MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G2/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.     

SET nuclear oncogene associates with microcephalin/MCPH1 and regulates chromosome condensation

Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.     

Microcephalin: A Causal Link Between Impaired Damage Response Signalling and Microcephaly

Seckel Syndrome (SS) and Primary Microcephaly (MCPH) are disorders exhibiting marked microcephaly with a head circumference less than 3 standard deviations below the mean. ATR-Seckel Syndrome is conferred by mutations in ataxia and telangiectasia and Rad3 related (ATR), a kinase that activates a DNA damage signalling response. Cell lines from additional SS patients, who are normal for ATR, show defective ATR signalling, suggesting that they carry mutations in other components of the ATR pathway. Primary Microcephaly is distinct from SS since patients displaying solely microcephaly without accompanying growth delay. MCPH1, the first Primary Microcephaly causative gene identified, encodes three BRCT domains, similar to other damage response proteins. Recent studies employing MCPH1 siRNA or exploiting cell lines from MCPH1 patients have shown that MCPH1 functions in the ATR-dependent DNA damage response pathway. Additionally, MCPH1 has a function in the regulation of mitotic entry that is ATR-independent and confers a characteristic phenotype of premature chromosome condensation. Recent studies will be reviewed and their relationship to the aetiology of microcephaly discussed.     

Mutations in microcephalin cause aberrant regulation of chromosome condensation

Microcephalin (MCPH1) is a gene mutated in primary microcephaly, an autosomal recessive neurodevelopmental disorder in which there is a marked reduction in brain size. PCC syndrome is a recently described disorder of microcephaly, short stature, and misregulated chromosome condensation. Here, we report the finding that MCPH1 primary microcephaly and PCC syndrome are allelic disorders, both having mutations in the MCPH1 gene. The two conditions share a common cellular phenotype of premature chromosome condensation in the early G2 phase of the cell cycle, which, therefore, appears to be a useful diagnostic marker for individuals with MCPH1 gene mutations. We demonstrate that an siRNA-mediated depletion of MCPH1 is sufficient to reproduce this phenotype and also show that MCPH1-deficient cells exhibit delayed decondensation postmitosis. These findings implicate microcephalin as a novel regulator of chromosome condensation and link the apparently disparate fields of neurogenesis and chromosome biology. Further characterization of MCPH1 is thus likely to lead to fundamental insights into both the regulation of chromosome condensation and neurodevelopment.     

Establishment of a Mouse Model with Misregulated Chromosome Condensation due to Defective Mcph1 Function

Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1^gt/gt mice, the overall survival rates of the Mcph1^gt/gt animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function.     

A novel homozygous frameshift variant in the <Emphasis Type="Italic">MCPH1 gene causes</Emphasis> primary microcephaly in a consanguineous Saudi family

Primary microcephaly (MCPH) is a rare developmental defect characterized by impaired cognitive functions, retarded neurodevelopment and reduced brain size. It is genetically heterogeneous and so far more than 17 genes associated with this disease have been identified. Primary microcephaly type 1 (MCPH1) gene encodes a protein called microcephalin, which is implicated in chromosome condensation and DNA damage induced cellular responses. It is suggested to play a role in neurogenesis and regulation of the size of the cerebral cortex. Whole exome sequencing revealed a novel, homozygous frameshift mutation (c.373_374delAA) in MCPH1 gene in exon 5 resulting in frameshift change from p.Lys125Glusfs*7. Our report presents the results of the simultaneous analysis of the trio exome data of both unaffected parents and their affected son. A homozygous frameshift variant in the MCPH1 gene was identified as a plausible candidate causal variant for the clinical phenotype in this family.     

MCPH1 functions in an H2AX-dependent but MDC1-independent pathway in response to DNA damage

Microcephalin (MCPH1) is one of the causative genes for the autosomal recessive disorder, primary microcephaly, characterized by dramatic reduction in brain size and mental retardation. MCPH1 also functions in the DNA damage response, participating in cell cycle checkpoint control. However, how MCPH1 is regulated in the DNA damage response still remains unknown. Here we report that the ability of MCPH1 to localize to the sites of DNA double-strand breaks depends on its C-terminal tandem BRCT domains. Although MCPH1 foci formation depends on H2AX phosphorylation after DNA damage, it can occur independently of MDC1. We also show that MCPH1 binds to a phospho-H2AX peptide in vitro with an affinity similar to that of MDC1, and overexpression of wild type, but not C-BRCT mutants of MCPH1, can interfere with the foci formation of MDC1 and 53BP1. Collectively, our data suggest MCPH1 is recruited to double-strand breaks via its interaction with gammaH2AX, which is mediated by MCPH1 C-terminal BRCT domains. These observations support that MCPH1 acts early in DNA damage responsive pathways.     

DNA damage response in microcephaly development of MCPH1 mouse model

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.     

Never-in-mitosis related Kinase 1 functions in DNA damage response and checkpoint control

Nek1, the first mammalian ortholog of the fungal protein kinase never in mitosis A, is involved early in the DNA damage sensing/repair pathway after ionizing radiation. Here we extend this finding by showing that Nek1 localizes to nuclear foci of DNA damage in response to many different types of damage in addition to IR. Untransformed cells established from kat2J/Nek1 -/- mice fail to arrest properly at G1/S and M-phase checkpoints in response to DNA damage. G1-S-phase checkpoint control can be rescued by ectopically overexpressing wild-type Nek1. In Nek1-/- murine cells and in human cells with Nek1 expression silenced by siRNA, the checkpoint kinases Chk1 and Chk2 fail to be activated properly in response to ionizing or UV radiation. In cells without functional Nek1, DNA is not repaired properly, double-stranded DNA breaks persist long after low dose IR, and excessive numbers of chromosome breaks are observed. These data show that Nek1 is important for efficient DNA damage checkpoint control and for proper DNA damage repair.     

Mutations in Centrosomal Protein CEP152 in Primary Microcephaly Families Linked to MCPH4

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.     

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G₂/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G₂/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G₂/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G₂/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.     

Induced Pluripotent Stem Cells Are Sensitive to DNA Damage

Induced pluripotent stem cells(iPSCs)resemble embryonic stem cells(ESCs)in morphology,gene expression and in vitro differentiation,raising new hope for personalized clinical therapy.While many efforts have been made to improve reprogramming effciency,signifcant problems such as genomic instability of iPSCs need to be addressed before clinical therapy.In this study,we try to fgure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation(IR).We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fbroblast(MEF)cells,from which 3FB4-1line was derived.After the introduction of DNA damage by IR,the number of c-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF,albeit both signifcantly(P<0.01).In addition,whole-genome sequencing analysis showed that after IR,3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes.These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity.The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.     

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G₀ and G₁ phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.     

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G₁/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild-type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1^−/− murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.Key words: checkpoint control, DNA damage response, Nek1, ATM, ATR     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Emerging roles of MCPH1: Expedition from primary microcephaly to cancer

Genetic mutations in microcephalin1 (MCPH1) cause primary autosomal recessive microcephaly which is characterized by a marked reduction in brain size. MCPH1 encodes a centrosomal protein with three BRCT (BRCA1 C-terminal) domains. Also, it is a key regulator of DNA repair pathway and cell cycle checkpoints. Interestingly, in the past few years, many research studies have explored the role of MCPH1, a neurodevelopmental gene in several cancers and its tumor suppressor functions have been elucidated. Given the diverse new emerging roles, it becomes critical to review and summarize the multiple roles of MCPH1 that is currently lacking in the literature. In this review after systematic analysis of literature, we summarise the multiple functional roles of MCPH1 in centrosomal, DNA repair and apoptotic pathways. Additionally, we discuss the considerable efforts taken to understand the implications of MCPH1 in diseases such as primary microcephaly and its other emerging association with cancer and otitis media. The promising view is that MCPH1 has distinct roles and its clinical associations in various diseases makes it an attractive therapeutic target.