Roles of mammalian Rap1 in TERT recruitment to chromatin, subtelomeric gene silencing and transcriptional regulation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE19011 | GEO | 2010/07/10
SECONDARY ACCESSION(S): PRJNA121075
REPOSITORIES: GEO
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Project description:Repressor-activator protein 1 (scRap1) is the major binding activity at Saccharomyces cerevisiae telomeres, with roles in telomere length regulation and establishment of subtelomeric silencing by recruiting the Sir proteins. scRap1 also acts as a transcription factor controlling the expression of ribosomal proteins and glycolytic enzymes. A homolog of scRap1 exist in mammals, Rap1 (also known as Terf2ip), however, its roles in telomere biology and transcriptional regulation are largely unknown. We have employed microarrays to obtain gene expression signatures related to Rap1-deleted mice when compared to the wild-type. Construction of the Rap1 targeting vector: The strategy for disrupting the Rap1 locus was designed to conditionallty delete exon 3 (E3) through a Cre-mediated excision. Deletion of E3 leads to the removal of coding sequences encoding for the RCT domain and the NLS sequence, as well as the whole 3’UTR region and poly-adenylation signal (Fig.1A,B). Deletion of E3 was expected to destabilize the Rap1 transcript and thereby to prevent translation of any truncated RAP1 protein. The targeting vector contains homology regions isogenic with the ES cell line used (129Sv/Pas). The short homology region (SA) harbors a 2.4 kb DNA fragment encompassing exon 2 (E2) and intron 2 (Fig. 1A). The long homology region (LA) is a 5.6 kb DNA fragment downstream the end of E3 (Fig. 1A). The central part contains E3 flanked by two loxP sites and a positive selection neomycin gene (PGK-Neo) flanked by two Frt sites (Fig. 1A). At the 3’-end of the LA a Diphteria Toxin (DTA) selection marker was cloned (Fig. 1A). The vector contains a unique NruI linearization site. The targeting vector was quality controlled by sequencing of the coding exons, the junctions between the homology arms and the selection cassettes, the selection cassettes and the junctions between the homology arms and the plasmid backbone. The analysis of the sequencing results showed no polymorphis between the C57BL/6 and 129Sv/Pas genetic background within the isolated Rap1 sequences. The sequence of the primers used for the PCR amplification of the different targeting vector parts and the details of the cloning steps are available upon request. The construction of the targeting vector was performed by genOway (www.genoway.com; Lyon, France). Generation of conditional Rap1 knockout mice 129Sv/Pas ES cells were transfected with 40 µg of linearized plasmid. Positive selection was started 48 hours after electroporation by addition of 200 µg/ML G418. Approximately 230 positive resistant clones were isolated and amplified. They were PCR screened for homologous recombination first at the 5’ end of the Rap1 locus. The 6 positive identified 5’ targeted ES cells were further investigated by PCR amplification over the 3’ long homology arm to amplify the region of the targeted locus containing the distal loxP site. The direct sequencing of the PCR products amplified from the 6 ES clones revealed that 3 of them contained the distal loxP site. The recombinant clones identified by PCR were further verified by Southern blot analysis of AflII and PciI restricted genomic DNA using a 5’-internal and a 3’-external probes, respectively (data not shown). The Southern blots confirmed the correct homologous integration over the 5’ and the 3’ homology arm and the absence of any additional randomly integrated copies of the targeting vector. Chimeric mice were generated by microinjection of three independently targeted ES clones into C57BL/6J host blastocyst. The resulting offspring showed a high level of chimerism as shown by coat color, and were mated to C57BL/6J mice to assess germ line transmission. The resulting heterozygous Rap1+/flox-Neo mice were then bred to transgenic mice expressing the Flpe recombinase (Rodriguez et al., 2000) to induce excision of the Neo marker. The Rap1+/flox heterozygous mice were then intercrossed to generate Rap1flox/flox, and Rap1+/flox mice. Homozygous Rap1flox/flox mice were crossed with transgenic mice expressing the Cre recombinase under the control of the keratine 5 promoter (Tarutani et al., 1997) (Fig. 5A). Heterozygous Rap1+/D K5-Cre were crossed either to Rap1flox/flox or Rap1+/flox to generate Rap1Δ/Δ K5-Cre. The removal of exon 3 by Cre-mediated recombination was confirmed by PCR analysis using primers F and R (Fig. 1A,C and Fig. 5A). Amplification of the wild type, flox and knockout alleles renders a 3.2 kb, 3.3 kb and 0.5 kb fragments, respectively. The breeding to the F1 generation and characterization of heterozygous Rap1+/flox-Neo F1 animals was performed by geneOway (www.genoway.com; Lyon, France). All mice were generated and maintained at the Spanish National Cancer Centre (CNIO) under specific pathogen-free conditions in accordance with the recommendation of the Federation of European Laboratory Animal Science Associations.
2010-07-10 | E-GEOD-19011 | biostudies-arrayexpress
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
Project description:The conserved Rap1 protein is part of the shelterin complex that plays critical roles in chromosome end protection and telomere length homeostasis. Previous studies addressed how fission yeast Rap1 contributes to telomere length maintenance, but the mechanism by which the protein inhibits end fusions has remained elusive. Here, we use a genetic screen in combination with high throughput sequencing to identify several amino acid positions in Rap1 that have a key role in end protection. Interestingly, mutations at these sites render cells susceptible to genome instability in a conditional manner with longer telomeres being prone to undergoing end fusions, while short telomeres are sufficiently protected. The protection of long telomeres requires their nuclear envelope attachment mediated by the Rap1-Bqt4 interaction. Our data demonstrates that longer telomeres pose an additional challenge for the maintenance of genome integrity and provides an explanation for a species-specific upper limit in telomere length.
2022-08-10 | GSE190759 | GEO
Project description:By performing chromatin immunoprecipitation coupled with ultra-high-throughput sequencing (ChIP-seq), we find that RAP1 binds to telomeres as well as to extra-telomeric sites through the (TTAGGG)2 consensus motif. Extra-telomeric RAP1 binding sites are particularly abundant at subtelomeric regions, and this is in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. Significantly, more than 70% of extratelomeric RAP1 binding sites are located in the vicinity of known genes and about 40% of the genes deregulated in Rap1-null cells contain binding sites for RAP1, suggesting a role of RAP1 in transcriptional control.
2010-07-10 | GSE20867 | GEO
Project description:Transcriptionally silent heterochromatin preferentially localizes at the nuclear periphery, but, despite this, certain budding yeast genes relocate to the nuclear periphery following gene activation, implicating the nuclear envelope in both transcriptional activation and silencing. It is unclear how these distinct chromatin domains are established, maintained and distinguished from one another at the nuclear envelope. Here we report that nuclear pore complexes (NPCs) facilitate the transition between chromatin states by providing a platform to which chromatin-remodeling and chromatin-modifying complexes bind. In particular, we show that the RSC chromatin-remodeling complex associates with NPCs and that the nucleoporin Nup170p nucleates heterochromatin formation at telomeres through recruitment of Sir4p. Deletion of NUP170 altered subtelomeric chromatin structure, reduced SIR complex binding at telomeres, impaired telomeric silencing and abated telomere tethering. These results support a model in which telomeric heterochromatin formation occurs through telomere-NPC interactions that both promote Sir4p binding at telomeres and permit chromatin-remodeling complexes to mediate the transition between chromatin states. Examination of genome-wide nucleosome positions in WT and nup170∆ cells via next-generation sequencing of mononucleosomal DNA.
2013-02-28 | E-GEOD-36792 | biostudies-arrayexpress
Project description:By performing chromatin immunoprecipitation coupled with ultra-high-throughput sequencing (ChIP-seq), we find that RAP1 binds to telomeres as well as to extra-telomeric sites through the (TTAGGG)2 consensus motif. Extra-telomeric RAP1 binding sites are particularly abundant at subtelomeric regions, and this is in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. Significantly, more than 70% of extratelomeric RAP1 binding sites are located in the vicinity of known genes and about 40% of the genes deregulated in Rap1-null cells contain binding sites for RAP1, suggesting a role of RAP1 in transcriptional control. Examination of RAP1 binding by CHIP-seq in two independent wild-type mefs using as negative control two independent RAP1-deleted mefs
2010-07-10 | E-GEOD-20867 | biostudies-arrayexpress
Project description:To determine whether telomere shortening in thyroid tumors delineates gene expression of subtelomeric regions, we performed RNA sequencing in a set of tumors (n = 15) and compare gene expression profiles of subtelomeric and non-subtelomeric genes between short telomere tumors and normal telomere tumors. We defined as subtelomeric genes, those located within the 5 megabase windows (Mb) adjacent to the telomeres.
2022-08-24 | GSE193623 | GEO
Project description:Short telomeres induce a DNA damage response (DDR) that evokes apoptosis or senescence in human cells. An extant question is the contribution of this DDR to the phenotypes observed in telomeropathies. One exemplar is RAP1, a telomere-associated protein that also controls transcription at extratelomeric regions. To distinguish these roles, we generated a knock-in mouse containing a Rap1 variant unable to bind telomeres but which did not result in eroded telomeres or a DDR. Rap1 variant MEFs exhibited decreased RAP1 expression and re-localization away from telomeres, with an increased cytosolic distribution akin to that observed in aging human fibroblasts. Rap1 knock-in mice were viable, but exhibited transcriptional alterations, proinflammatory cytokine/chemokine signaling, decreased lifespans, increased body weight/fasting blood glucose levels, increased spontaneous tumor incidence, and behavioral deficits. Taken together, our data indicate that some of the underlying mechanisms that drive telomeropathies may arise from phenotypes distinct from a telomere-induced DDR.
2022-11-04 | GSE193523 | GEO
Project description:Short telomeres induce a DNA damage response (DDR) that evokes apoptosis or senescence in human cells. An extant question is the contribution of this DDR to the phenotypes observed in telomeropathies. One exemplar is RAP1, a telomere-associated protein that also controls transcription at extratelomeric regions. To distinguish these roles, we generated a knock-in mouse containing a Rap1 variant unable to bind telomeres but which did not result in eroded telomeres or a DDR. Rap1 variant MEFs exhibited decreased RAP1 expression and re-localization away from telomeres, with an increased cytosolic distribution akin to that observed in aging human fibroblasts. Rap1 knock-in mice were viable, but exhibited transcriptional alterations, proinflammatory cytokine/chemokine signaling, decreased lifespans, increased body weight/fasting blood glucose levels, increased spontaneous tumor incidence, and behavioral deficits. Taken together, our data indicate that some of the underlying mechanisms that drive telomeropathies may arise from phenotypes distinct from a telomere-induced DDR.
2022-11-04 | GSE193520 | GEO
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