Publications by authors named "Sandy Chang"

91 Publications

Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly.

Nat Commun 2020 11 17;11(1):5861. Epub 2020 Nov 17.

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT, 06520, USA.

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1-TRF2 complex reveals that this interaction is mediated by the MCPH1 YRLSP motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.
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http://dx.doi.org/10.1038/s41467-020-19674-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672075PMC
November 2020

Shelterin and the replisome: at the intersection of telomere repair and replication.

Curr Opin Genet Dev 2020 02 12;60:77-84. Epub 2020 Mar 12.

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Department of Pathology, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA. Electronic address:

Telomeres are G-rich repetitive sequences that are difficult to replicate, resulting in increased replication stress that can threaten genome stability. Shelterin protects telomeres from engaging in aberrant DNA repair and dictates the choice of DNA repair pathway at dysfunctional telomeres. Recently, shelterin has been shown to participate in telomere replication. Here we review the most recent discoveries documenting the mechanisms by which shelterin represses DNA repair pathways at telomeres while assisting its replication. The interplay between shelterin and the replisome complex highlights a novel connection between telomere maintenance and repair.
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http://dx.doi.org/10.1016/j.gde.2020.02.016DOI Listing
February 2020

The Replisome Mediates A-NHEJ Repair of Telomeres Lacking POT1-TPP1 Independently of MRN Function.

Cell Rep 2019 12 10;29(11):3708-3725.e5. Epub 2019 Dec 10.

Departments of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Department of Pathology, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA. Electronic address:

Telomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5' C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PK-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5' end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.
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http://dx.doi.org/10.1016/j.celrep.2019.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001145PMC
December 2019

WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Nature 2019 04 10;568(7753):551-556. Epub 2019 Apr 10.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
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http://dx.doi.org/10.1038/s41586-019-1102-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580861PMC
April 2019

Internal medicine residents' evaluation of fevers overnight.

Diagnosis (Berl) 2019 06;6(2):157-163

Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.

Background Scant data exists to guide the work-up for fever in hospitalized patients, and little is known about what diagnostic tests medicine residents order for such patients. We sought to analyze how cross-covering medicine residents address fever and how sign-out systems affect their response. Methods We conducted a prospective cohort study to evaluate febrile episodes that residents responded to overnight. Primary outcomes included diagnostic tests ordered, if an in-person evaluation occurred, and the effect of sign-out instructions that advised a "full fever work-up" (FFWU). Results Investigators reviewed 253 fevers in 155 patients; sign-out instructions were available for 204 fevers. Residents evaluated the patient in person in 29 (11%) episodes. The most common tests ordered were: blood cultures (48%), urinalysis (UA) with reflex culture (34%), and chest X-ray (30%). If the sign-out advised an FFWU, residents were more likely to order blood cultures [odds ratio (OR) 14.75, 95% confidence interval (CI) 7.52-28.90], UA with reflex culture (OR 12.07, 95% CI 5.56-23.23), chest X-ray (OR 16.55, 95% CI 7.03-39.94), lactate (OR 3.33, 95% CI 1.47-7.55), and complete blood count (CBC) (OR 3.16, 95% CI 1.17-8.51). In a multivariable regression, predictors of the number of tests ordered included hospital location, resident training level, timing of previous blood culture, in-person evaluation, escalation to a higher level of care, and sign-out instructions. Conclusions Sign-out instructions and a few patient factors significantly impacted cross-cover resident diagnostic test ordering for overnight fevers. This practice can be targeted in resident education to improve diagnostic reasoning and stewardship.
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http://dx.doi.org/10.1515/dx-2018-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541517PMC
June 2019

CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length.

Aging Cell 2018 08 17;17(4):e12783. Epub 2018 May 17.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut.

Coats plus (CP) is a rare autosomal recessive disorder caused by mutations in CTC1, a component of the CST (CTC1, STN1, and TEN1) complex important for telomere length maintenance. The molecular basis of how CP mutations impact upon telomere length remains unclear. The CP CTC1 mutation has been previously shown to disrupt telomere maintenance. In this study, we used CRISPR/Cas9 to engineer this mutation into both alleles of HCT116 and RPE cells to demonstrate that CTC1:STN1 interaction is required to repress telomerase activity. CTC1 interacts poorly with STN1, leading to telomerase-mediated telomere elongation. Impaired interaction between CTC1 :STN1 and DNA Pol-α results in increased telomerase recruitment to telomeres and further telomere elongation, revealing that C:S binding to DNA Pol-α is required to fully repress telomerase activity. CP CTC1 mutants that fail to interact with DNA Pol-α resulted in loss of C-strand maintenance and catastrophic telomere shortening. Our findings place the CST complex as an important regulator of both G-strand extensions by telomerase and C-strand synthesis by DNA Pol-α.
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http://dx.doi.org/10.1111/acel.12783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052479PMC
August 2018

Structural and functional analyses of the mammalian TIN2-TPP1-TRF2 telomeric complex.

Cell Res 2017 Dec 21;27(12):1485-1502. Epub 2017 Nov 21.

State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China.

Telomeres are nucleoprotein complexes that play essential roles in protecting chromosome ends. Mammalian telomeres consist of repetitive DNA sequences bound by the shelterin complex. In this complex, the POT1-TPP1 heterodimer binds to single-stranded telomeric DNAs, while TRF1 and TRF2-RAP1 interact with double-stranded telomeric DNAs. TIN2, the linchpin of this complex, simultaneously interacts with TRF1, TRF2, and TPP1 to mediate the stable assembly of the shelterin complex. However, the molecular mechanism by which TIN2 interacts with these proteins to orchestrate telomere protection remains poorly understood. Here, we report the crystal structure of the N-terminal domain of TIN2 in complex with TIN2-binding motifs from TPP1 and TRF2, revealing how TIN2 interacts cooperatively with TPP1 and TRF2. Unexpectedly, TIN2 contains a telomeric repeat factor homology (TRFH)-like domain that functions as a protein-protein interaction platform. Structure-based mutagenesis analyses suggest that TIN2 plays an important role in maintaining the stable shelterin complex required for proper telomere end protection.
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http://dx.doi.org/10.1038/cr.2017.144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717407PMC
December 2017

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

Nat Commun 2017 07 17;8:16081. Epub 2017 Jul 17.

GlaxoSmithKline, 830 Winter Street, Waltham, Massachusetts 02451, USA.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.
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http://dx.doi.org/10.1038/ncomms16081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520047PMC
July 2017

GPIHBP1 autoantibodies in a patient with unexplained chylomicronemia.

J Clin Lipidol 2017 Jul - Aug;11(4):964-971. Epub 2017 Jun 13.

Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA. Electronic address:

Background: GPIHBP1, a glycolipid-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the interstitial spaces and transports it to the capillary lumen. GPIHBP1 deficiency prevents LPL from reaching the capillary lumen, resulting in low intravascular LPL levels, impaired intravascular triglyceride processing, and severe hypertriglyceridemia (chylomicronemia). A recent study showed that some cases of hypertriglyceridemia are caused by autoantibodies against GPIHBP1 ("GPIHBP1 autoantibody syndrome").

Objective: Our objective was to gain additional insights into the frequency of the GPIHBP1 autoantibody syndrome in patients with unexplained chylomicronemia.

Methods: We used enzyme-linked immunosorbent assays to screen for GPIHBP1 autoantibodies in 33 patients with unexplained chylomicronemia and then used Western blots and immunocytochemistry studies to characterize the GPIHBP1 autoantibodies.

Results: The plasma of 1 patient, a 36-year-old man with severe hypertriglyceridemia, contained GPIHBP1 autoantibodies. The autoantibodies, which were easily detectable by Western blot, blocked the ability of GPIHBP1 to bind LPL. The plasma levels of LPL mass and activity were low. The patient had no history of autoimmune disease, but his plasma was positive for antinuclear antibodies.

Conclusions: One of 33 patients with unexplained chylomicronemia had the GPIHBP1 autoantibody syndrome. Additional studies in large lipid clinics will be helpful for better defining the frequency of this syndrome and for exploring the best strategies for treatment.
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http://dx.doi.org/10.1016/j.jacl.2017.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568906PMC
February 2018

Structural insights into POT1-TPP1 interaction and POT1 C-terminal mutations in human cancer.

Nat Commun 2017 04 10;8:14929. Epub 2017 Apr 10.

National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China.

Mammalian shelterin proteins POT1 and TPP1 form a stable heterodimer that protects chromosome ends and regulates telomerase-mediated telomere extension. However, how POT1 interacts with TPP1 remains unknown. Here we present the crystal structure of the C-terminal portion of human POT1 (POT1C) complexed with the POT1-binding motif of TPP1. The structure shows that POT1C contains two domains, a third OB fold and a Holliday junction resolvase-like domain. Both domains are essential for binding to TPP1. Notably, unlike the heart-shaped structure of ciliated protozoan Oxytricha nova TEBPα-β complex, POT1-TPP1 adopts an elongated V-shaped conformation. In addition, we identify several missense mutations in human cancers that disrupt the POT1C-TPP1 interaction, resulting in POT1 instability. POT1C mutants that bind TPP1 localize to telomeres but fail to repress a DNA damage response and inappropriate repair by A-NHEJ. Our results reveal that POT1 C terminus is essential to prevent initiation of genome instability permissive for tumorigenesis.
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http://dx.doi.org/10.1038/ncomms14929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394241PMC
April 2017

Probing the Telomere Damage Response.

Methods Mol Biol 2017 ;1587:133-138

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT, 06520, USA.

Telomere dysfunctions, rendered through replicative attrition of telomeric DNA or due to the removal of shelterin components, are recognized as DNA double-stranded breaks (DSBs) by the DNA damage repair (DDR) pathway. This leads to the activation of DNA damage checkpoint sensors, including the Mre11-Rad50-Nbs1 (MRN) complex, γ-H2AX and 53BP1, the ATM and ATR signal-transducing kinases, and downstream effectors, including Chk1, Chk2, and p53. Robust DNA damage response signals at dysfunctional telomeres, achieved by the complete deletion of TRF2 or by expressing dominant-negative mutant TPP1ΔRD, can be detected by their association with γ-H2AX and 53BP1 forming "telomere dysfunction induced foci (TIFs)." Induction of TIFs at telomeres provides an opportunity to quantify the extent of telomere dysfunction and monitor downstream signaling pathways.
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http://dx.doi.org/10.1007/978-1-4939-6892-3_13DOI Listing
February 2018

Cytogenetic Analysis of Telomere Dysfunction.

Methods Mol Biol 2017 ;1587:127-131

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT, 06520, USA.

Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by the removal of telomere-binding proteins are recognized as double-stranded breaks (DSBs). Repair of DSBs is crucial for the maintenance of genome stability. In mammals, DSBs are repaired by either error-prone nonhomologous end joining (NHEJ) or error-free homologous recombination (HR) and can be visualized as chromosomal fusions.
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http://dx.doi.org/10.1007/978-1-4939-6892-3_12DOI Listing
February 2018

NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres.

Mol Cell 2017 Mar 16;65(5):801-817.e4. Epub 2017 Feb 16.

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar Street, New Haven, CT 06520, USA; Department of Pathology, Yale University School of Medicine, 330 Cedar Street, New Haven, CT 06520, USA; Molecular Biophysics and Biochemistry, Yale University School of Medicine, 330 Cedar Street, New Haven, CT 06520, USA. Electronic address:

Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 YQLSP motif interacts specifically with the TRF2 domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1 to activate ATM, while interaction of de-phosphorylated NBS1 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2 domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1 dictates repair pathway choice of dysfunctional telomeres.
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http://dx.doi.org/10.1016/j.molcel.2017.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639704PMC
March 2017

Dysfunctional telomeres induce p53-dependent and independent apoptosis to compromise cellular proliferation and inhibit tumor formation.

Aging Cell 2016 08 26;15(4):646-60. Epub 2016 Apr 26.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA.

Aging is associated with progressive telomere shortening, resulting in the formation of dysfunctional telomeres that compromise tissue proliferation. However, dysfunctional telomeres can limit tumorigenesis by activating p53-dependent cellular senescence and apoptosis. While activation of both senescence and apoptosis is required for repress tumor formation, it is not clear which pathway is the major tumor suppressive pathway in vivo. In this study, we generated Eμ-myc; Pot1b(∆/∆) mouse to directly compare tumor formation under conditions in which either p53-dependent apoptosis or senescence is activated by telomeres devoid of the shelterin component Pot1b. We found that activation of p53-dependent apoptosis plays a more critical role in suppressing lymphoma formation than p53-dependent senescence. In addition, we found that telomeres in Pot1b(∆/∆) ; p53(-/-) mice activate an ATR-Chk1-dependent DNA damage response to initiate a robust p53-independent, p73-dependent apoptotic pathway that limited stem cell proliferation but suppressed B-cell lymphomagenesis. Our results demonstrate that in mouse models, both p53-dependent and p53-independent apoptosis are important to suppressing tumor formation.
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http://dx.doi.org/10.1111/acel.12476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933665PMC
August 2016

TRF2-RAP1 is required to protect telomeres from engaging in homologous recombination-mediated deletions and fusions.

Nat Commun 2016 Mar 4;7:10881. Epub 2016 Mar 4.

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar Street, New Haven, Connecticut 06520, USA.

Repressor/activator protein 1 (RAP1) is a highly conserved telomere-interacting protein. Yeast Rap1 protects telomeres from non-homologous end joining (NHEJ), plays important roles in telomere length control and is involved in transcriptional gene regulation. However, a role for mammalian RAP1 in telomere end protection remains controversial. Here we present evidence that mammalian RAP1 is essential to protect telomere from homology directed repair (HDR) of telomeres. RAP1 cooperates with the basic domain of TRF2 (TRF2(B)) to repress PARP1 and SLX4 localization to telomeres. Without RAP1 and TRF2(B), PARP1 and SLX4 HR factors promote rapid telomere resection, resulting in catastrophic telomere loss and the generation of telomere-free chromosome fusions in both mouse and human cells. The RAP1 Myb domain is required to repress both telomere loss and formation of telomere-free fusions. Our results highlight the importance of the RAP1-TRF2 heterodimer in protecting telomeres from inappropriate processing by the HDR pathway.
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http://dx.doi.org/10.1038/ncomms10881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785230PMC
March 2016

Monitoring the DNA Damage Response at Dysfunctional Telomeres.

Methods Mol Biol 2015 ;1343:175-80

Department of Laboratory Medicine, Yale School of Medicine, 330 Cedar St, New Haven, CT, 06520, USA.

Telomeres are repetitive DNA repeats that cap the ends of all eukaryotic chromosomes. Their proper maintenance is essential for genomic stability and cellular viability. Dysfunctional telomeres could arise through natural attrition of telomeric DNA or due to the removal of shelterin components. These uncapped chromosomal ends are recognized as DSBs by the DDR pathway, leading to the accumulation of DNA damage sensors at telomeres. The association of these DDR proteins with dysfunctional telomeres forms telomere dysfunction induced DNA damage foci (TIFs). Detection of TIFs at telomeres provides an opportunity to quantify the extent of telomere dysfunction and monitor downstream DNA damage signaling pathways. Here we describe a method for the detection of TIFs using a fluorescent in situ hybridization (FISH) approach.
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http://dx.doi.org/10.1007/978-1-4939-2963-4_14DOI Listing
June 2016

Ketogenic Diet as a Therapeutic Option in Super-refractory Status Epilepticus.

Pediatr Neonatol 2015 Dec 14;56(6):429-31. Epub 2015 May 14.

Dietetics Department, Prince of Wales Hospital, Hong Kong, China.

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http://dx.doi.org/10.1016/j.pedneo.2015.01.010DOI Listing
December 2015

First-Line Antimicrobial Resistance Patterns of Escherichia coli in Children With Urinary Tract Infection in Emergency Department and Primary Care Clinics.

Clin Pediatr (Phila) 2016 Jan 2;55(1):19-28. Epub 2015 Jun 2.

Southern Illinois University Medical Center, Springfield, IL, USA.

Objective: To identify risk factors for antibiotic resistance to Escherichia coli (E. coli) in children with urinary tract infections (UTIs) in emergency room and primary care clinics.

Method: This is a cross-sectional study of children 0 to 18 years of age reported to have E coli-positive UTIs whose medical and laboratory records were systematically reviewed.

Result: Compared with girls, boys were 2.29 times (confidence interval [CI] = 1.30-4.02) more likely to have E coli isolates resistant to ampicillin and 2 times more likely (CI = 1.13-3.62) to have isolates resistant to trimethoprim-sulfamethoxazole (TMP/SMX). Patients with genitourinary abnormalities were 1.57 times more likely to be resistant to ampicillin (CI = 1.03-2.41) and 1.86 times to TMP/SMX (CI = 1.18-2.94).

Conclusion: Higher rates of ampicillin and TMP/SMX resistant urinary E coli isolates were observed among boys and children with a history of genitourinary abnormality. Age and recent antibiotic prescription are also potential risk factors for resistance.
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http://dx.doi.org/10.1177/0009922815588822DOI Listing
January 2016

Synergistic tumor suppression by combined inhibition of telomerase and CDKN1A.

Proc Natl Acad Sci U S A 2014 Jul 14;111(30):E3062-71. Epub 2014 Jul 14.

Departments of Pathology and

Tumor suppressor p53 plays an important role in mediating growth inhibition upon telomere dysfunction. Here, we show that loss of the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21(WAF1/CIP1)) increases apoptosis induction following telomerase inhibition in a variety of cancer cell lines and mouse xenografts. This effect is highly specific to p21, as loss of other checkpoint proteins and CDK inhibitors did not affect apoptosis. In telomerase, inhibited cell loss of p21 leads to E2F1- and p53-mediated transcriptional activation of p53-upregulated modulator of apoptosis, resulting in increased apoptosis. Combined genetic or pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simultaneous inhibition of telomerase and p21 also suppresses growth of tumors containing mutant p53 following pharmacological restoration of p53 activity. Collectively, our results establish that inactivation of p21 leads to increased apoptosis upon telomerase inhibition and thus identify a genetic vulnerability that can be exploited to treat many human cancers containing either wild-type or mutant p53.
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http://dx.doi.org/10.1073/pnas.1411370111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121806PMC
July 2014

Pot1a prevents telomere dysfunction and ATM-dependent neuronal loss.

J Neurosci 2014 Jun;34(23):7836-44

Department of Genetics, St Jude Children's Research Hospital, Memphis, Tennessee 38105,

Genome stability is essential for neural development and the prevention of neurological disease. Here we determined how DNA damage signaling from dysfunctional telomeres affects neurogenesis. We found that telomere uncapping by Pot1a inactivation resulted in an Atm-dependent loss of cerebellar interneurons and granule neuron precursors in the mouse nervous system. The activation of Atm by Pot1a loss occurred in an Atr-dependent manner, revealing an Atr to Atm signaling axis in the nervous system after telomere dysfunction. In contrast to telomere lesions, Brca2 inactivation in neural progenitors also led to ablation of cerebellar interneurons, but this did not require Atm. These data reveal that neural cell loss after DNA damage selectively engages Atm signaling, highlighting how specific DNA lesions can dictate neuropathology arising in human neurodegenerative syndromes.
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http://dx.doi.org/10.1523/JNEUROSCI.4245-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4044246PMC
June 2014

p16(INK4a) protects against dysfunctional telomere-induced ATR-dependent DNA damage responses.

J Clin Invest 2013 Oct 16;123(10):4489-501. Epub 2013 Sep 16.

Dysfunctional telomeres limit cellular proliferative capacity by activating the p53-p21- and p16(INK4a)-Rb-dependent DNA damage responses (DDRs). The p16(INK4a) tumor suppressor accumulates in aging tissues, is a biomarker for cellular senescence, and limits stem cell function in vivo. While the activation of a p53-dependent DDR by dysfunctional telomeres has been well documented in human cells and mouse models, the role for p16(INK4a) in response to telomere dysfunction remains unclear. Here, we generated protection of telomeres 1b p16-/- mice (Pot1bΔ/Δ;p16-/-) to address the function of p16(INK4a) in the setting of telomere dysfunction in vivo. We found that deletion of p16(INK4a) accelerated organ impairment and observed functional defects in highly proliferative organs, including the hematopoietic system, small intestine, and testes. Pot1bΔ/Δ;p16-/- hematopoietic cells exhibited increased telomere loss, increased chromosomal fusions, and telomere replication defects. p16(INK4a) deletion enhanced the activation of the ATR-dependent DDR in Pot1bΔ/Δ hematopoietic cells, leading to p53 stabilization, increased p21-dependent cell cycle arrest, and elevated p53-dependent apoptosis. In contrast to p16(INK4a), deletion of p21 did not activate ATR, rescued proliferative defects in Pot1bΔ/Δ hematopoietic cells, and significantly increased organismal lifespan. Our results provide experimental evidence that p16(INK4a) exerts protective functions in proliferative cells bearing dysfunctional telomeres.
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http://dx.doi.org/10.1172/JCI69574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784543PMC
October 2013

SLX4 assembles a telomere maintenance toolkit by bridging multiple endonucleases with telomeres.

Cell Rep 2013 Sep 5;4(5):861-9. Epub 2013 Sep 5.

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China; National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China; Howard Hughes Medical Institute, University of Michigan Medical School, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA; Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.

SLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM) in complex with the TRFH domain of TRF2 (TRF2TRFH) and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.
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http://dx.doi.org/10.1016/j.celrep.2013.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334113PMC
September 2013

The mINO80 chromatin remodeling complex is required for efficient telomere replication and maintenance of genome stability.

Cell Res 2013 Dec 27;23(12):1396-413. Epub 2013 Aug 27.

Department of Laboratory Medicine and Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.

The INO80 (inositol requiring mutant 80) chromatin remodeling complex plays important roles in transcriptional regulation and DNA replication and repair, and consists of several functional protein subunits, including the critical Ino80 ATPase catalytic subunit. While the function of INO80 has been studied in yeast and mammalian cell lines, we do not know how mIno80 contributes to the maintenance of genome stability to prevent cancer development in mice. Here, we use a conditional knockout approach to explore the cellular and organismal functions of mIno80. Deletion of mIno80 results in profound cellular proliferative defects and activation of p21-dependent cellular senescence. While mIno80 is required for efficient repair of DNA double strand breaks, its depletion did not impact upon the formation of γ-H2AX and 53BP1 DNA damage foci, or the activation of the ATM-CHK2-dependent DNA damage response. mIno80 deletion inhibited the generation of single-strand DNA, resulting in defects in homology-directed DNA repair (HDR) at telomeres. Fragile telomeres were prominent in mIno80(Δ/Δ) MEFs, suggesting that chromatin remodeling is required for efficient telomere replication. mIno80(-/-) mouse embryos die early during embryogenesis, while conditional deletion of mIno80 in adult mice results in weight loss and premature death. In a p53(-/-) tumor-prone background, mIno80 haploinsufficiency favored the development of sarcomas. Our studies suggest that the mIno80 chromatin remodeling complex plays important roles in telomere replication, HDR-mediated repair of dysfunctional telomeres, and maintenance of genome stability.
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http://dx.doi.org/10.1038/cr.2013.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847565PMC
December 2013

Functional characterization of human CTC1 mutations reveals novel mechanisms responsible for the pathogenesis of the telomere disease Coats plus.

Aging Cell 2013 Dec 4;12(6):1100-9. Epub 2013 Sep 4.

Department of Laboratory Medicine, Yale University School of Medicine, 330 Cedar Street, New Haven, CT, 06520, USA.

Coats plus is a rare recessive disorder characterized by intracranial calcifications, hematological abnormalities, and retinal vascular defects. This disease results from mutations in CTC1, a member of the CTC1-STN1-TEN1 (CST) complex critical for telomere replication. Telomeres are specialized DNA/protein structures essential for the maintenance of genome stability. Several patients with Coats plus display critically shortened telomeres, suggesting that telomere dysfunction plays an important role in disease pathogenesis. These patients inherit CTC1 mutations in a compound heterozygous manner, with one allele encoding a frameshift mutant and the other a missense mutant. How these mutations impact upon telomere function is unknown. We report here the first biochemical characterization of human CTC1 mutations. We found that all CTC1 frameshift mutations generated truncated or unstable protein products, none of which were able to form a complex with STN1-TEN1 on telomeres, resulting in progressive telomere shortening and formation of fused chromosomes. Missense mutations are able to form the CST complex at telomeres, but their expression levels are often repressed by the frameshift mutants. Our results also demonstrate for the first time that CTC1 mutations promote telomere dysfunction by decreasing the stability of STN1 to reduce its ability to interact with DNA Polα, thus highlighting a previously unknown mechanism to induce telomere dysfunction.
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http://dx.doi.org/10.1111/acel.12139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083614PMC
December 2013

Cancer chromosomes going to POT1.

Authors:
Sandy Chang

Nat Genet 2013 May;45(5):473-5

Department of Laboratory Medicine and Pathology at Yale University School of Medicine, New Haven, Connecticut, USA.

Alterations in the single-stranded telomere-binding protein POT1 have recently been identified in chronic lymphocytic leukemia. This discovery provides novel insights into how genomic instability induced by dysfunctional telomeres contributes to tumorigenesis.
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http://dx.doi.org/10.1038/ng.2617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040961PMC
May 2013

Single strand DNA binding proteins 1 and 2 protect newly replicated telomeres.

Cell Res 2013 May 5;23(5):705-19. Epub 2013 Mar 5.

Department of Laboratory Medicine and Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.

Human single-strand (ss) DNA binding proteins 1 and 2 (hSSB1 and 2) are components of the hSSB1/2-INTS3-C9orf80 heterotrimeric protein complex shown to participate in DNA damage response and maintenance of genome stability. However, their roles at telomeres remain unknown. Here, we generated murine SSB1 conditional knockout mice and cells and found that mSSB1 plays a critical role in telomere end protection. Both mSSB1 and mSSB2 localize to a subset of telomeres and are required to repair TRF2-deficient telomeres. Deletion of mSSB1 resulted in increased chromatid-type fusions involving both leading- and lagging-strand telomeric DNA, suggesting that it is required for the protection of G-overhangs. mSSB1's interaction with INTS3 is required for its localization to damaged DNA. mSSB1 interacts with Pot1a, but not Pot1b, and its association with telomeric ssDNA requires Pot1a. mSSB1(Δ/Δ) mice die at birth with developmental abnormalities, while mice with the hypomorphic mSSB1(F/F) allele are born alive and display increased sensitivity to ionizing radiation (IR). Our results suggest that mSSB1 is required to maintain genome stability, and document a previously unrecognized role for mSSB1/2 in the protection of newly replicated leading- and lagging-strand telomeres.
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http://dx.doi.org/10.1038/cr.2013.31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641597PMC
May 2013

Effect of coexisting chronic obstructive pulmonary disease and cognitive impairment on health outcomes in older adults.

J Am Geriatr Soc 2012 Oct 4;60(10):1839-46. Epub 2012 Oct 4.

Section of Geriatrics, Department of Internal Medicine, School of Medicine, Yale University, New Haven, CT 06520, USA.

Objectives: To determine the extent to which the co-occurrence of chronic obstructive pulmonary disease (COPD) and cognitive impairment affect adverse health outcomes in older adults.

Design: Multicenter longitudinal cohort study.

Setting: California, Pennsylvania, Maryland, and North Carolina.

Participants: Three thousand ninety-three community-dwelling adults aged 65 and older from the Cardiovascular Health Study. Four hundred thirty-one had chronic obstructive pulmonary disease (COPD) at study baseline.

Measurements: Follow-up began at the second CHS visit and continued for 3 years. Spirometric criteria for airflow limitation served to establish COPD using the Lambda-Mu-Sigma method, which accounts for age-related changes in lung function. Cognitive impairment was evaluated using the modified Mini-Mental State Examination and claims data. Outcomes were respiratory-related and all-cause hospitalizations and death.

Results: Participants with coexisting COPD and cognitive impairment had the highest rates of respiratory-related (adjusted hazard ratio (aHR) = 4.10, 95% confidence interval (CI) = 1.86-9.05) and all-cause hospitalizations (aHR = 1.34, 95% CI = 1.00-1.80) and death (aHR = 2.29, 95% CI = 1.18-4.45). In particular, individuals with both conditions had a 48% higher rate of all-cause hospitalizations (adjusted synergy index (aSI) = 1.48, 95% CI = 0.19-11.31) and a rate of death nearly three times as high (aSI = 2.74, 95% CI = 0.43-17.32) as the sum of risks for each respective outcome associated with having COPD or cognitive impairment alone. Nevertheless, tests for interaction were not statistically significant for the presence of synergism between the two conditions contributing to each of the outcomes. Therefore, it cannot be concluded that the combined effect of COPD and cognitive impairment is greater than additive.

Conclusion: Coexisting COPD and cognitive impairment have an additive effect on respiratory-related and all-cause hospitalizations and death. Optimizing outcomes in older adults with COPD and cognitive impairment will require that how to improve concurrent management of both conditions be determined.
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http://dx.doi.org/10.1111/j.1532-5415.2012.04171.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3470752PMC
October 2012

Chromosome ends teach unexpected lessons on DNA damage signalling.

Authors:
Sandy Chang

EMBO J 2012 Aug 27;31(16):3380-1. Epub 2012 Jul 27.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA.

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http://dx.doi.org/10.1038/emboj.2012.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419931PMC
August 2012

CTC1 deletion results in defective telomere replication, leading to catastrophic telomere loss and stem cell exhaustion.

EMBO J 2012 May 24;31(10):2309-21. Epub 2012 Apr 24.

Department of Laboratory Medicine and Pathology, Yale University School of Medicine, New Haven, CT 06520-8035, USA.

The proper maintenance of telomeres is essential for genome stability. Mammalian telomere maintenance is governed by a number of telomere binding proteins, including the newly identified CTC1-STN1-TEN1 (CST) complex. However, the in vivo functions of mammalian CST remain unclear. To address this question, we conditionally deleted CTC1 from mice. We report here that CTC1 null mice experience rapid onset of global cellular proliferative defects and die prematurely from complete bone marrow failure due to the activation of an ATR-dependent G2/M checkpoint. Acute deletion of CTC1 does not result in telomere deprotection, suggesting that mammalian CST is not involved in capping telomeres. Rather, CTC1 facilitates telomere replication by promoting efficient restart of stalled replication forks. CTC1 deletion results in increased loss of leading C-strand telomeres, catastrophic telomere loss and accumulation of excessive ss telomere DNA. Our data demonstrate an essential role for CTC1 in promoting efficient replication and length maintenance of telomeres.
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http://dx.doi.org/10.1038/emboj.2012.96DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364752PMC
May 2012