found that TRF2 was phosphorylated by ATR at S410 after induction of DNA damage at telomeres and this modification was increased in cells lacking PPM1D or after PPM1D inhibition. Phosphorylation of TRF2 stimulated its interaction with TIN2 at telomeres and in vitro. Conversely, overexpression of PPM1D impaired localisation of TIN2 at telomeres. Finally, inhibition of PPM1D impaired recruitment of 53BP1 and RAD51 to DNA double strand breaks at telomeres. Expression of TRF2-S410A mutant fully rescued the recruitment of 53BP1 to telomeric breaks. These results suggest that TRF2 phosphorylation promotes association of TIN2 with the shelterin complex and regulates DNA repair at telomeres.

2023-03-11 | PXD035268 | Pride

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA repair at damaged telomeres.

Project description:Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D colocalized with TRF2 at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

2023-03-11 | PXD035273 | Pride

A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere protection

Project description:RAP1 is one of the components of mammalian shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere protection has remained elusive. RAP1 binds along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres and its deficiency causes a premature aging in mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact in mouse survival, mice lacking both RAP1 and telomerase show a progressive decreased survival with increasing mouse generation as compared to telomerase single mutants. Telomere shortening was more pronounced in Rap1-/- Terc-/- than in Terc-/- counterparts, leading to an earlier onset of DNA damage and its consequent DNA damage response as well as accelerated degenerative pathologies in the intestines. In its turn, telomerase deficiency abolishes RAP1-mediated obesity and liver pathologies. Mouse embryonic fibroblasts with shorten telomeres present less amount of telomere-bound RAP1 but in contrast show higher numbers of RAP1 bound extratelomeric sites genomewide. Absence of RAP1 leads to deregulation of several metabolic pathways, and these changes were more pronounce in cells with short telomeres suggesting that RAP1 release from telomere foci could constitute a coordinated genomic response to telomere shortening. Our findings also demonstrate that although RAP1 is not a key factor in telomere capping under normal conditios, under stress situation such as critical telomere shortening RAP1 exerts an important function for telomere protection and justify its evolutionary conservation as a shelterin component in mammalian cells.

2016-11-16 | GSE79996 | GEO

Target enrichment followed by high throughput sequencing of telomeric DDRNAs in telomere-deprotected mouse cells

Project description:Telomeres are the nucleoprotein complexes located at the tips of eukaryotic chromosomes, composed of repetitive DNA (TTAGGG in vertebrates), and coated by a set of proteins collectively known as the shelterin complex. Dysfunctional telomeres resemble DSBs and they have been observed during ageing and cancer and a number of pathological conditions. Apart from telomeric repeat-containing RNA (TERRA), a non-coding UUAGGG-rich transcript starting from promoters located in the subtelomeric region, in mammals no other transcripts at telomeres have been characterized so far. Here we show that in mammals telomere dysfunction (analysed using TRF2+/- and TRF2-/- mouse embryonic fibroblasts) induces the transcription of telomeric DDRNAs (tDDRNAs) from both DNA strands. To characterize the length and sequence of tDDRNAs, we devised and employed an innovative method for target enrichment of RNA, based on in solution capture of low abundance small RNA species followed by next generation sequencing, developed in our laboratories. By this approach, we observed that telomere deprotection induced the accumulation of small RNA species generated from the transcription of both telomere strands and including the expected DDRNA size range products. Importantly, 20-23 nucleotide RNAs displayed a base bias at both 5’ and 3’ ends significantly different from the telomeric locus suggesting a regulated processing.

2017-05-10 | GSE86964 | GEO

A cell cycle switch in TRF2 controls t-loop dynamics and DNA secondary structure resolution across the genome

Project description:Telomere end-protection by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3’ single-stranded telomere end can assemble into a lasso-like t-loop configuration, which has been proposed to safeguard chromosome ends from being recognized as DNA double strand breaks. Mechanisms must also exist to transiently disassemble t-loops to allow faithful telomere replication and to permit telomerase access to the 3’-end to solve the end replication problem. However, the regulation and physiological importance of t-loops in end-protection remains uncertain. Here, we identify a CDK phosphorylation site in the shelterin subunit, TRF2 (Ser365), whose dephosphorylation in S-phase by the PP6C/R3 phosphatase provides a narrow window during which the helicase RTEL1 is able to transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 on Ser365 outside of S-phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate ATM activation, but also counteracts replication conflicts at DNA secondary structures arising within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.

2019-11-25 | PXD014843 | Pride

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