The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo

Jamie L. Rickmyre 2 Shamik DasGupta 1 Danny Liang-Yee Ooi 0 Jessica Keel 2 Ethan Lee 2 Marc W. Kirschner 0 Scott Waddell 1 Laura A. Lee 2 0 Department of Systems Biology, Harvard Medical School , 200 Longwood Avenue, Boston, MA 02115 , USA 1 Department of Neurobiology, University of Massachusetts Medical School , 364 Plantation Street, Worcester, MA 01605 , USA 2 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4200 MRBIII , 465 21st Avenue South, Nashville, TN 37232-8240 , USA Summary Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. We identified mcph1, the Drosophila homolog of MCPH1, in a genetic screen for regulators of S-M cycles in ce the early embryo. Embryos of null mcph1 female flies n undergo mitotic arrest with barrel-shaped spindles lacking e ic centrosomes. Mutation of Chk2 suppresses these defects, S indicating that they occur secondary to a previously lle described Chk2-mediated response to mitotic entry with C unreplicated or damaged DNA. mcph1 embryos exhibit fo genomic instability as evidenced by frequent chromatin l bridging in anaphase. In contrast to studies of human na MCPH1, the ATR/Chk1-mediated DNA checkpoint is ru intact in Drosophila mcph1 mutants. Components of this Jo checkpoint, however, appear to cooperate with MCPH1 to - Introduction Drosophila melanogaster is an ideal model organism for study of the cell cycle during development (reviewed by Foe et al., 1993; Lee and Orr-Weaver, 2003). Drosophila achieves rapid embryogenesis by using a streamlined cell cycle that is not dependent on transcription or growth. The first 13 embryonic cell cycles are nearly synchronous nuclear divisions without cytokinesis occurring in the shared cytoplasm of the syncytial blastoderm. These cycles differ from canonical G1-S-G2-M cycles in that they have no intervening gaps; instead DNA replication and mitosis rapidly oscillate. Maternal RNA and protein stockpiles drive these abbreviated S-M cycles (~10 minutes each). In mammalian embryos, rapid peri-gastrulation divisions that occur later in development share many features and have been proposed to be related by evolutionary descent to early embryonic divisions of flies and frogs (OFarrell et al., 2004). Thus, advances gained from studies of these streamlined cycles in simple model organisms likely have relevance for understanding mammalian cell cycles. In a genetic screen for regulators of embryonic S-M cycles, we identified the Drosophila homolog of a human disease gene, MCPH1 (microcephalin). Mutation of human MCPH1 causes autosomal recessive primary microcephaly, a developmental disorder characterized by severe reduction of regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. We propose a model 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. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/20/3565/DC1 cerebral cortex size (Jackson et al., 2002). Mcph1 is highly expressed in the developing forebrain of fetal mice, consistent with its proposed role in regulating the number neuronal precursor cell divisions and, ultimately, brain size (Jackson et al., 2002). Human MCPH1 protein is predicted to contain three BRCA1 C-terminal (BRCT) domains (reviewed by Glover et al., 2004; Huyton et al., 2000), which mediate phosphorylation-dependent protein-protein interactions in cellcycle checkpoint and DNA repair functions. 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 et al., 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 et al., 2005; Xu et al., 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 et al., 2006). Another report (Rai et al., 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). 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 et al., 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 MCPH1deficient cells (Alderton et al., 2006). It is not clear whether MCPH1s 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 ce normal division (Kramer et al., 2004). MCPH1 (also called n Brit1) was independently identified in a screen for negative e ic regulators of telomerase, suggesting that it may function as a S tumor suppressor (Lin and Elledge, 2003). Further evidence for lle such a role comes from a study showing that gene copy number C and expression of MCPH1 is reduced in human breast cancer fo cell lines and epithelial tumors (Rai et al., 2006). l We report here the identification and phenotypic a n characterization of Drosophila mutants null for mcph1. We ru show that syncytial embryos from mcph1 females exhibit o genomic instability and undergo mitotic arrest due to activation J of a DNA checkpoint kinase, Chk2. We find that, in contrast to reports of MCPH1 function in human cells, the ATR/Chk1mediated DNA checkpoint is intact in Dr (...truncated)