[Show abstract][Hide abstract] ABSTRACT: 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.
The EMBO Journal 03/2015; DOI:10.15252/embj.201490070 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 06/2014; 9(6):e99956. DOI:10.1371/journal.pone.0099956 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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. GLIA 2014;62:133-144.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR
in vitro. All three can also independently activate Mec1ATR
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
DNA repair 05/2013; 12(8). DOI:10.1016/j.dnarep.2013.04.017 · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 11/2012; 7(11):e49687. DOI:10.1371/journal.pone.0049687 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cell Research 01/2012; 22(5):859-72. DOI:10.1038/cr.2012.3 · 12.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
DNA repair 11/2011; 11(2):210-21. DOI:10.1016/j.dnarep.2011.10.021 · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nijmegen breakage syndrome (NBS) is a genomic instability disorder caused by hypomorphic mutations in the Nbs1 gene. When Nbs1 is conditionally inactivated in the central nervous system of mice (Nbs1-CNS-Δ), they suffer from severe cerebellar atrophy, ataxia, and white matter damage. Here, we show that conditional inactivation of the murine Nbs1 gene has a profound effect on the integrity and the functionality of the glial cells, which suggests their crucial role in the pathogenesis of NBS. Interestingly, in Nbs1-CNS-Δ mice, the dramatic reduction in the numbers of Purkinje and granule cells was also linked to a reduction of microglial cells but not to astrocytes (GFAP+), suggesting an impairment in astrocytic functionality. Nbs1 levels were dramatically reduced in adult astrocyte isolated from Nbs1-CNS-Δ mice, suggesting a major role in cerebellar pathology. In order to investigate the effect of Nbs1 deletion on astrocyte activity, we investigated glutamine synthetase levels in astrocyte and discovered 40% reduction as compared to WT. Furthermore, we found a significant reduction in the secretion of neurotrophic factors, such as brain-derived neurotrophic factor and neurotrophin 3. Understanding the contribution of malfunctioning astrocytes to the etiology of NBS can elucidate a hitherto unknown aspect of this disorder.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/2011; 31(27):10088-100. DOI:10.1523/JNEUROSCI.1324-11.2011 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Mechanisms of ageing and development 06/2011; 132(8-9):382-8. DOI:10.1016/j.mad.2011.05.001 · 3.40 Impact Factor