20 results found
Innes A, Sun B, Wagner V, et al., 2021, XPO7 is a tumor suppressor regulating p21CIP1-dependent senescence, Genes and Development, Vol: 35, Pages: 379-391, ISSN: 0890-9369
Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin dependent kinase inhibitor p21CIP1 during OIS. Deletion of XPO7 correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21CIP1 expression to control senescence and tumorigenesis.
Vilar R, Lewis BW, Bisballe N, et al., 2021, Assessing the key photophysical properties of triangulenium dyes for DNA binding by alteration of the fluorescent core, Chemistry: A European Journal, Vol: 27, Pages: 2523-2536, ISSN: 0947-6539
Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, we expand the library of available triangulenium probes to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.
Vilar Compte R, Summers P, Lewis B, et al., 2021, Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy, Nature Communications, Vol: 12, ISSN: 2041-1723
Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.
Herrera-Moyano E, Porreca RM, Ranjha L, et al., 2020, The human SKI complex prevents DNA-RNA hybrid-associated telomere instability
<jats:title>Abstract</jats:title><jats:p>Super killer (SKI) complex is a well-known cytoplasmic 3′ to 5′ mRNA decay complex that functions with the exosome to degrade excessive and aberrant mRNAs. Recently, SKIV2L, the 3′ to 5′ RNA helicase of the human SKI (hSKI) complex has been implicated in the degradation of nuclear non-coding RNAs escaping to the cytoplasm. Here, we show that hSKI is present in the nucleus, on chromatin and in particular at telomeres during the G2 cell cycle phase. In cells, SKIV2L prevents telomeric loss, and DNA damage response activation, and its absence leads to DNA-RNA hybrid-mediated telomere fragility. Moreover, we demonstrate that purified hSKI complex preferentially unwinds telomeric DNA-RNA hybrids <jats:italic>in vitro</jats:italic>. Taken together, our results provide a nuclear function of the hSKI complex in overcoming replication stress caused by aberrant processing of telomeric DNA-RNA hybrids and thus maintaining telomere stability.</jats:p>
Summers PA, Lewis BW, Gonzalez-Garcia J, et al., 2020, Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy
<jats:title>ABSTRACT</jats:title><jats:p>Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form <jats:italic>in vivo</jats:italic> and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (<jats:bold>DAOTA-M2</jats:bold>) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that <jats:bold>DAOTA-M2</jats:bold> can be used to study G4 stability in live cells. Reduction of <jats:italic>FancJ</jats:italic> and <jats:italic>RTEL1</jats:italic> expression in mammalian cells increases the <jats:bold>DAOTA-M2</jats:bold> lifetime and therefore suggests an increased number of G4 in these cells, implying that <jats:italic>FancJ</jats:italic> and <jats:italic>RTEL1</jats:italic> play a role in resolving G4 structures <jats:italic>in cellulo</jats:italic>.</jats:p>
Porreca RM, Herrera-Moyano E, Skourti E, et al., 2020, TRF1 averts chromatin remodelling, recombination and replication dependent-break induced replication at mouse telomeres, ELIFE, Vol: 9, ISSN: 2050-084X
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Porreca RM, Law PP, Herrera-Moyano E, et al., 2019, TRF1 prevents permissive DNA damage response, recombination and Break Induced Replication at telomeres
<jats:title>Abstract</jats:title><jats:p>Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that telomeres lacking TRF1 undergo protein composition reorganisation associated with a DNA damage response and chromatin remodelers. Surprisingly, TRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for TRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.</jats:p>
León-Ortiz AM, Panier S, Sarek G, et al., 2018, A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination., Mol Cell, Vol: 69, Pages: 292-305.e6
Erroneous DNA repair by heterologous recombination (Ht-REC) is a potential threat to genome stability, but evidence supporting its prevalence is lacking. Here we demonstrate that recombination is possible between heterologous sequences and that it is a source of chromosomal alterations in mitotic and meiotic cells. Mechanistically, we find that the RTEL1 and HIM-6/BLM helicases and the BRCA1 homolog BRC-1 counteract Ht-REC in Caenorhabditis elegans, whereas mismatch repair does not. Instead, MSH-2/6 drives Ht-REC events in rtel-1 and brc-1 mutants and excessive crossovers in rtel-1 mutant meioses. Loss of vertebrate Rtel1 also causes a variety of unusually large and complex structural variations, including chromothripsis, breakage-fusion-bridge events, and tandem duplications with distant intra-chromosomal insertions, whose structure are consistent with a role for RTEL1 in preventing Ht-REC during break-induced replication. Our data establish Ht-REC as an unappreciated source of genome instability that underpins a novel class of complex genome rearrangements that likely arise during replication stress.
Speckmann C, Sahoo SS, Rizzi M, et al., 2017, Corrigendum: Clinical and Molecular Heterogeneity of RTEL1 Deficiency., Front Immunol, Vol: 8, ISSN: 1664-3224
[This corrects the article on p. 449 in vol. 8, PMID: 28507545.].
Speckmann C, Sahoo SS, Rizzi M, et al., 2017, Clinical and Molecular Heterogeneity of RTEL1 Deficiency., Front Immunol, Vol: 8, ISSN: 1664-3224
Typical features of dyskeratosis congenita (DC) resulting from excessive telomere shortening include bone marrow failure (BMF), mucosal fragility, and pulmonary or liver fibrosis. In more severe cases, immune deficiency and recurring infections can add to disease severity. RTEL1 deficiency has recently been described as a major genetic etiology, but the molecular basis and clinical consequences of RTEL1-associated DC are incompletely characterized. We report our observations in a cohort of six patients: five with novel biallelic RTEL1 mutations p.Trp456Cys, p.Ile425Thr, p.Cys1244ProfsX17, p.Pro884_Gln885ins53X13, and one with novel heterozygous mutation p.Val796AlafsX4. The most unifying features were hypocellular BMF in 6/6 and B-/NK-cell lymphopenia in 5/6 patients. In addition, three patients with homozygous mutations p.Trp456Cys or p.Ile425Thr also suffered from immunodeficiency, cerebellar hypoplasia, and enteropathy, consistent with Hoyeraal-Hreidarsson syndrome. Chromosomal breakage resembling a homologous recombination defect was detected in patient-derived fibroblasts but not in hematopoietic compartment. Notably, in both cellular compartments, differential expression of 1243aa and 1219/1300aa RTEL1 isoforms was observed. In fibroblasts, response to ionizing irradiation and non-homologous end joining were not impaired. Telomeric circles did not accumulate in patient-derived primary cells and lymphoblastoid cell lines, implying alternative pathomechanisms for telomeric loss. Overall, RTEL1-deficient cells exhibited a phenotype of replicative exhaustion, spontaneous apoptosis and senescence. Specifically, CD34+ cells failed to expand in vitro, B-cell development was compromised, and T-cells did not proliferate in long-term culture. Finally, we report on the natural history and outcome of our patients. While two patients died from infections, hematopoietic stem cell transplantation (HSCT) resulted in sustained engraftment in two patients. Whether chemotherapy
Sarek G, Vannier J-B, Panier S, et al., 2016, TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding., Mol Cell, Vol: 61, Pages: 788-789
Sarek G, Vannier J-B, Panier S, et al., 2015, TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding., Mol Cell, Vol: 57, Pages: 622-635
The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase.
Vannier J-B, Sarek G, Boulton SJ, 2014, RTEL1: functions of a disease-associated helicase., Trends Cell Biol, Vol: 24, Pages: 416-425
DNA secondary structures that arise during DNA replication, repair, and recombination (3R) must be processed correctly to prevent genetic instability. Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles a variety of DNA secondary structures to facilitate 3R processes and to maintain telomere integrity. The past few years have witnessed the emergence of RTEL1 variants that confer increased susceptibility to high-grade glioma, astrocytomas, and glioblastomas. Mutations in RTEL1 have also been implicated in Hoyeraal-Hreidarsson syndrome, a severe form of the bone-marrow failure and cancer predisposition disorder, dyskeratosis congenita. We review these recent findings and highlight its crucial link between DNA secondary-structure metabolism and human disease.
Vannier J-B, Sandhu S, Petalcorin MIR, et al., 2013, RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication., Science, Vol: 342, Pages: 239-242
Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.
Choi HJC, Lin J-R, Vannier J-B, et al., 2013, NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies., Mol Cell, Vol: 51, Pages: 423-439
Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.
Ballew BJ, Joseph V, De S, et al., 2013, A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome., PLoS Genet, Vol: 9
Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.
Chapman JR, Barral P, Vannier J-B, et al., 2013, RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection., Mol Cell, Vol: 49, Pages: 858-871
The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5' end resection, but how this is achieved remains uncertain. Here we report that Rif1(-/-) mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.
Vannier J-B, Pavicic-Kaltenbrunner V, Petalcorin MIR, et al., 2012, RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity., Cell, Vol: 149, Pages: 795-806
T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere.
Vannier J-B, Depeiges A, White C, et al., 2009, ERCC1/XPF protects short telomeres from homologous recombination in Arabidopsis thaliana., PLoS Genet, Vol: 5
Many repair and recombination proteins play essential roles in telomere function and chromosome stability, notwithstanding the role of telomeres in "hiding" chromosome ends from DNA repair and recombination. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and is the subject of considerable interest in studies of recombination. In contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants mutated for either XPF (AtRAD1) or ERCC1 (AtERCC1) orthologs, which develop normally and show wild-type telomere length. However, in the absence of telomerase, mutation of either of these two genes induces a significantly earlier onset of chromosomal instability. This early appearance of telomere instability is not due to a general acceleration of telomeric repeat loss, but is associated with the presence of dicentric chromosome bridges and cytologically visible extrachromosomal DNA fragments in mitotic anaphase. Such extrachromosomal fragments are not observed in later-generation single-telomerase mutant plants presenting similar frequencies of anaphase bridges. Extensive FISH analyses show that these DNAs are broken chromosomes and correspond to two specific chromosome arms. Analysis of the Arabidopsis genome sequence identified two extensive blocks of degenerate telomeric repeats, which lie at the bases of these two arms. Our data thus indicate a protective role of ERCC1/XPF against 3' G-strand overhang invasion of interstitial telomeric repeats. The fact that the Atercc1 (and Atrad1) mutants dramatically potentiate levels of chromosome instability in Attert mutants, and the absence of such events in the presence of telomerase, have important implications for models of the roles of recombination at telomeres and is a striking illustration of the impact of genome structure on the outcomes of equivalent recombination processes
Vannier J-B, Depeiges A, White C, et al., 2006, Two roles for Rad50 in telomere maintenance., EMBO J, Vol: 25, Pages: 4577-4585, ISSN: 0261-4189
We describe two roles for the Rad50 protein in telomere maintenance and the protection of chromosome ends. Using fluorescence in situ hybridisation (FISH) and fibre-FISH analyses, we show that absence of AtRad50 protein leads to rapid shortening of a subpopulation of chromosome ends and subsequently chromosome-end fusions lacking telomeric repeats. In the absence of telomerase, mutation of atrad50 has a synergistic effect on the number of chromosome end fusions. Surprisingly, this 'deprotection' of the shortened telomeres does not result in increased exonucleolytic degradation, but in a higher proportion of anaphase bridges containing telomeric repeats in atrad50/tert plants, compared to tert mutant plants. Absence of AtRad50 thus facilitates the action of recombination on these shortened telomeres. We propose that this protective role of Rad50 protein on shortened telomeres results from its action in constraining recombination to sister chromatids and thus avoiding end-to-end interactions.
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