Publications by authors named "Zhao-Qi Wang"

119 Publications

Mre11 Is Essential for the Removal of Lethal Topoisomerase 2 Covalent Cleavage Complexes.

Mol Cell 2016 11;64(3):580-592

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11) and nuclease-deficient Mre11 (MRE11) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons. The defects in Mre11 compromise the repair of etoposide-induced Top2-DNA covalent complexes, and MRE11 and MRE11 cells accumulate high levels of Top2 covalent conjugates even in the absence of exogenous damage. We demonstrate that both the genome instability and mortality of MRE11 and MRE11 cells are significantly reversed by overexpression of Tdp2, an enzyme that eliminates covalent Top2 conjugates; thus, the essential role of Mre11 nuclease activity is likely to remove these lesions.
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http://dx.doi.org/10.1016/j.molcel.2016.10.011DOI Listing
November 2016

PARPing for balance in the homeostasis of poly(ADP-ribosyl)ation.

Semin Cell Dev Biol 2017 03 21;63:81-91. Epub 2016 Sep 21.

Leibniz Institute on Aging - Fritz-Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Fürstengraben 1, 07743 Jena, Germany. Electronic address:

Despite more than 50 years of research, the vast majority of the biology of poly(ADP-ribosyl)ation (PARylation) still remains a gross mystery. Originally described to be a part of the DNA repair machinery, poly(ADP-ribose) (PAR) is synthesized immediately by poly(ADP-ribose) polymerases (PARPs, also known as ARTDs) upon DNA damage and then rapidly removed by degrading enzymes. PAR provides a delicate and spatiotemporal interaction scaffold for numerous target proteins. Thus, the multifaceted PARylation system, consisting of PAR itself and its synthesizers and erasers, plays diverse roles in the DNA damage response (DDR), in DNA repair, transcription, replication, chromatin remodelling, metabolism and cell death. In this review, we summarize the current understanding of the biology of PARylation, focusing on the functionality and the activities of the PARPs' founding member PARP1/ARTD1, which is modulated by a variety of posttranslational modifications. We also discuss the homeostasis of PAR - a process which is maintained by the balance of PAR synthesizers and erasers. We aim to sensitize the scientific community to the complexity of PAR homeostasis. Finally, we provide some perspective on how future research could try to disentangle the biology of PARylation - perhaps the most sophisticated, but still intricate posttranslational modification described to date.
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http://dx.doi.org/10.1016/j.semcdb.2016.09.011DOI Listing
March 2017

DNA Damage Response in Hematopoietic Stem Cell Ageing.

Genomics Proteomics Bioinformatics 2016 06 21;14(3):147-154. Epub 2016 May 21.

Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena D-07745, Germany; Faculty of Biology and Pharmacy, Friedrich-Schiller University of Jena, Jena D-07745, Germany.

Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity. Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic system. They divide asymmetrically and give rise to daughter cells with HSC identity (self-renewal) and progenitor progenies (differentiation), which further proliferate and differentiate into full hematopoietic lineages. Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation. Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process. The DNA damage response (DDR) in the cells involves an orchestrated signaling pathway, consisting of cell cycle regulation, cell death and senescence, transcriptional regulation, as well as chromatin remodeling. Recent studies employing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically regulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system. In this review, we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing.
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http://dx.doi.org/10.1016/j.gpb.2016.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936660PMC
June 2016

The DNA damage response molecule MCPH1 in brain development and beyond.

Acta Biochim Biophys Sin (Shanghai) 2016 Jul 19;48(7):678-85. Epub 2016 May 19.

Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany Faculty of Biology and Pharmacy, Friedrich-Schiller University of Jena, Jena, Germany

Microcephalin (MCPH1) is identified as being responsible for the neurodevelopmental disorder primary microcephaly type 1, which is characterized by a smaller-than-normal brain size and mental retardation. MCPH1 has originally been identified as an important regulator of telomere integrity and of cell cycle control. Genetic and cellular studies show that MCPH1 controls neurogenesis by coordinating the cell cycle and the centrosome cycle and thereby regulating the division mode of neuroprogenitors to prevent the exhaustion of the progenitor pool and thereby microcephaly. In addition to its role in neurogenesis, MCPH1 plays a role in gonad development. MCPH1 also functions as a tumor suppressor in several human cancers as well as in mouse models. Here, we review the role of MCPH1 in DNA damage response, cell cycle control, chromosome condensation and chromatin remodeling. We also summarize the studies on the biological functions of MCPH1 in brain size determination and in pathologies, including infertility and cancer.
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http://dx.doi.org/10.1093/abbs/gmw048DOI Listing
July 2016

Deficiency in mTORC1-controlled C/EBPβ-mRNA translation improves metabolic health in mice.

EMBO Rep 2015 Aug 25;16(8):1022-36. Epub 2015 Jun 25.

Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena, Germany European Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E-binding proteins (4E-BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1-associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPβ) mRNA into the C/EBPβ-LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E-BPs is required for suppression of LIP. Intriguingly, mice lacking the cis-regulatory upstream open reading frame (uORF) in the C/EBPβ-mRNA, which is required for mTORC1-stimulated translation into C/EBPβ-LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPβ-isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.
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http://dx.doi.org/10.15252/embr.201439837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552494PMC
August 2015

Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.

EMBO J 2015 May 27;34(10):1371-84. Epub 2015 Mar 27.

Leibniz Institute of Age Research, Fritz Lipmann Institute e.V., Jena, Germany

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy-controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb-dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase-deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy-induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.
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http://dx.doi.org/10.15252/embj.201490070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491997PMC
May 2015

Smg6/Est1 licenses embryonic stem cell differentiation via nonsense-mediated mRNA decay.

EMBO J 2015 Jun 14;34(12):1630-47. Epub 2015 Mar 14.

Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany Faculty of Biology and Pharmacy, Friedrich-Schiller University of Jena, Jena, Germany

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional mechanism that targets aberrant transcripts and regulates the cellular RNA reservoir. Genetic modulation in vertebrates suggests that NMD is critical for cellular and tissue homeostasis, although the underlying mechanism remains elusive. Here, we generate knockout mice lacking Smg6/Est1, a key nuclease in NMD and a telomerase cofactor. While the complete loss of Smg6 causes mouse lethality at the blastocyst stage, inducible deletion of Smg6 is compatible with embryonic stem cell (ESC) proliferation despite the absence of telomere maintenance and functional NMD. Differentiation of Smg6-deficient ESCs is blocked due to sustained expression of pluripotency genes, normally repressed by NMD, and forced down-regulation of one such target, c-Myc, relieves the differentiation block. Smg6-null embryonic fibroblasts are viable as well, but are refractory to cellular reprograming into induced pluripotent stem cells (iPSCs). Finally, depletion of all major NMD factors compromises ESC differentiation, thus identifying NMD as a licensing factor for the switch of cell identity in the process of stem cell differentiation and somatic cell reprograming.
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http://dx.doi.org/10.15252/embj.201489947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475398PMC
June 2015

Systematic characterization of cell cycle phase-dependent protein dynamics and pathway activities by high-content microscopy-assisted cell cycle phenotyping.

Genomics Proteomics Bioinformatics 2014 Dec 29;12(6):255-65. Epub 2014 Nov 29.

Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), 07745 Jena, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, 07745 Jena, Germany. Electronic address:

Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically powerful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular localization at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3-4-day protocol, which can be adjusted to any other cell cycle stage-dependent analysis.
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http://dx.doi.org/10.1016/j.gpb.2014.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411490PMC
December 2014

RPLP1, a crucial ribosomal protein for embryonic development of the nervous system.

PLoS One 2014 24;9(6):e99956. Epub 2014 Jun 24.

Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, Jena, Germany.

Ribosomal proteins are pivotal to development and tissue homeostasis. RP Large P1 (Rplp1) overexpression is associated with tumorigenesis. However, the physiological function of Rplp1 in mammalian development remains unknown. In this study, we disrupted Rplp1 in the mouse germline and central nervous system (Rplp1CNSΔ). Rplp1 heterozygosity caused body size reductions, male infertility, systemic abnormalities in various tissues and a high frequency of early postnatal death. Rplp1CNSΔ newborn mice exhibited perinatal lethality and brain atrophy with size reductions of the neocortex, midbrain and ganglionic eminence. The Rplp1 knockout neocortex exhibited progenitor cell proliferation arrest and apoptosis due to the dysregulation of key cell cycle and apoptosis regulators (cyclin A, cyclin E, p21CIP1, p27KIP1, p53). Similarly, Rplp1 deletion in pMEFs led to proliferation arrest and premature senescence. Importantly, Rplp1 deletion in primary mouse embryonic fibroblasts did not alter global protein synthesis, but did change the expression patterns of specific protein subsets involved in protein folding and the unfolded protein response, cell death, protein transport and signal transduction, among others. Altogether, we demonstrated that the translation "fine-tuning" exerted by Rplp1 is essential for embryonic and brain development and for proper cell proliferation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099956PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069005PMC
November 2015

Trrap-dependent histone acetylation specifically regulates cell-cycle gene transcription to control neural progenitor fate decisions.

Cell Stem Cell 2014 May;14(5):632-43

Leibniz Institute for Age Research, Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, Fuerstengraben 26, 07743 Jena, Germany. Electronic address:

Fate decisions in neural progenitor cells are orchestrated via multiple pathways, and the role of histone acetylation in these decisions has been ascribed to a general function promoting gene activation. Here, we show that the histone acetyltransferase (HAT) cofactor transformation/transcription domain-associated protein (Trrap) specifically regulates activation of cell-cycle genes, thereby integrating discrete cell-intrinsic programs of cell-cycle progression and epigenetic regulation of gene transcription in order to control neurogenesis. Deletion of Trrap impairs recruitment of HATs and transcriptional machinery specifically to E2F cell-cycle target genes, disrupting their transcription with consequent cell-cycle lengthening specifically within cortical apical neural progenitors (APs). Consistently, Trrap conditional mutants exhibit microcephaly because of premature differentiation of APs into intermediate basal progenitors and neurons, and overexpressing cell-cycle regulators in vivo can rescue these premature differentiation defects. These results demonstrate an essential and highly specific role for Trrap-mediated histone regulation in controlling cell-cycle progression and neurogenesis.
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http://dx.doi.org/10.1016/j.stem.2014.04.001DOI Listing
May 2014

The essential function of the MRN complex in the resolution of endogenous replication intermediates.

Cell Rep 2014 Jan 2;6(1):182-95. Epub 2014 Jan 2.

Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena 07745, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University Jena, Jena 07743, Germany. Electronic address:

The MRN complex (Mre11/Rad50/Nbs1) is important in double-strand break (DSB) recognition, end resection, replication fork stabilization, and ATM and ATR activation. Complete deletion of MRN is incompatible with cell and organism life, presumably due to replication-born DSBs; however, the underlying mechanism remains unknown. We devised a noninvasive high-content assay, termed high-content microscopy-assisted cell-cycle phenotyping (hiMAC), to investigate the fate of cells lacking Nbs1. Surprisingly, deletion of Nbs1 does not kill cells during replication. The primary lesions in Nbs1-deleted cells are replication intermediates that result from defective resolution rather than fork destabilization. These lesions are converted to DSBs in the subsequent G2 phase, which subsequently activate Chk1, delay G2 progression, and lead to chromosome instability. Nbs1-deleted cells establish a DSB equilibrium that permits cell cycling but activates p53, causing G1 and G2 arrest, and cell death. Thus, we identify a physiological role of Nbs1 in the resolution of stalled replication forks.
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http://dx.doi.org/10.1016/j.celrep.2013.12.018DOI Listing
January 2014

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation.

Nat Commun 2013 ;4:2993

1] Leibniz Institute For Age Research-Fritz Lipmann Institute (FLI), Beutenberger strasse 11, 07745 Jena, Germany [2] Faculty of Biology and Pharmacy, Friedrich-Schiller-University Jena, Beutenberger strasse 11, 07745 Jena, Germany.

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.
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http://dx.doi.org/10.1038/ncomms3993DOI Listing
July 2014

Nbn gene inactivation in the CNS of mouse inhibits the myelinating ability of the mature cortical oligodendrocytes.

Glia 2014 Jan;62(1):133-44

Department of Pathology, Center for Experimental Animal Research, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Nijmegen Breakage Syndrome (NBS) is a recessive genetic disorder characterized by immunodeficiency, elevated sensitivity to ionizing radiation, chromosomal instability, microcephaly, and high predisposition to malignancies. Since the underlying molecular mechanisms of the NBS microcephaly are still obscure, thus our group previously inactivated the Nbn gene in the central nervous system (CNS) of mice by nestin-Cre targeting gene system, and generated Nbn(CNS-del) mice. Interestingly, the newborn Nbn(CNS-del) mice exhibit obvious microcephaly, which is accompanied by severe ataxia and balance deficiency. In this study presented here, we report that Nbn-deficiency induces the enhanced apoptosis of the mature oligodendrocytes at postnatal day 7, which further affects the myelination of the nerve fibers of cerebrum and corpus callosum.The distinct regulatory roles of Ataxia telangiectasia mutated (ATM) signaling and protein kinase B(Akt)/the mammalian target of Rapamycin (AKT/mTOR) signaling are responsible for the enhanced apoptosis of the Nbn-deficient oligodendrocytes. In addition, a series of transcriptional factors including histonedeacetylase (HDAC), zinc finger protein 191 (ZFP-191) and myelin sheath regulatory factor (MRF) play distinct roles in regulating the myelination of the Nbn-deficient oligodendrocytes. Based on these results, it concludes that ATM-Chk2-P53-P21 signaling pathway and the AKT/mTOR signaling pathway are both responsible for the enhanced apoptosis of the Nbn-deficient oligodendrocytes. HDAC, ZFP-191, and MRF are also involved in the pathogenesis of the hypomyelination of the Nbn-deficient oligodendrocytes.
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http://dx.doi.org/10.1002/glia.22593DOI Listing
January 2014

PARP-1 controls immunosuppressive function of regulatory T cells by destabilizing Foxp3.

PLoS One 2013 19;8(8):e71590. Epub 2013 Aug 19.

Mucosal Immunology Unit, OIIB, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland, United States of America.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme and transcription factor that is involved in inflammatory response, but its role in T cell response remains largely unknown. We show here that PARP-1 regulates the suppressive function of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Specifically, Tregs in mice with a null mutation of the PARP-1 gene (PARP-1(-/-)) showed significantly stronger suppressive activity than did wild-type Tregs in culture. We elucidate that this enhanced suppressive function is attributed to sustained higher expression of Foxp3 and CD25 in PARP-1(-/-) Tregs. Furthermore, in PARP-1(-/-) Tregs, Foxp3 protein shows substantially higher levels of binding to the conserved non-coding DNA sequence 2 (CNS2) at the foxp3 gene, a region important in maintaining Foxp3 gene expression in Tregs. Thus, our data reveal a role for PARP-1 in controlling the function of Tregs through modulation of the stable expression of Foxp3.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071590PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747222PMC
April 2014

An essential function for the ATR-activation-domain (AAD) of TopBP1 in mouse development and cellular senescence.

PLoS Genet 2013 8;9(8):e1003702. Epub 2013 Aug 8.

Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany.

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins - Dpb11(TopBP1), Ddc1(Rad9) and Dna2 - all interact with and activate Mec1(ATR). Each contains an ATR activation domain (ADD) that interacts directly with the Mec1(ATR):Ddc2(ATRIP) complex. Any of the Dpb11(TopBP1), Ddc1(Rad9) or Dna2 ADDs is sufficient to activate Mec1(ATR) in vitro. All three can also independently activate Mec1(ATR) in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.
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http://dx.doi.org/10.1371/journal.pgen.1003702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738440PMC
March 2014

PARP-1 regulates expression of TGF-β receptors in T cells.

Blood 2013 Sep 12;122(13):2224-32. Epub 2013 Aug 12.

Mucosal Immunology Section, Oral Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD;

Transforming growth factor-β (TGF-β) receptors (TβRs) are essential components for TGF-β signal transduction in T cells, yet the mechanisms by which the receptors are regulated remain poorly understood. We show here that Poly(ADP-ribose) polymerase-1 (PARP-1) regulates TGF-β receptor I (TβRI) and II (TβRII) expression in CD4(+) T cells and subsequently affects Smad2/3-mediated TGF-β signal transduction. Inhibition of PARP-1 led to the upregulation of both TβRI and TβRII, yet the underlying molecular mechanisms were distinct. PARP-1 selectively bound to the promoter of TβRII, whereas the enzymatic activity of PARP-1 was responsible for the inhibition of TβRI expression. Importantly, inhibition of PARP-1 also enhanced expression of TβRs in human CD4(+) T cells. Thus, PARP-1 regulates TβR expression and TGF-β signaling in T cells.
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http://dx.doi.org/10.1182/blood-2013-05-503250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785120PMC
September 2013

DNA damage response in microcephaly development of MCPH1 mouse model.

DNA Repair (Amst) 2013 Aug 15;12(8):645-55. Epub 2013 May 15.

Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany.

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV-VI), while generate less late-born neurons that form a thinner outer layer (II-III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.
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http://dx.doi.org/10.1016/j.dnarep.2013.04.017DOI Listing
August 2013

Histone acetyltransferase cofactor Trrap maintains self-renewal and restricts differentiation of embryonic stem cells.

Stem Cells 2013 May;31(5):979-91

International Agency for Research on Cancer (IARC), Lyon, France.

Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs.
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http://dx.doi.org/10.1002/stem.1341DOI Listing
May 2013

Cell-type, dose, and mutation-type specificity dictate mutant p53 functions in vivo.

Cancer Cell 2012 Dec;22(6):751-64

Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11, Hospital Drive, Singapore 169610, Singapore.

The specific roles of mutant p53's dominant-negative (DN) or gain-of-function (GOF) properties in regulating acute response and long-term tumorigenesis is unclear. Using "knockin" mouse strains expressing varying R246S mutant levels, we show that the DN effect on transactivation is universally observed after acute p53 activation, whereas the effect on cellular outcome is cell-type specific. Reducing mutant p53 levels abrogated the DN effect. Mutant p53's DN effect protected against radiation-induced death but did not accentuate tumorigenesis. Furthermore, the R246S mutant did not promote tumorigenesis compared to p53(-/-) mice in various models, even when MDM2 is absent, unlike the R172H mutant. Together, these data demonstrate that mutant p53's DN property only affects acute responses, whereas GOF is not universal, being mutation-type specific.
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http://dx.doi.org/10.1016/j.ccr.2012.10.022DOI Listing
December 2012

Competition between NBS1 and ATMIN controls ATM signaling pathway choice.

Cell Rep 2012 Dec 6;2(6):1498-504. Epub 2012 Dec 6.

Mammalian Genetics Laboratory, Cancer Research UK, London Research Institute, London, UK.

Ataxia telangiectasia mutated (ATM) protein kinase activation by DNA double-strand breaks (DSBs) requires the Mre11-Rad50-NBS1 (MRN) complex, whereas ATM interactor (ATMIN) protein is required for ATM signaling induced by changes in chromatin structure. We show here that NBS1 and ATMIN proteins compete for ATM binding and that this mechanism controls ATM function. DSB-induced ATM substrate phosphorylation was increased in atmin mutant cells. Conversely, NBS1 deficiency resulted in increased ATMIN-dependent ATM signaling. Thus, the absence of one cofactor increased flux through the alternative pathway. Notably, ATMIN deficiency rescued the cellular lethality of NBS1-deficient cells, and NBS1/ATMIN double deficiency resulted in complete abrogation of ATM signaling and profound radiosensitivity. Hence, ATMIN and NBS1 mediate all ATM signaling by DSBs, and increased ATMIN-dependent ATM signaling explains the different phenotypes of nbs1- and atm-mutant cells. Thus, the antagonism and redundancy of ATMIN and NBS1 constitute a crucial regulatory mechanism for ATM signaling and function.
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http://dx.doi.org/10.1016/j.celrep.2012.11.002DOI Listing
December 2012

A novel role of human holliday junction resolvase GEN1 in the maintenance of centrosome integrity.

PLoS One 2012 16;7(11):e49687. Epub 2012 Nov 16.

Disease Genomics and Individualized Medicine Laboratory, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, P.R. China.

The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity in vitro and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase in vivo as well as in vitro. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049687PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500319PMC
June 2013

A distinct response to endogenous DNA damage in the development of Nbs1-deficient cortical neurons.

Cell Res 2012 May 3;22(5):859-72. Epub 2012 Jan 3.

Department of Pathology and Center for Experimental Animal Research, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.

Microcephaly is a clinical characteristic for human nijmegen breakage syndrome (NBS, mutated in NBS1 gene), a chromosomal instability syndrome. However, the underlying molecular pathogenesis remains elusive. In the present study, we demonstrate that neuronal disruption of NBS (Nbn in mice) causes microcephaly characterized by the reduction of cerebral cortex and corpus callosum, recapitulating neuronal anomalies in human NBS. Nbs1-deficient neocortex shows accumulative endogenous DNA damage and defective activation of Ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway upon DNA damage. Notably, in contrast to massive apoptotic cell death in Nbs1-deficient cerebella, activation of p53 leads to a defective neuroprogenitor proliferation in neocortex, likely via specific persistent induction of hematopoietic zinc finger (Hzf) that preferentially promotes p53-mediated cell cycle arrest whilst inhibiting apoptosis. Moreover, Trp53 mutations substantially rescue the microcephaly in Nbs1-deficient mice. Thus, the present results reveal the first clue that developing neurons at different regions of brain selectively respond to endogenous DNA damage, and underscore an important role for Nbs1 in neurogenesis.
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http://dx.doi.org/10.1038/cr.2012.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3343649PMC
May 2012

Differential function of NBS1 and ATR in neurogenesis.

DNA Repair (Amst) 2012 Feb 9;11(2):210-21. Epub 2011 Nov 9.

Leibniz Institute for Age Research, Fritz Lipmann Institute, Beutenbergstrasse 11, Jena, Germany.

MRN (MRE11/RAD50/NBS) helps to activate ATM in response to DNA double strand breaks (DSBs) and also facilitates ATR activation by catalyzing the formation and extension of DNA single strand breaks (SSBs). Mutations of NBS1 and ATR cause human genomic instability syndrome NBS and ATR-Seckel, respectively, both of which feature neurodevelopmental defects. Whether these two DNA damage response components interact to prevent neuropathology is largely unknown. Here we show that a deletion of Nbs1 or Atr in the mouse central nervous system (CNS) results in neurodevelopmental defects characterized by reduced proliferation and increased apoptosis in embryonic brains. In contrast to Nbs1, deletion of Atr alone and both Nbs1 and Atr in the CNS causes early postnatal lethality, indicating a wider function of Atr. Importantly, deletion of Nbs1 and Atr together results in dramatic proliferation defects in neuroprogenitors. Whereas most apoptosis in the Nbs1-deleted cortex is restricted to the highly proliferating progenitors, Atr knockout induces apoptosis in both proliferating and non-proliferating neural cells. Consistently, an inducible deletion of Atr or Nbs1-Atr, but not of Nbs1, triggers a p53-independent cell death pathway in differentiated neurons, albeit elevated DNA damage in Nbs1 null neurons. Altogether, we identify a distinct function of Nbs1 and Atr in neurogenesis, namely a specific function of Nbs1 in proliferating neuroprogenitors and of Atr in both proliferating and non-dividing cells.
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http://dx.doi.org/10.1016/j.dnarep.2011.10.021DOI Listing
February 2012

Cancer, senescence, and aging: translation from basic research to clinics.

J Aging Res 2011 20;2011:692301. Epub 2011 Oct 20.

Oncología y Patología Grupo, Vall d'Hebron Institut de Recerca, 08035 Barcelona, Spain.

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http://dx.doi.org/10.4061/2011/692301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199198PMC
November 2011

MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1-Cdc25 pathway.

Nat Cell Biol 2011 Sep 25;13(11):1325-34. Epub 2011 Sep 25.

Leibniz Institute for Age Research-Fritz Lipmann Institute, Beurtenbergstrasse 11, 07745 Jena, Germany.

Primary microcephaly 1 is a neurodevelopmental disorder caused by mutations in the MCPH1 gene, whose product MCPH1 (also known as microcephalin and BRIT1) regulates DNA-damage response. Here we show that Mcph1 disruption in mice results in primary microcephaly, mimicking human MCPH1 symptoms, owing to a premature switching of neuroprogenitors from symmetric to asymmetric division. MCPH1-deficiency abrogates the localization of Chk1 to centrosomes, causing premature Cdk1 activation and early mitotic entry, which uncouples mitosis and the centrosome cycle. This misorients the mitotic spindle alignment and shifts the division plane of neuroprogenitors, to bias neurogenic cell fate. Silencing Cdc25b, a centrosome substrate of Chk1, corrects MCPH1-deficiency-induced spindle misalignment and rescues the premature neurogenic production in Mcph1-knockout neocortex. Thus, MCPH1, through its function in the Chk1-Cdc25-Cdk1 pathway to couple the centrosome cycle with mitosis, is required for precise mitotic spindle orientation and thereby regulates the progenitor division mode to maintain brain size.
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http://dx.doi.org/10.1038/ncb2342DOI Listing
September 2011

Malfunctioning DNA damage response (DDR) leads to the degeneration of nigro-striatal pathway in mouse brain.

J Mol Neurosci 2012 Mar 16;46(3):554-68. Epub 2011 Sep 16.

Department of Neurobiology, George S. Wise, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel.

Pronounced neuropathology is a feature of ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS), which are both genomic instability syndromes. The Nbs1 protein, which is defective in NBS, is a component of the Mre11/RAD50/NBS1 (MRN) complex. This complex plays a major role in the early phase of the cellular response to double strand breaks (DSBs) in the DNA. Among others, MRN is required for timely activation of the protein kinase ATM (A-T mutated), which is disrupted in patients with A-T. Earlier reports show that Atm-deficient mice exhibit severe degeneration of tyrosine hydroxylase (TH)-positive dopaminergic nigro-striatal neurons and their terminals in the striatum. This cell loss is accompanied by a large reduction in immunoreactivity for the dopamine transporter protein (DAT) in the striatum. To test whether Nbs1 inactivation also affects the integrity of the nigro-striatal pathway, we examined this pathway in a murine model with conditional inactivation of the Nbs1 gene in central nervous system (Nbs1-CNS-Δ). We report that this model has a reduction in TH-positive cells in the substantia nigra. This phenomenon was seen at very early age, while Atm-/- mice showed a progressive age-dependent reduction. Furthermore, we observed an age-dependent increase in the level of TH in the striatum of Atm-/- and Nbs1-CNS-Δ mice. In addition to the altered expression of TH, we also found a reduction of DAT in the striatum of both Atm-/- and Nbs1-CNS-Δ mice at 60 days of age. Finally, microglial recruitment and alterations in the levels of various neurotrophic factors were also observed. These results indicate that malfunctioning DNA damage response severely affects the integrity of the nigro-striatal pathway and suggest a new neurodegenerative pathway in Parkinsonian syndromes.
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http://dx.doi.org/10.1007/s12031-011-9643-yDOI Listing
March 2012

Requirement for DNA ligase IV during embryonic neuronal development.

J Neurosci 2011 Jul;31(27):10088-100

Development and Stem Cell Institute, Key Laboratory of Ministry of Education, Department of Paediatrics, West China Second University Hospital, Sichuan University, Chengdu, China.

The embryonic ventricular and subventricular zones (VZ/SVZ) contain the neuronal stem and progenitor cells and undergo rapid proliferation. The intermediate zone (IZ) contains nonreplicating, differentiated cells. The VZ/SVZ is hypersensitive to radiation-induced apoptosis. Ablation of DNA non-homologous end-joining (NHEJ) proteins, XRCC4 or DNA ligase IV (LigIV), confers ataxia telangiectasia mutated (ATM)-dependent apoptosis predominantly in the IZ. We examine the mechanistic basis underlying these distinct sensitivities using a viable LigIV (Lig4(Y288C)) mouse, which permits an examination of the DNA damage responses in the embryonic and adult brain. Via combined analysis of DNA breakage, apoptosis, and cell-cycle checkpoint control in tissues, we show that apoptosis in the VZ/SVZ and IZ is activated by low numbers of DNA double-strand breaks (DSBs). Unexpectedly, high sensitivity in the VZ/SVZ arises from sensitive activation of ATM-dependent apoptosis plus an ATM-independent process. In contrast, the IZ appears to be hypersensitive to persistent DSBs. NHEJ functions efficiently in both compartments. The VZ/SVZ and IZ regions incur high endogenous DNA breakage, which correlates with VZ proliferation. We demonstrate a functional G(2)/M checkpoint in VZ/SVZ cells and show that it is not activated by low numbers of DSBs, allowing damaged VZ/SVZ cells to transit into the IZ. We propose a novel model in which microcephaly in LIG4 syndrome arises from sensitive apoptotic induction from persisting DSBs in the IZ, which arise from high endogenous breakage in the VZ/SVZ and transit of damaged cells to the IZ. The VZ/SVZ, in contrast, is highly sensitive to acute radiation-induced DSB formation.
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http://dx.doi.org/10.1523/JNEUROSCI.1324-11.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154654PMC
July 2011

Point mutation at the Nbs1 Threonine 278 site does not affect mouse development, but compromises the Chk2 and Smc1 phosphorylation after DNA damage.

Mech Ageing Dev 2011 Aug 2;132(8-9):382-8. Epub 2011 Jun 2.

Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena, Germany.

NBS1, mutated in Nijmegen breakage syndrome (NBS), senses the DNA double strand breaks (DSBs) and initiates the DNA damage response (DDR) by activating ATM kinase. Meanwhile, NBS1 is phosphorylated by ATM at Serine 278 and Serine 343 and thereby assists the activation of the ATM downstream targets. To study the physiological function of the Nbs1 phosphorylation, we have knocked in a point mutation in the moue genome that results in the replacement of Threonine 278 (equivalent to the human Serine 278) by Alanine. The Nbs1(T278A) knock-in mice develop normally and show no gross defects. The mutation of this phosphorylation site does not affect the proliferation or genomic stability. Ionizing radiation (IR) of primary Nbs1(T278A) MEFs reveals no obvious defects in the Chk2 phosphorylation at 1Gy, but a delayed phosphorylation of Chk2 and Smc1 only at intermediate (4.5Gy) and high (10Gy) doses, respectively. In contrast to Serine 343 mutant, Threonine 278 mutation has no effect on the HU-induced ATR-Chk1 activation. Our study thus shows that Nbs1 phosphorylation at the Threonine 278 is dispensable for mouse development and plays a differential function in assisting the DDR of downstream effectors in vivo, depending on the doses of DNA damage.
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http://dx.doi.org/10.1016/j.mad.2011.05.001DOI Listing
August 2011

Progress in the function and regulation of ADP-Ribosylation.

Sci Signal 2011 May 24;4(174):mr5. Epub 2011 May 24.

Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse, Switzerland.

Adenosine 5'-diphosphate (ADP)-ribosylation is a protein posttranslational modification that is catalyzed by ADP-ribosyltransferases (ARTs), using nicotinamide adenine dinucleotide (NAD(+)) as a substrate. Mono-ribosylation can be extended into polymers of ADP-ribose (PAR). Poly(ADP-ribosyl)polymerase (PARP) 1, the best-characterized cellular enzyme catalyzing this process, is the prototypical member of a family of mono- and poly(ADP-ribosyl)transferases. The physiological consequences of ADP-ribosylation are inadequately understood. PARP2010, the 18th International Conference on ADP-Ribosylation, attracted scientists from all over the world to Zurich, Switzerland. Highlights from this meeting include promising clinical trials with PARP inhibitors and new insights into cell, structural, and developmental biology of ARTs and the (glyco)hydrolase proteins that catalyze de-ADP-ribosylation of mono- or poly-ADP-ribosylated proteins. Moreover, potential links to the NAD-dependent sirtuin family were explored on the basis of a shared dependence on cellular NAD(+) concentrations and the relationship of ADP-ribosylation with intermediary metabolism and cellular energetics.
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http://dx.doi.org/10.1126/scisignal.2001645DOI Listing
May 2011