TALEN gene knockouts reveal no requirement for the shelterin protein Rap1 in telomere protection and length regulation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE61900 | GEO | 2014/09/30
SECONDARY ACCESSION(S): PRJNA262641
REPOSITORIES: GEO
Similar Datasets
Project description:Gene expression of human knockouts of telomere-protein Rap1 were compared to their wildtype counterparts in mutliple cell lines (available from the ATCC repository). We discovered that Rap1, desite being mostly associated with the telomere, performed a conserved role in transcriptional regulation, as evidenced by a number of genes that were differentially regulated upon deletion of Rap1. Total RNA was obtained from multiple knockout and wildtype clones generated in three different human parental cell lines obtained from ATCC. Datasets of multiple wild type and knockout clones were compared within cell lines, and not across cell lines, as differences in origin of cell lines led to extremely different transcriptional profiles.
2014-09-30 | E-GEOD-61900 | biostudies-arrayexpress
Project description:The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3' telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.
| S-EPMC4254571 | biostudies-literature
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:RAP1 is well known as a telomere-binding protein; yet its functions in human stem cells remain unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by CRISPR/Cas9 and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 negatively regulated telomere length. In addition, RAP1 acted as a transcriptional regulator of RLEN by tuning the methylation status of its promoter region. Phenotypically, RAP1 deficiency resulted in enhanced self-renewal and delayed senescence in hMSCs rather than in hNSCs, suggesting a complex role of RAP1 in regulating lineage specific stem cell homeostasis. Altogether, these results indicate that RAP1 plays both telomeric and non-telomeric roles in regulating the homeostasis of human stem cells.
2018-11-20 | GSE122654 | 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: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:Telomere-Dependent and Telomere-Independent Roles of RAP1 in Regulating Human Stem Cell Homeostasis
| PRJNA505881 | ENA
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: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
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.
2023-10-21 | PXD024630 | Pride