Publications by authors named "Andres Canela"

24 Publications

  • Page 1 of 1

Neuronal enhancers are hotspots for DNA single-strand break repair.

Nature 2021 Mar 25. Epub 2021 Mar 25.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
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http://dx.doi.org/10.1038/s41586-021-03468-5DOI Listing
March 2021

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.
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http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells.

Methods Mol Biol 2021 ;2153:9-31

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA.

DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.
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http://dx.doi.org/10.1007/978-1-0716-0644-5_2DOI Listing
March 2021

Intra-Vκ Cluster Recombination Shapes the Ig Kappa Locus Repertoire.

Cell Rep 2019 12;29(13):4471-4481.e6

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.
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http://dx.doi.org/10.1016/j.celrep.2019.11.088DOI Listing
December 2019

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

Mol Cell 2020 01 22;77(1):26-38.e7. Epub 2019 Oct 22.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA153BP1 mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA153BP1 cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.
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http://dx.doi.org/10.1016/j.molcel.2019.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993210PMC
January 2020

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Mol Cell 2019 07 12;75(2):252-266.e8. Epub 2019 Jun 12.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
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http://dx.doi.org/10.1016/j.molcel.2019.04.030DOI Listing
July 2019

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Cell 2018 08 2;174(5):1127-1142.e19. Epub 2018 Aug 2.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.
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http://dx.doi.org/10.1016/j.cell.2018.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591735PMC
August 2018

The Energetics and Physiological Impact of Cohesin Extrusion.

Cell 2018 05 26;173(5):1165-1178.e20. Epub 2018 Apr 26.

Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD 20892, USA; Center of Cancer Research, NCI, NIH, Bethesda, MD 20892, USA. Electronic address:

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.
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http://dx.doi.org/10.1016/j.cell.2018.03.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065110PMC
May 2018

Genome Organization Drives Chromosome Fragility.

Cell 2017 Jul 20;170(3):507-521.e18. Epub 2017 Jul 20.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2017.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133249PMC
July 2017

DNA Breaks and End Resection Measured Genome-wide by End Sequencing.

Mol Cell 2016 09 28;63(5):898-911. Epub 2016 Jul 28.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice.
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http://dx.doi.org/10.1016/j.molcel.2016.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299834PMC
September 2016

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

Nat Med 2015 Oct 14;21(10):1199-208. Epub 2015 Sep 14.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center (MSKCC), New York, New York, USA.

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.
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http://dx.doi.org/10.1038/nm.3943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4676270PMC
October 2015

Collateral DNA damage produced by genome-editing drones: exception or rule?

Mol Cell 2015 May;58(4):565-7

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

In the recent issue of Nature Biotechnology, Frock et al. (2015) developed an elegant technique to capture translocation partners that can be utilized to determine off-target regions of genome-editing endonucleases as well as endogenous mutators at nucleotide resolution.
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http://dx.doi.org/10.1016/j.molcel.2015.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289255PMC
May 2015

Single-cell telomere-length quantification couples telomere length to meristem activity and stem cell development in Arabidopsis.

Cell Rep 2015 May 30;11(6):977-989. Epub 2015 Apr 30.

Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona 08193, Spain. Electronic address:

Telomeres are specialized nucleoprotein caps that protect chromosome ends assuring cell division. Single-cell telomere quantification in animals established a critical role for telomerase in stem cells, yet, in plants, telomere-length quantification has been reported only at the organ level. Here, a quantitative analysis of telomere length of single cells in Arabidopsis root apex uncovered a heterogeneous telomere-length distribution of different cell lineages showing the longest telomeres at the stem cells. The defects in meristem and stem cell renewal observed in tert mutants demonstrate that telomere lengthening by TERT sets a replicative limit in the root meristem. Conversely, the long telomeres of the columella cells and the premature stem cell differentiation plt1,2 mutants suggest that differentiation can prevent telomere erosion. Overall, our results indicate that telomere dynamics are coupled to meristem activity and continuous growth, disclosing a critical association between telomere length, stem cell function, and the extended lifespan of plants.
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http://dx.doi.org/10.1016/j.celrep.2015.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827700PMC
May 2015

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

Nature 2014 Oct 27;514(7520):107-11. Epub 2014 Jul 27.

Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.
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http://dx.doi.org/10.1038/nature13483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410707PMC
October 2014

Accumulation of cells with short telomeres is associated with impaired zinc homeostasis and inflammation in old hypertensive participants.

J Gerontol A Biol Sci Med Sci 2009 Jul 9;64(7):745-51. Epub 2009 Apr 9.

Immunology Center, Research Department Istituto Nazionale Riposo e Cura per Anziani, Ancona, Italy.

Critical shortening of telomeres, likely associated with a considerable increase of senescent cells, can be observed in PBMC of individuals aged 80 and older. We investigated the relationship between critical telomere shortening and zinc status in healthy or hypertensive participants with or without cardiovascular disease in old and very old participants. Telomere shortening and accumulation of cells with short telomeres (percent of cells with short telomeres) in advancing age was evident in patients and healthy controls, but exacerbated in those patients aged 80 and older. Moreover, in very old patients, the accumulation of % CST may impair intracellular zinc homeostasis and metallothioneins expression, which itself is linked to an increased number of inflammatory agents, thereby suggesting the existence of a possible causal relationship between % CST and zinc homeostasis. The determination of % CST could be a more reliable means than the simple measure of telomere length as fundamental parameter in ageing to determine whether individuals are still able to respond to stress.
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http://dx.doi.org/10.1093/gerona/glp048DOI Listing
July 2009

Telomerase deficiency promotes oxidative stress by reducing catalase activity.

Free Radic Biol Med 2008 Nov 30;45(9):1243-51. Epub 2008 Jul 30.

Departamento Fisiología, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

Telomere shortening and redox imbalance have been related to the aging process. We used cultured mouse embryonic fibroblasts (MEF) isolated from mice lacking telomerase activity (Terc(-/-)) to analyze the redox balance and the functional consequences promoted by telomerase deficiency. Comparison with wild-type (WT) MEF showed that Terc(-/-) MEF had greater oxidant damage, showing higher superoxide anion and hydrogen peroxide production and lower catalase activity. Restoration of telomerase activity in Terc(-/-) MEF increased catalase expression and activity. TGF-beta1 and collagen type IV levels were higher in Terc(-/-) than in WT MEF. TGF-beta1 promoter activity decreased when Terc(-/-) MEF were incubated with exogenous catalase, suggesting that catalase deficiency is the cause of the TGF-beta1 increase. Similar results were obtained in vivo. Homogenized renal cortex from 6-month-old Terc(-/-) showed higher oxidant capacity, lower catalase activity, greater oxidative damage, and higher TGF-beta1 and fibronectin levels than that from WT mice. In summary, telomerase deficiency reduces catalase activity, determining a redox imbalance that promotes overexpression of TGF-beta1 and extracellular matrix proteins.
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http://dx.doi.org/10.1016/j.freeradbiomed.2008.07.017DOI Listing
November 2008

Centromere mitotic recombination in mammalian cells.

J Cell Biol 2008 Jun 9;181(6):885-92. Epub 2008 Jun 9.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre, 28029 Madrid, Spain.

Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.
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http://dx.doi.org/10.1083/jcb.200803042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426939PMC
June 2008

The longest telomeres: a general signature of adult stem cell compartments.

Genes Dev 2008 Mar 18;22(5):654-67. Epub 2008 Feb 18.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre, Madrid E-28029, Spain.

Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age.
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http://dx.doi.org/10.1101/gad.451008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2259034PMC
March 2008

Telomere length analysis.

Methods Mol Biol 2007 ;371:45-72

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Center, Madrid, Spain.

Most somatic cells of long-lived species undergo telomere shortening throughout life. Critically short telomeres trigger loss of cell viability in tissues, which has been related to alteration of tissue function and loss of regenerative capabilities in aging and aging-related diseases. Hence, telomere length is an important biomarker for aging and can be used in the prognosis of aging diseases. These facts highlight the importance of developing methods for telomere length determination that can be employed to evaluate telomere length during the human aging process. Telomere length quantification methods have improved greatly in accuracy and sensitivity since the development of the conventional telomeric Southern blot. Here, we describe the different methodologies recently developed for telomere length quantification, as well as their potential applications for human aging studies.
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http://dx.doi.org/10.1007/978-1-59745-361-5_5DOI Listing
September 2007

High-throughput telomere length quantification by FISH and its application to human population studies.

Proc Natl Acad Sci U S A 2007 Mar 16;104(13):5300-5. Epub 2007 Mar 16.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre, 3 Melchor Fernández Almagro, Madrid E-28029, Spain.

A major limitation of studies of the relevance of telomere length to cancer and age-related diseases in human populations and to the development of telomere-based therapies has been the lack of suitable high-throughput (HT) assays to measure telomere length. We have developed an automated HT quantitative telomere FISH platform, HT quantitative FISH (Q-FISH), which allows the quantification of telomere length as well as percentage of short telomeres in large human sample sets. We show here that this technique provides the accuracy and sensitivity to uncover associations between telomere length and human disease.
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http://dx.doi.org/10.1073/pnas.0609367104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828130PMC
March 2007

Effectors of mammalian telomere dysfunction: a comparative transcriptome analysis using mouse models.

Carcinogenesis 2005 Sep 28;26(9):1613-26. Epub 2005 Apr 28.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Center (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain.

Critical telomere shortening in the absence of telomerase in late generation Terc-/- mice (G3 Terc-/-) or loss of telomere capping due to abrogation of the DNA repair/telomere binding protein Ku86 (Ku86-/- mice) results in telomere dysfunction and organismal premature aging. Here, we report on genome-wide transcription in mouse G3 Terc-/-, Ku86-/- and G3 Terc-/-/Ku86-/- germ cells using high-density oligonucleotide microarrays. Although a few transcripts are modulated specifically in Ku86- or Terc-deficient cells, the observed transcriptional response is mainly inductive and qualitatively similar for all three genotypes, with highest transcriptional induction observed in double mutant G3 Terc-/-/Ku86-/- cells compared with either single mutant. Analysis of 92 known genes induced in G3 Terc-/-/Ku86-/- germ cells compared with wild-type cells shows predominance of genes involved in cell adhesion, cell-to-cell and cell-to-matrix communication, as well as increased metabolic turnover and augmented antioxidant responses. In addition, the data presented in this study support the view that telomere dysfunction induces a robust compensatory response to rescue impaired germ cell function through the induction of survival signals related to the PI3-kinase pathway, as well as by the coordinated upregulation of transcripts that are essential for mammalian spermatogenesis.
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http://dx.doi.org/10.1093/carcin/bgi107DOI Listing
September 2005

Constitutive expression of tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice.

Mol Cell Biol 2004 May;24(10):4275-93

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), 28029 Madrid, Spain.

Here we describe a new mouse model with constitutive expression of the catalytic subunit of telomerase (Tert) targeted to thymocytes and peripheral T cells (Lck-Tert mice). Two independent Lck-Tert mouse lines showed higher incidences of spontaneous T-cell lymphoma than the corresponding age-matched wild-type controls, indicating that constitutive expression of Tert promotes lymphoma. Interestingly, T-cell lymphomas in Lck-Tert mice were more disseminated than those in wild-type controls and affected both lymphoid and nonlymphoid tissues, while nonlymphoid tissues were never affected with lymphoma in age-matched wild-type controls. Importantly, these roles of Tert constitutive expression in promoting tumor progression and dissemination were independent of the role of telomerase in telomere length maintenance, since telomere length distributions on a single-cell basis were identical in Lck-Tert and wild-type thymocytes. Finally, Tert constitutive expression did not interfere with telomere capping in Lck-Tert primary thymocytes, although it resulted in greater chromosomal instability upon gamma irradiation in Lck-Tert primary lymphocytes than in controls, suggesting that Tert overexpression may interfere with the cellular response to DNA damage.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC400466PMC
http://dx.doi.org/10.1128/mcb.24.10.4275-4293.2004DOI Listing
May 2004

Massive telomere loss is an early event of DNA damage-induced apoptosis.

J Biol Chem 2003 Jan 29;278(2):836-42. Epub 2002 Oct 29.

Unidad de Investigación, Hospital Universitario Reina Sofia, 14004 Córdoba, Spain.

Chromosomal stability and cell viability require a proficient telomeric end-capping function. In particular, telomere dysfunction because of either critical telomere shortening or because of mutation of telomere-binding proteins results in increased apoptosis and/or cell arrest. Here, we show that, in turn, DNA damage-induced apoptosis results in a dramatic telomere loss. In particular, using flow cytometry for simultaneous detection of telomere length and apoptosis, we show that cells undergoing apoptosis upon DNA damage also exhibit a rapid and dramatic loss of telomeric sequences. This telomere loss occurs at early stages of apoptosis, because it does not require caspase-3 activation, and it is induced by loss of the mitochondrial membrane potential (Deltapsi(m)) and production of reactive oxygen species. These observations suggest a direct effect of mitochondrial dysfunction on telomeres.
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http://dx.doi.org/10.1074/jbc.M206818200DOI Listing
January 2003