Microcephalin: Biological Overview | References
Gene name - Microcephalin
Cytological map position-48C5-48C5
Keywords - cell cycle, co-ordination of centrosome and nuclear division cycles, regulation and maintenance of chromosome condensation
Symbol - MCPH1
FlyBase ID: FBgn0260959
Genetic map position - 2R: 7,784,347..7,791,419 [-]
Classification - Breast Cancer Suppressor Protein (BRCA1), carboxy-terminal domain
Cellular location - nuclear
Microcephalin (MCPH1) is mutated in primary microcephaly, an autosomal recessive human disorder of reduced brain size. It encodes a protein with three BRCT domains that has established roles in DNA damage signalling and the cell cycle, regulating chromosome condensation. Significant adaptive evolutionary changes in primate MCPH1 sequence suggest that changes in this gene could have contributed to the evolution of the human brain. To understand the developmental role of microcephalin its function in Drosophila has been studied. Drosophila MCPH1 is cyclically localised during the cell cycle, co-localising with DNA during interphase, but not with mitotic chromosomes. mcph1 mutant flies have a maternal effect lethal phenotype, due to mitotic arrest occurring in early syncytial cell cycles. Mitotic entry is slowed from the very first mitosis in such embryos, with prolonged prophase and metaphase stages; and frequent premature separation as well as detachment of centrosomes. As a consequence, centrosome and nuclear cycles become uncoordinated, resulting in arrested embryonic development. Phenotypic similarities with abnormal spindle (asp) and centrosomin (cnn) mutants (whose human orthologues are also mutated in primary microcephaly), suggest that further studies in the Drosophila embryo may establish a common developmental and cellular pathway underlying the human primary microcephaly phenotype (Brunk, 2007).
Mutation in microcephalin (MCPH1) causes primary microcephaly (MIM#251200), an autosomal recessive disorder of human brain size in which brain volume is reduced to a third of normal, a size comparable with that of early hominids (McCreary, 1996; Wood, 1999; Woods, 2005). Comparative genomic sequencing has established that significant adaptive evolutionary changes have occurred in microcephalin in primates, in the lineage from the last common simian ancestor to man (Evans, 2004; Wang, 2004). It has thus been suggested that changes in this gene could have contributed to the evolution of the human brain (Brunk, 2007).
Microcephalin (MCPH1) is the first of four disease genes to be identified for this condition (Jackson, 2002) and encodes an 835 amino acid protein containing 3 BRCA1 C-terminal (BRCT) domains (Huyton, 2000). Such domains can bind other proteins in a phosphorylation-dependent manner (Yu, 2003), explaining their presence in many proteins involved in DNA repair and regulation of cell cycle (Huyton, 2000), processes dependent on phosphorylation signals. Subsequently microcephalin has been found to have multiple roles in cell cycle and DNA repair. Microcephalin has been implicated in the timing of cell cycle events and specifically in the regulation of chromosome condensation (Neitzel, 2002; Trimborn, 2004) and Cdk1 phosphorylation (Alderton, 2006). Both RNAi depletion and mutation of microcephalin result in a cellular phenotype of premature chromosome condensation (Neitzel, 2002; Trimborn, 2004). This is the consequence of premature onset of chromosome condensation, mediated by condensin II in G2 and delayed decondensation after mitosis (Trimborn, 2004; Trimborn, 2006), and correlates with the loss of inhibitory Cdk1 phosphorylation in S and G2 in microcephalin-deficient cells (Alderton, 2006). In the context of DNA repair, microcephalin has been found to be a chromatin-associated protein that also colocalizes with DNA repair proteins at ionizing radiation-induced DNA repair foci (Lin, 2005; Rai, 2006; Xu, 2004). It is required for intra-S and G2-M checkpoints (Lin, 2005; Xu, 2004), and acts downstream of Chk1 in the ATR signalling cascade regulating Cdc25A stability, and consequently G2-M transition (Brunk, 2007 and references therein).
Three other primary microcephaly genes have been identified, ASPM, CENPJ and CDK5RAP2 (Bond, 2002; Bond, 2005). All their proteins have a centrosomal localization, and Drosophila orthologues that are required for centrosomal function: Abnormal spindle (Asp), Sas-4 and Centrosomin (Cnn), respectively. Asp is required for the integrity of centrosomes as microtubule-organizing centres, focusing the spindle poles at mitosis. Cnn is essential for the localisation of other centrosomal components, gamma tubulin, CP190 and CP60, whereas Sas-4 is necessary for centriole duplication, with mutant flies consequently lacking centrosomes, cilia and flagella (Basto, 2006). Despite these important cellular functions, all three mutants can generate morphologically normal adults, though sas-4 mutants die shortly after birth as they lack sensory neurons. asp and cnn have maternal-effect lethal alleles in which mitotic arrest occurs in embryonic cell cycles prior to cellularisation. Defects in localisation of asymmetric determinants have also been observed in cnn and sas-4 neuroblasts (Brunk, 2007 and references therein).
Overall, there is a unifying theme for primary microcephaly of genes functioning in processes of cell division. It has therefore been proposed that reduced brain size is the result of reduced 'neurogenic' mitosis, perhaps as a consequence of perturbed neural progenitor asymmetric cell division. However, microcephalin's function has appeared to be distinct from the other genes, and so this study has generated a Drosophila model for microcephalin, to characterise its developmental role and to examine its mitotic functions. mcph1 mutants are shown to have a maternal effect lethal phenotype; a short isoform of microcephalin is required for the co-ordination of syncytial centrosomal and nuclear division cycles (Brunk, 2007).
The Drosophila melanogaster homologue of microcephalin was identified by TBLASTN searches of the Drosophila genomic and EST databases using the human protein sequence. A transcript, mcph1(L), is predicted corresponds to the human microcephalin protein that combines two adjacent annotated genes (CG8981 and CG30038) on chromosome 2R at 48C5. This transcript was confirmed by RT-PCR and sequencing of adult testis mRNA. The nine exon mcph1 gene encodes a 1028 amino acid protein sharing 18% amino acid identity with human microcephalin. Like its human counterpart, Drosophila MCPH1 has a unique pattern of BRCT domains, with a single N-terminal and tandem C-terminal BRCT domains, and is predicted (PSORT II) to be a nuclear protein (Brunk, 2007).
mcph1 has two sites of alternative splicing at intron 1 and 7, potentially resulting in four processed transcripts. The fully spliced transcript encodes the longest protein isoform, MCPH1(L), that contains all three BRCT domains. This transcript was experimentally validated. A second transcript, mcph1(S), encodes a short (S) variant of MCPH1 containing only one functional, N-terminal BRCT domain. This transcript contains an unspliced intron 7, resulting in a premature stop codon). This transcript was confirmed through re-sequencing of a full-length cDNA clone from the Drosophila Genomic Collection (Brunk, 2007).
EST data also suggests the presence of other mcph1 transcripts with an unspliced intron 1 and an alternative translational start site that results in a different, shorter N-terminal coding sequence. In combination with alternative splicing of intron 7, these would encode two further proteins. These proteins would have domain structures similar to MCPH1(S) and MCPH1(L), but be shorter by 47 amino acids at their N termini. The existence of unspliced intron 1 transcripts, which appear to be present in all developmental stages, was experimentally confirmed. Unlike the intron 7 alternative splicing, the intron 1 splicing is not predicted to alter domain structure; therefore subsequent analysis focused on the 'full length' MCPH1(L) and MCPH1(S) proteins (Brunk, 2007).
RT-PCR experiments established that mcph1 transcripts are present at all developmental stages from embryo to adults. Whole-mount in situ hybridisation of wild-type embryos was performed using a riboprobe common to both L and S transcripts. This detected mcph1 mRNA at its highest level in syncytial stages, distributed throughout the embryo. By the cellular blastoderm stage minimal mcph1 transcript was apparent, consistent with mcph1 being maternally derived until cellularisation and midblastula transition. During gastrulation, mcph1 transcript is again detectable, presumably as a result of zygotic expression of the gene (Brunk, 2007).
To characterise MCPH1 protein expression, polyclonal rabbit antibodies were raised against an N-terminal fragment of the protein. The affinity purified antibody detected a single specific 115 kDa band in immunoblots performed on extracts of syncytial embryos that is not present in embryos derived from mothers homozygous for a mcph1 deletion (mcph1d2/d2), hereafter referred to as mcph1d2/d2 embryos. This protein was also present in ovaries and at reduced levels in larval brain. This band corresponds to the predicted molecular mass of the L isoform; however, human microcephalin protein migrates significantly slower than predicted, so the 115 kDa band could represent a shorter isoform (Brunk, 2007).
Drosophila MCPH1 localisation is mitotically regulated and mcph1 mutant flies have a maternal effect lethal phenotype, as a result of mitotic arrest occurring in early syncytial cell cycles. S short isoform of MCPH1 lacking C-terminal BRCT domains rescues this phenotype, indicating that this protein has an essential role in these embryonic cell cycles. GFP-MCPH1(S) localises to the centrosomes during mitosis, suggesting that it might have centrosomally related functions, relevant to the primary microcephaly phenotype. No centrosomal or spindle abnormalities during neuroblast mitosis have been observed in the larval brains of mcph1 mutants and the asymmetric determinants Inscuteable and Miranda also appear to be appropriately localised. Therefore, the centrosomal abnormalities appear to be specific to the early embryonic cell cycles. These data, along with the slowed rate of nuclear division in embryos, have led to the conclusion that the embryonic phenotype is the result of nuclear division cycle being slowed relative to the centrosome cycle. Additional centrosomes are consequently generated, perturbing subsequent nuclear divisions, leading to nuclear fragmentation and terminal mitotic arrest or catastrophe. In the syncytial embryo even a small reduction in nuclear division rate may have major effects, as cell cycle is very rapid, comprising rapid synchronous S and M phases without intervening G1 and G2 phases. A subtle effect on cell division timing in all cells could therefore be sufficient to explain the essential requirement for MCPH1 in the syncytial embryos, but not in other cell cycles. Alternatively, MCPH1, and in particular the S isoform may have a specialised function, which is important for these cycles. It is important to note that human MCPH1 has been reported to localize to the nucleus (Lin, 2005) as well as to centrosomes (Jeffers, 2007; Zhong, 2006; Brunk, 2007).
What causes the nuclear cycle to slow? The nuclear cycle is retarded prior to metaphase in cycle 1. This might be because of prolonged mitotic entry, similar to the perturbed cell cycle regulation observed in human cells where there is an extended prophase-like state of 'premature chromosome condensation' (Alderton, 2006; Trimborn, 2004). In this view, the loss of MCPH1 could affect the coordination of chromosome condensation with the rest of cell cycle entry. The larger, less compacted nuclei seen at pronuclear fusion and prophase could suggest that chromosome compaction or condensation might be delayed relative to other events at mitotic entry. However, live imaging of mutant embryos would be required to confirm this, but has not been technically possible in such early embryos. Immunofluorescence and live imaging studies of GFP-tagged and endogenous MCPH1 protein in wild-type embryos revealed an inverse correlation of MCPH1 localisation with mitotically condensed chromosomes, consistent with a role for this chromatin-associated protein (Lin, 2005) in maintaining decondensed chromosomes. Reduced Cdk1 inhibitory phosphorylation (P-Cdk1tyr15) has been observed in S and G2 phases of human MCPH1 cell lines (Alderton, 2006), and suggested to underlie the early onset of chromosome condensation in human cells relative to other mitotic events. P-Cdk1tyr15 is also reduced in mcph1d2/d2 embryos, which could explain the asynchrony of centrosomal and nuclear events observed. However, it cannot be ruled out that this is a secondary consequence of mitotic arrest in these embryos (Brunk, 2007).
A second possibility is that S phase is prolonged in mcph1 embryos. Embryos in which DNA synthesis is blocked by aphidicolin injection, have similarly slowed cell cycles, with particularly slowed (paradoxically) M phase. Such embryos still undergo cycles of chromosome condensation and decondensation and exhibit centrosome overduplication. In Drosophila the ATR/Chk1 pathway prevents premature mitotic entry while S phase is still ongoing, only becoming developmentally required at mid-blastula transition when both ATR and Chk1 become essential. The mcph1 phenotype, however, is manifest much earlier than either of these mutants, inconsistent with its being initiated by defective ATR/Chk1 signalling. Additionally, though RNAi experiments have suggested that human microcephalin might transcriptionally regulate CHK1 (Lin, 2005; Xu, 2004), Chk1 protein levels are unaltered in mcph1 embryos. Rather it seems more likely, that there is a separate function for Drosophila MCPH1 in cell cycle regulation, independent of its downstream role in ATR/Chk1 signalling, as has been previously concluded (Alderton, 2006) from analysis of human MCPH1 patient cell lines (Brunk, 2007).
Nuclear cell cycle can also be slowed if cyclins fail to degrade, and is the cause of mitotic arrest in the Cks30A mutant. A global accumulation of Cyclin A, B or B3 does not, however, occur in mcph1d2/d2 embryos. In the embryo, cell cycle progression is regulated by localised Cyclin B degradation on the mitotic spindle. Degradation occurs from spindle poles inwards, and appears to be mediated by the centrosome. Therefore, both the poorly formed mitotic spindles and detachment of centrosomes observed in mcph1 embryos could also delay progression in the nuclear cycle through inhibition of local Cyclin B degradation. Consistent with this, Cyclin B immunostaining was observed on terminally arrested mutant nuclei (Brunk, 2007).
In summary, any of these abnormalities -- localised failure in cyclin degradation, perturbed regulation of chromosome condensation, or prolonged DNA synthesis -- could account for the mcph1 maternal effect lethal phenotype. Each would delay mitotic entry through slowing the nuclear cycle while the centrosomal cycle would continue unchecked, resulting in premature separation and detachment of centrosomes. Lost connections of centrosomes with spindles would then delay mitosis further, through impaired Cyclin B degradation, and insubstantial mitotic spindle formation. Further centrosome-nuclear incoordination subsequently would result in mitotic catastrophe and arrested embryo development (Brunk, 2007).
An alternative, more speculative, interpretation is that the mcph1 phenotype has a centrosomal aetiology. Firstly, there is a correlation between phenotypic rescue with MCPH1(S), and the apparent centrosomal localisation of this isoform during mitosis. Furthermore, the early separation of centrosomes observed could reflect a requirement for microcephalin in maintaining the two centrioles together during mitosis and attachment of centrosomes to the spindle poles. Early separation and centrosome detachment would result in failure of localised Cyclin B degradation and a consequently delayed nuclear cycle, culminating in mitotic catastrophe and arrested embryo development (Brunk, 2007).
The cerebral cortex is reduced to a third of normal volume in primary microcephaly. The marked reduction in neural cell number is presumed to be caused by inefficient neurogenic mitosis (Woods, 2005). Primary microcephaly brain size is similar to that of early hominids, and the Darwinian adaptive evolutionary changes observed in primate microcephalin suggest that changes in this gene could have contributed to the evolutionary expansion of the human brain. Given the evolutionary scaling of the brain, it is unsurprising that the invertebrate mcph1 fly brain is not grossly reduced in size. Nevertheless, the finding of this study that mcph1 is required for mitosis in the Drosophila embryo is pertinent to understanding the disease phenotype. Although a complete elucidation of the cell cycle function of MCPH1 will be necessary for unravelling its role in brain development and evolution, the finding that the short isoform, lacking C-terminal BRCT domains, functions in coordinating mitosis is likely to be relevant to the pathogenesis of primary microcephaly. Moreover, as BRCT domains are important in phosphoserine/threonine-dependent protein binding (Manke, 2003; Yu, 2003) it is predicted that this protein lacking functional C-terminal tandem BRCT domains will have significantly different functions from the full-length protein. In this context, it is notable that a recent analysis of MCPH1 in DT40 cells has demonstrated that the C-terminal BRCT domains are required for microcephalin localisation at ionising radiation-induced DNA damage repair foci (Jeffers, 2007). The fly, therefore, provides an ideal opportunity for analysis of the role of microcephalin in DNA damage repair, having physiological isoforms of microcephalin, with and without these C-terminal BRCT domains (Brunk, 2007).
Two of the other primary microcephaly homologue mutants (asp, cnn) also exhibit maternal effect lethal phenotypes with mitotic arrest and free centrosomes and are therefore also required for mitosis in the early fly embryo. Furthermore, the mitotic centrosomal localisation of GFP-MCPH1(S) is intriguing, as it suggests that Microcephalin could co-localise with the other primary microcephaly proteins during mitosis. In contrast to the nuclear localisation generally reported for Microcephalin (Lin, 2005; Xu, 2004), all other primary microcephaly proteins, ASPM, CDK5RAP2 and CENPJ, and their Drosophila homologues (Asp, Cnn and Sas-4, respectively) are centrosomally localised. Therefore Drosophila MCPH1 could still act in the same biochemical pathway as other primary microcephaly orthologs (Brunk, 2007).
In summary, this study has shown that MCPH1 is a mitotically regulated protein, which co-localises with decondensed chromosomes, with a short isoform, MCPH1(S), localising to the centrosome and spindle during mitosis. MCPH1(S) is essential for the early rapid syncytial nuclear divisions, where it is required to co-ordinate centrosome and nuclear division cycles, prior to the physiological requirement for ATR-Chk1 signalling in the blastoderm embryo. The mcph1 fly therefore provides a relevant model for further genetic and biochemical characterisation of microcephalin's role in cell cycle regulation during development. Given that MCPH1 may co-localise with other primary microcephaly proteins at the spindle and centrosome during mitosis, this model also provides the opportunity to examine whether they may act in a common developmental and cellular pathway underlying the human phenotype (Brunk, 2007).
Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. mcph1, the Drosophila homolog of MCPH1, has been identified in a genetic screen for regulators of S-M cycles in the early embryo. Embryos of null mcph1 female flies undergo mitotic arrest with barrel-shaped spindles lacking centrosomes. Mutation of Chk2 suppresses these defects, indicating that they occur secondary to a previously described Chk2-mediated response to mitotic entry with unreplicated or damaged DNA. mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. In contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. Components of this checkpoint, however, appear to cooperate with MCPH1 to regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. A model is proposed in which MCPH1 coordinates the S-M transition in fly embryos: in the absence of mcph1, premature chromosome condensation results in mitotic entry with unreplicated DNA, genomic instability, and Chk2-mediated mitotic arrest. Finally, brains of mcph1 adult male flies have defects in mushroom body structure, suggesting an evolutionarily conserved role for MCPH1 in brain development (Rickmyre, 2007).
Several studies have implicated human MCPH1 in the cellular response to DNA damage. The DNA checkpoint is engaged at critical cell-cycle transitions in response to DNA damage or incomplete replication and serves as a mechanism to preserve genomic integrity (reviewed by Nyberg, 2002). Triggering of this checkpoint causes cell-cycle delay, presumably to allow time for correction of DNA defects. When a cell senses DNA damage or incomplete replication, a kinase cascade is activated. Activated ATM and ATR kinases phosphorylate their targets, including the checkpoint kinase Chk1, which is activated to phosphorylate its targets. The first clue that MCPH1 plays a role in the DNA damage response came from siRNA-mediated knockdown studies in cultured mammalian cells demonstrating a requirement for MCPH1 in the intra-S phase and G2-M checkpoints in response to ionizing radiation (Lin, 2005; Xu, 2004). Two recent reports have further implicated MCPH1 in the DNA checkpoint, although puzzling discrepancies remain to be resolved (reviewed by Bartek, 2006). One report indicates that MCPH1 functions far downstream in the pathway, at a level between Chk1 and one of its targets, Cdc25 (Alderton, 2006). Another report (Rai, 2006) suggests that MCPH1 is a proximal component of the DNA damage response required for radiation-induced foci formation (i.e. recruitment of checkpoint and repair proteins to damaged chromatin) (Rickmyre, 2007).
Additional functions have been reported for MCPH1. MCPH1- lymphocytes of microcephalic patients exhibit premature chromosome condensation (PCC) characterized by an abnormally high percentage of cells in a prophase-like state, suggesting that MCPH1 regulates chromosome condensation and/or cell-cycle timing (Trimborn, 2004). A possible explanation for the PCC phenotype is that MCPH1-deficient cells have high Cdk1-cyclin B activity, which drives mitotic entry; decreased inhibitory phosphorylation of Cdk1 was found to be responsible for elevated Cdk1 activity in MCPH1-deficient cells (Alderton, 2006). It is not clear whether MCPH1's role in regulating mitotic entry in unperturbed cells is related to its checkpoint function; intriguingly, Chk1 has similarly been reported to regulate timing of mitosis during normal division. MCPH1 (also called Brit1) was independently identified in a screen for negative regulators of telomerase, suggesting that it may function as a tumor suppressor (Lin, 2003). Further evidence for such a role (Rai, 2006) comes from a study showing that gene copy number and expression of MCPH1 is reduced in human breast cancer cell lines and epithelial tumors (Rickmyre, 2007).
This study reports the identification and phenotypic characterization of Drosophila mutants null for mcph1. Syncytial embryos from mcph1 females exhibit genomic instability and undergo mitotic arrest due to activation of a DNA checkpoint kinase, Chk2. On contrast to reports of MCPH1 function in human cells, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. It is propose that Drosophila MCPH1, like its human counterpart, is required for proper coordination of cell-cycle events; in early embryos lacking mcph1, chromosome condensation prior to completion of DNA replication causes genomic instability and Chk2-mediated mitotic arrest (Rickmyre, 2007).
Drosophila mcph1 was identified in a genetic screen for cell-cycle regulators, and it is required for genomic stability in the early embryo. Three additional primary microcephaly (MCPH) genes have been identified in humans: ASPM, CDK5RAP2, and CENPJ. Much of the understanding of the biological functions of the proteins encoded by human MCPH genes has come from studies of their Drosophila counterparts. Mutation of abnormal spindle (asp), the Drosophila ortholog of ASPM, results in cytokinesis defects and spindles with poorly focused poles. The Drosophila ortholog of CDK5RAP2, centrosomin (cnn), is required for proper localization of other centrosomal components. Sas-4, the Drosophila ortholog of CENPJ, is essential for centriole production, and the mitotic spindle is often misaligned in asymmetrically dividing neuroblasts of Sas-4 larvae (Basto, 2006). Whereas all of these primary microcephaly genes are critical regulators of spindle and centrosome functions, mitotic defects in Drosophila mcph1 mutants are largely secondary to Chk2 activation in response to DNA defects; thus, mcph1 probably represents a distinct class of primary microcephaly genes (Rickmyre, 2007).
MCPH1 is a BRCT domain-containing protein, suggesting that it plays a role in the DNA damage response. Conflicting models of MCPH1 function, however, have emerged from studies of human cells as it has been proposed to function at various levels in this pathway: upstream, at the level of damage-induced foci formation (Rai, 2006) and further downstream, to augment phosphorylation of targets by the effector Chk1 (Alderton, 2006). The phenotype of embryos from null mcph1 females is more severe than that of embryos from null grp females, suggesting that enhancement of phosphorylation of GRP (Chk1) substrates is not the sole function of MCPH1. Furthermore, both the DNA checkpoint in larval stages and its developmentally regulated use at the MBT are intact in mcph1 mutants, suggesting a requisite role for MCPH1 in the DNA checkpoint evolved in higher organisms (Rickmyre, 2007).
Studies of human cells suggest a role for MCPH1 in regulation of chromosome condensation. Microcephalic patients homozygous for a severely truncating mutation in MCPH1 show increased frequency of G2-like cells displaying premature chromosome condensation (PCC) with an intact nuclear envelope (Alderton, 2006; Trimborn, 2004). Depletion of Condensin II subunits by RNAi in MCPH1-deficient cells leads to reduction in the frequency of PCC, suggesting that MCPH1 is a negative regulator of chromosome condensation (Trimborn, 2006). Alderton (2006) observed a decreased level of inhibitory phosphates on Cdk1 that correlated with PCC in MCPH1-deficient cells. That study proposed that MCPH1 maintains Cdk1 phosphorylation in an ATR-independent manner because PCC is not seen in cells of patients with Seckel syndrome, which is caused by mutation of ATR; residual ATR present in these cells, however, may be sufficient to prevent PCC. Furthermore, in several experimental systems, ATR and Chk1 have been implicated in an S-M checkpoint that prevents premature mitotic entry with unreplicated DNA (Rickmyre, 2007).
This study has shown that embryos from grp (Chk1) females occasionally undergo mcph1-like arrest in early syncytial cycles, prior to the time at which inhibitory phosphorylation of Cdk1 is thought to control mitotic entry. Thus, decreased signaling through the DNA checkpoint resulting in less Cdk1 phosphorylation is unlikely to explain this mcph1-like arrest. In contrast to studies of MCPH1-deficient human cells, no decrease is detected in pY15-Cdk1 levels in mcph1 embryos allowed to progress beyond their normal arrest point by mutation of mnk (Chk2). Based on these data and the PCC phenotype associated with loss of MCPH1 in humans, a model is proposed in which MEI-41/GRP cooperate with MCPH1 in syncytial embryos in a Cdk1-independent manner to delay chromosome condensation until DNA replication is complete. In the absence of mcph1, it is hypothesized that embryos condense chromosomes before finishing S phase, resulting in DNA defects (bridging chromatin), Chk2 activation, and mitotic arrest. It was not possible to directly monitor chromosome condensation in mnk mcph1 embryos expressing Histone-GFP, because fly stocks could not be established carrying this transgene in the mnk background. Live imaging of mcph1 embryos was not technically feasible because they arrest prior to cortical stages, and yolk proteins obscure more interior nuclei in early embryos. grp embryos have been reported to initiate chromosome condensation with normal kinetics, although a subtle PCC phenotype might be difficult to detect (Rickmyre, 2007).
Support for a model that MCPH1 allows completion of S phase by delaying chromosome condensation comes from the observation that inhibition of DNA replication in syncytial embryos (via injection of aphidicolin or HU) results in phenotypes similar to those observed in mcph1 embryos, including chromatin bridging, which is presumably a direct consequence of progressing through mitosis with unreplicated chromosomes, and Chk2 activation. Alternatively, mcph1 might be required during S phase for timely completion of DNA synthesis; in this case, mcph1 embryos would initiate chromosome condensation with normal kinetics prior to completing replication. Coordination of S-phase completion and mitotic entry may be particularly critical in the rapid cell cycles of the early embryo that lack gap phases and may explain why loss of Drosophila mcph1 is most apparent at this developmental stage. Interestingly, even in the absence of exogenous genotoxic stress, MCPH1-deficient human cells also exhibit a high frequency of chromosomal aberrations (Rai, 2006), which may be a consequence of PCC (Rickmyre, 2007).
An evolutionary role for mcph1 in expansion of brain size along primate lineages has emerged in recent years (reviewed by Woods, 2005). In brains of Drosophila mcph1 males, low-penetrance defects in MB structure were found. Both MCPH1 isoforms are expressed in larval brains, and all mcph1 mutations described in this study affect both isoforms, so it is unclear whether MB formation requires one or both isoforms. The lack of MB defects in mcph1 females is puzzling because both isoforms are found in male and female larval brains; other sex-specific factors are probably involved. Larval brains of mcph1 males show no obvious aneuploidy or spindle orientation defects, so the cellular basis for these defects remains to be determined. It will be interesting to test in future studies whether mei-41 and grp, which cooperate with mcph1 to regulate early embryogenesis, are similarly required in Drosophila males for brain development (Rickmyre, 2007).
In conclusion, this study has demonstrated an essential role for Drosophila MCPH1 in maintaining genomic integrity in the early embryo. The data suggest that, in contrast to the mammalian protein, Drosophila MCPH1 is not required for the DNA checkpoint, although its role in regulating other processes (e.g. chromosome condensation) may be conserved. It is predicted that the early embryo of Drosophila will continue to be an important model genetic system for unraveling the biological functions of MCPH1, a critical determinant of brain size in humans (Rickmyre, 2007).
Search PubMed for articles about Drosophila Microcephalin
Alderton, G. K., Galbiati, L., Griffith, E., Surinya, K. H., Neitzel, H., Jackson, A. P., Jeggo, P. A. and O'Driscoll, M. (2006). Regulation of mitotic entry by microcephalin and its overlap with ATR signalling. Nat. Cell Biol. 8: 725-733. PubMed ID: 16783362
Bartek, J. (2006). Microcephalin guards against small brains, genetic instability, and cancer. Cancer Cell 10: 91-93. PubMed ID: 16904606
Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C. G., Khodjakov, A. and Raff, J. W. (2006). Flies without centrioles. Cell 125: 1375-1386. PubMed ID: 16814722
Bond, J., Roberts, E., Mochida, G. H., Hampshire, D. J., Scott, S., Askham, J. M., Springell, K., Mahadevan, M., Crow, Y. J. and Markham, A. F. (2002). ASPM is a major determinant of cerebral cortical size. Nat. Genet. 32: 316-320. PubMed ID: 12355089
Bond, J., Roberts, E., Springell, K., Lizarraga, S., Scott, S., Higgins, J., Hampshire, D. J., Morrison, E. E., Leal, G. F., Silva and E. O. (2005). A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat. Genet. 37: 353-355. PubMed ID: 15793586
Brunk, K., et al. (2007). Microcephalin coordinates mitosis in the syncytial Drosophila embryo. J Cell Sci. 120(Pt 20): 3578-88. PubMed ID: 17895363
Evans, P. D., Anderson, J. R., Vallender, E. J., Choi, S. S. and Lahn, B. T. (2004). Reconstructing the evolutionary history of microcephalin, a gene controlling human brain size. Hum. Mol. Genet. 13: 1139-1145. PubMed ID: 15056607
Huyton, T., Bates, P. A., Zhang, X., Sternberg, M. J. and Freemont, P. S. (2000). The BRCA1 C-terminal domain: structure and function. Mutat. Res. 460: 319-332. PubMed ID: 10946236
Jackson, A. P., Eastwood, H., Bell, S. M., Adu, J., Toomes, C., Carr, I. M., Roberts, E., Hampshire, D. J., Crow, Y. J., Mighell, A. J. (2002). Identification of microcephalin, a protein implicated in determining the size of the human brain. Am. J. Hum. Genet. 71: 136-142. PubMed ID: 12046007
Jeffers, L. J., Coull, B. J., Stack, S. J. and Morisson, C. G. (2008). Distinct BRCT domains in Mcph1/Brit1 mediate ionizing radiation-induced focus formation and centrosomal localization. Oncogene 27(1): 139-44. PubMed ID: 17599047
Lin, S. Y., Rai, R., Li, K., Xu, Z. X. and Elledge, S. J. (2005). BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly. Proc. Natl. Acad. Sci. 102: 15105-15109. PubMed ID: 16217032
Manke, I. A., Lowery, D. M., Nguyen, A. and Yaffe, M. B. (2003). BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 302: 636-639. PubMed ID: 14576432
McCreary, B. D., Rossiter, J. P. and Robertson, D. M. (1996). Recessive (true) microcephaly: a case report with neuropathological observations. J. Intellect. Disabil. Res. 40: 66-70. PubMed ID: 8930059
Neitzel, H., Neumann, L. M., Schindler, D., Wirges, A., Tonnies, H., Trimborn, M., Krebsova, A., Richter, R. and Sperling, K. (2002). Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition. Am. J. Hum. Genet. 70: 1015-1022. PubMed ID: 11857108
Nyberg, K. A., Michelson, R. J., Putnam, C. W. and Weinert, T. A. (2002). Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36: 617-656. PubMed ID: 12429704
Rai, R., Dai, H., Multani, A. S., Li, K., Chin, K., Gray, J., Lahad, J. P., Liang, J., Mills, G. B., Meric-Bernstam, F. (2006). BRIT1 regulates early DNA damage response, chromosomal integrity, and cancer. Cancer Cell 10: 145-157. PubMed ID: 16872911
Rickmyre, J. L., et al. (2006). The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo. J. Cell Sci. 120(Pt 20): 3565-77. PubMed ID: 17895362
Trimborn, M., Bell, S. M., Felix, C., Rashid, Y., Jafri, H., Griffiths, P. D., Neumann, L. M., Krebs, A., Reis, A., Sperling, K. (2004). Mutations in microcephalin cause aberrant regulation of chromosome condensation. Am. J. Hum. Genet. 75: 261-266. PubMed ID: 15199523
Trimborn, M., Schindler, D., Neitzel, H. and Hirano, T. (2006). Misregulated chromosome condensation in MCPH1 primary microcephaly is mediated by condensin II. Cell Cycle 5: 322-326. PubMed ID: 16434882
Wang, Y.-q. and Su, B. (2004). Molecular evolution of microcephalin, a gene determining human brain size. Hum. Mol. Genet. 13: 1131-1137. PubMed ID: 15056608
Wood, B. and Collard, M. (1999). The human genus. Science 284: 65-71. PubMed ID: 10102822
Woods, C. G., Bond, J. and Enard, W. (2005). Autosomal recessive primary microcephaly (MCPH): a review of clinical, molecular, and evolutionary findings. Am. J. Hum. Genet. 76: 717-728. PubMed ID: 15806441
Xu, X., Lee, J. and Stern, D. F. (2004). Microcephalin is a DNA damage response protein involved in regulation of CHK1 and BRCA1. J. Biol. Chem. 279: 34091-34094. PubMed ID: 15220350
Yu, X., Chini, C. C., He, M., Mer, G. and Chen, J. (2003). The BRCT domain is a phospho-protein binding domain. Science 302: 639-642. PubMed ID: 14576433
Zhong, X., Pfeifer, G. P. and Xu, X. (2006). Microcephalin encodes a centrosomal protein. Cell Cycle 5: 457-458. PubMed ID: 16479174
date revised: 6 January 2007
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