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Regulation of mitotic entry by microcephalin and its overlap with ATR signalling

Nature Cell Biology volume 8, pages 725–733 (2006)Cite this article

Abstract

Ataxia-telangiectasia mutated and Rad3 related (ATR)–Seckel syndrome and autosomal recessive primary microcephaly (MCPH) syndrome share clinical features. RNA interference (RNAi) of MCPH1 have implicated the protein it encodes as a DNA-damage response protein that regulates the transcription of Chk1 and BRCA1, two genes involved in the response to DNA damage1,2. Here, we report that truncating mutations observed in MCPH-syndrome patients do not impact on Chk1 or BRCA1 expression or early ATR-dependent damage-induced phosphorylation events. However, like ATR–Seckel syndrome cells, MCPH1-mutant cell lines show defective G2–M checkpoint arrest and nuclear fragmentation after DNA damage, and contain supernumerary mitotic centrosomes. MCPH1-mutant and ATR–Seckel cells also show impaired degradation of Cdc25A and fail to inhibit Cdc45 loading onto chromatin after replication arrest. Additionally, microcephalin interacts with Chk1. We conclude that MCPH1 has a function downstream of Chk1 in the ATR-signalling pathway. In contrast with ATR–Seckel syndrome cells, MCPH1-mutant cells have low levels of Tyr 15-phosphorylated Cdk1 (pY15-Cdk1) in S and G2 phases, which correlates with an elevated frequency of G2-like cells displaying premature chromosome condensation (PCC)3,4. Thus, MCPH1 also has an ATR-independent role in maintaining inhibitory Cdk1 phosphorylation, which prevents premature entry into mitosis.

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Figure 1: MCPH1-mutant cell lines are proficient in upstream activation of ATR-dependent damage signalling.
Figure 2: MCPH1-mutant cell lines show defects in downstream ATR-dependent phenotypes.
Figure 3: ATR–Seckel and MCPH1-mutant cells show impaired Chk1-dependent degradation of Cdc25A.
Figure 4: MCPH1-mutant cells but not ATR–Seckel cells show rapid loss of p-Y15 Cdk1 following S phase progression that correlates with PCC formation.
Figure 5: MCPH1 siRNA confers the phenotype observed in MCPH1 patient cells.

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Acknowledgements

We are grateful to S.-Y. Lin for the anti-MCPH1 antibody. The P.A.J. laboratory is supported by the Medical Research Council (MRC), the Human Frontiers Science Programme, the Leukaemia Research Fund, the International Agency for Cancer Research and an EU grant (FIGH-CT-200200207). A.P.J. is funded by an MRC Clinical Scientist Fellowship.

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Authors and Affiliations

  1. Genome Damage and Stability Centre, University of Sussex, BN1 9RQ, East Sussex, UK

    Gemma K. Alderton, Laura Galbiati, Penny A. Jeggo & Mark O'Driscoll

  2. Molecular Medicine Unit, University of Leeds, UK

    Katharina H. Surinya & Andrew P. Jackson

  3. Institute of Human Genetics, Charite´Universitary Medicine, Berlin, Germany

    Heidemarie Neitzel

  4. MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK

    Elen Griffith & Andrew P. Jackson

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  1. Gemma K. Alderton
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Contributions

The laboratories of P.A.J. and A.P.J. made the major contributions to this work. A.P.J. and members of his laboratory contributed both intellectually and practically to the inception and execution of the work. H.N. provided the MCPH1427insA cell line.

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Correspondence to Penny A. Jeggo or Mark O'Driscoll.

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The authors declare no competing financial interests.

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Supplementary Figures S1, S2, S3, S4 and Supplementary Table S1 (PDF 322 kb)

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Alderton, G., Galbiati, L., Griffith, E. et al. Regulation of mitotic entry by microcephalin and its overlap with ATR signalling. Nat Cell Biol 8, 725–733 (2006). https://doi.org/10.1038/ncb1431

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