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.     

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.Key words: chromosome condensation, DNA damage, G₂/M checkpoint, ionizing radiation, PCC syndrome, primary microcephaly, repair foci     

MCPH1 regulates chromosome condensation and shaping as a composite modulator of condensin II

Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, which is characterized by a marked reduction of brain size. Interestingly, hMCPH1 mutant patient cells display unique cellular phenotypes, including premature chromosome condensation (PCC), in G2 phase. To test whether hMCPH1 might directly participate in the regulation of chromosome condensation and, if so, how, we developed a cell-free assay using Xenopus laevis egg extracts. Our results demonstrate that an N-terminal domain of hMCPH1 specifically inhibits the action of condensin II by competing for its chromosomal binding sites in vitro. This simple and powerful assay allows us to dissect mutations causing primary microcephaly in vivo and evolutionary substitutions among different species. A complementation assay using patient cells revealed that, whereas the N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, its central domain plays an auxiliary role in shaping metaphase chromosomes by physically interacting with condensin II. Thus, hMCPH1 acts as a composite modulator of condensin II to regulate chromosome condensation and shaping.     

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.     

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.     

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.     

Novel Alternative Splice Variants of Mouse Cdk5rap2

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.     

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.     

A Pocket on the Surface of the N-Terminal BRCT Domain of Mcph1 Is Required to Prevent Abnormal Chromosome Condensation

Mcph1 is mutated in autosomal recessive primary microcephaly and premature chromosome condensation (PCC) syndrome. Increased chromosome condensation is a common feature of cells isolated from patients afflicted with either disease. Normal cells depleted of Mcph1 also exhibit PCC phenotype. Human Mcph1 contains three BRCA1-carboxyl terminal (BRCT) domains, the first of which (Mcph1N) is necessary for the prevention of PCC. The only known disease-associated missense mutation in Mcph1 resides in this domain (T27R). We have determined the X-ray crystal structure of human Mcph1N to 1.6 Å resolution. Compared with other BRCT domain structures, the most striking differences are an elongated, ordered β1-α1 loop and an adjacent hydrophobic pocket. This pocket is in the equivalent structural position to the phosphate binding site of BRCT domains that recognize phospho-proteins, although the phosphate-binding residues are absent in Mcph1N. Mutations in the pocket abrogate the ability of full-length Mcph1 to rescue the PCC phenotype of Mcph1^−/− mouse embryonic fibroblast cells, suggesting that it forms an essential part of a protein-protein interaction site necessary to prevent PCC.     

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

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.     

Identification of microcephalin,a protein implicated in determining the size of the human brain

Primary microcephaly (MIM 251200) is an autosomal recessive neurodevelopmental condition in which there is a global reduction in cerebral cortex volume, to a size comparable with that of early hominids. We previously mapped the MCPH1 locus, for primary microcephaly, to chromosome 8p23, and here we report that a gene within this interval, encoding a BRCA1 C-terminal domain-containing protein, is mutated in MCPH1 families sharing an ancestral 8p23 haplotype. This gene, microcephalin, is expressed in the developing cerebral cortex of the fetal brain. Further study of this and related genes may provide important new insights into neocortical development and evolution.     

What primary microcephaly can tell us about brain growth

Autosomal recessive primary microcephaly (MCPH) is a neuro-developmental disorder that causes a great reduction in brain growth in utero. MCPH is hypothesized to be a primary disorder of neurogenic mitosis, leading to reduced neuron number. Hence, MCPH proteins are likely to be important components of cellular pathways regulating human brain size. At least six genes can cause this disorder and four of these have recently been identified: autosomal recessive primary microcephaly 1 (MCPH1), abnormal spindle-like, microcephaly associated (ASPM), cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) and centromere protein J (CENPJ). Whereas aberration of ASPM is the most common cause of MCPH, MCPH1 patients can be more readily diagnosed by the finding of increased numbers of "prophase-like cells" on routine cytogenetic investigation. Three MCPH proteins are centrosomal components but have apparently diverse roles that affect mitosis. There is accumulating evidence that evolutionary changes to the MCPH genes have contributed to the large brain size seen in primates, particularly humans. The aim of this article is to review what has been learnt about the rare condition primary microcephaly and the information this provides about normal brain growth.     

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.     

Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B–Cdk1.