¹Department of Biochemistry and NCBES, National University of Ireland-Galway, Galway, Ireland

Correspondence: Dr CG Morrison, Department of Biochemistry and NCBES, National University of Ireland-Galway, University Road, Galway, Connacht, Ireland. E-mail: ciaran.morrison@nuigalway.ie

Received 14 August 2006; Revised 12 April 2007; Accepted 16 May 2007; Published online 25 June 2007.

Abstract

Microcephalin (MCPH1/BRIT1) forms ionizing radiation-induced nuclear foci (IRIF) and is required for DNA damage-responsive S and G₂-M-phase checkpoints. MCPH1 contains three BRCT domains. Here we report the cloning of chicken Mcph1 (cMcph1) and functional analysis of its individual BRCT domains. Full-length cMcph1 localized to centrosomes throughout the cell cycle and formed IRIF that colocalized with -H2AX. The tandem C-terminal BRCT2 and BRCT3 domains of cMcph1 were necessary for IRIF formation, while the N-terminal BRCT1 was required for centrosomal localization in irradiated cells. Centrosomal targeting of cMcph1 was independent of ATM, Brca1 or Chk1. cMcph1 formed IRIF in ATM- and Brca1-deficient cells, but not in H2AX-deficient cells. Inability to form cMcph1 IRIF impaired the cellular response to DNA damage. These results suggest that the role of microcephalin in the vertebrate DNA damage response is controlled by interaction of the C-terminal BRCT domains with -H2AX.

The tumour suppressor BRCA1 is involved in numerous activities that maintain genome stability (Starita and Parvin, 2003). Crucial to the functions of BRCA1 are a pair of C-terminal domains, which can recognize phosphopeptides and mediate protein–protein interactions in the response to DNA damage (Manke et al., 2003; Yu et al., 2003). These BRCA1 C-terminal (BRCT) repeats are found in a large superfamily of proteins involved in cellular responses to genotoxic stress (Glover et al., 2004).

One member of this BRCT domain-containing superfamily is microcephalin (MCPH1). MCPH1 is encoded by MCPH1/BRIT1 (hereafter MCPH1), a gene mutated in primary microcephaly (OMIM 251200) (Jackson et al., 2002) and in premature chromosome condensation syndrome (OMIM 606858) (Neitzel et al., 2002; Trimborn et al., 2004). MCPH1 has been described as a transcriptional repressor of human telomerase reverse transcriptase, from which the alternative gene name of BRIT1, for BRCT-repeat inhibitor of hTERT expression, was derived (Lin and Elledge, 2003). MCPH1 is involved in the DNA damage-responsive S and G₂-M-phase checkpoints, although the precise mechanism is not yet clear (Xu et al., 2004; Lin et al., 2005; Alderton et al., 2006). MCPH1 colocalizes in ionizing radiation-induced nuclear foci (IRIF) with MDC1/NFBD1 (Xu et al., 2004) and phosphorylated H2AX (-H2AX) (Lin et al., 2005), a chromatin modification that occurs in a large region around a DNA double-strand break (Rogakou et al., 1999). A recent study (Rai et al., 2006) found that depletion of MCPH1 in human cells blocked formation of IRIF by NBS1, 53BP1, phosphorylated ATM and MDC1, but not -H2AX. DNA damage-induced chromatin association of several DNA damage response proteins was also blocked by MCPH1 RNAi and a high level of chromosome aberrations was observed. MCPH1 aberrations were frequently detected in breast, ovarian and prostate cancer (Rai et al., 2006). Together, these data suggest an important role for MCPH1 in DNA damage responses and in preventing cellular transformation.

We identified the chicken MCPH1 orthologue by database analysis, cloned it by RT–PCR and confirmed the sequence (DDBJ/EMBL/Genbank accession no.DQ788861). Zebrafish Mcph1 sequence was derived by database analysis and these sequences were aligned with known mammalian Mcph1 sequences using ClustalW. As shown in Supplementary Figure S1, all vertebrate sequences analysed showed similar organization, with a highly conserved single N-terminal BRCT domain and a pair of C-terminal BRCT domains, which will be referred to here as BRCT1, BRCT2 and BRCT3, respectively. The conserved BRCT1 and BRCT2–BRCT3 regions are separated by a poorly conserved region that does not contain any distinct structural motifs, as determined by SMART domain search at http://smart.embl-heidelberg.de/. These findings suggest that key regions for microcephalin function lie within the BRCT domains.

We expressed recombinant, tagged cMcph1 in chicken DT40 cells and examined its subcellular localization. As shown in Figure 1a, N-terminally, EGFP-tagged cMcph1 localized to -tubulin-containing foci, which we confirmed as centrosomes by immunofluorescence microscopy with antibodies against the centrosome markers centrin, Aurora-A and the pericentriolar marker, Nedd1 (Haren et al., 2006). C-terminally tagged cMcph1-EGFP and myc-cMcph1 also localized to centrosomes (unpublished data and Figure 1b), and we saw a complete colocalization of EGFP-cMcph1 and myc-cMcph1 (Figure 1c), demonstrating that the observed localization was not a tagging artefact.

Figure 1.

Centrosomal localization of cMcph1. Cloning and primary antibodies are detailed in Supplementary Information. Culture of wild-type, Brca1^-/- cells (R Franklin and K Hiom, in preparation), Chk1^-/- (Zachos et al., 2003) and H2AX mutant (Sonoda et al., 2007) DT40 cells and stable transfections by electroporation were performed as described previously (Dodson et al., 2004). For transient transfections, 15 g of endotoxin-free DNA (Qiagen, Crawley, UK) was introduced into 5 10⁶ cells using nucleofection (Amaxa, Cologne, Germany, programme B-23). Cells were fixed and prepared for microscopy and imaging performed using an Olympus BX51 microscope as described (Dodson et al., 2004). Cells were counterstained with DAPI (blue) before fluorescence microscopy. Scale bars, 5 m. (a) EGFP-cMcph1 (green)-expressing DT40 cells stained with antibodies to the centrosomal or pericentrosomal proteins (red) indicated at left. (b) Myc-cMcph1-expressing (green) DT40 cell stained for -tubulin (red). (c) EGFP-cMcph1 (green)-expressing DT40 cell transiently transfected with a myc-cMcph1 expression construct and then stained with anti-myc antibodies (red) after 10 Gy irradiation (IR).

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To confirm our observations on cMcph1 localization, we raised an antiserum (11989) to the N-terminal part of cMcph1. In immunofluorescence microscopy experiments, this antiserum recognized overexpressed myc-tagged cMcph1 (Figure 2a), demonstrating its specificity for cMcph1. Using these anti-cMcph1 antibodies, we observed endogenous cMcph1 in IRIF that colocalized with those formed by -H2AX (Figure 2b) and the centrosomal localization of cMcph1 throughout the cell cycle (Figure 2c). While a centrosomal signal was consistently observed with antiserum 11989, we saw IRIF in only a small fraction of irradiated cells. As 11989 was raised and affinity-purified against an epitope-tagged fragment of cMcph1, it is likely that reactivity to this epitope reduces the anti-cMcph1-specific titre of the antiserum. We are generating additional antisera to explore this issue further. Together with published data on Mcph1 localization (Xu et al., 2004; Lin et al., 2005), these observations indicate that the localization of tagged, overexpressed cMcph1 reflects the behaviour of the endogenous protein.

Figure 2.

Immunofluorescence microscopy of cMcph1. The N-terminal 248 amino acids of cMcph1 were bacterially expressed as a His-tagged fusion protein by cloning into pET30a (Novagen, Madison, WI, USA), then purified over a HIS-Select nickel affinity gel (Sigma, Dublin, Ireland) and used as an immunogen to generate rabbit polyclonal anti-cMcph1 serum 11989 (Harlan Seralabs, Loughborough, UK). (a) Myc-cMcph1 (red)-expressing DT40 cells were stained with affinity-purified anti-cMcph1 11989 (green). Micrograph shows recognition by the anti-cMcph1 antiserum of the myc-cMcph1 IRIF after 10 Gy irradiation (IR). (b) Immunofluorescence micrograph of a wild-type DT40 cell showing colocalization of cMcph1 (green) with -H2AX foci (red) induced by 10 Gy IR. (c) Immunofluorescence micrograph of a wild-type DT40 cells showing centrosomal colocalization of cMcph1 (green) with -tubulin (red) in mitotic, S/G₂ and G₁-phase cells (upper, middle and lower panels, respectively). Cells were counterstained with DAPI (blue) before fluorescence microscopy. Scale bars, 5 m.

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Central to the signalling cascades that control cell cycle arrests after DNA damage are the ATM and ATR kinases, members of a family of large, phosphatidylinositol 3-OH kinase-related serine-threonine kinases (PIKKs; Shiloh, 2003) that activate the Chk1 and Chk2 effector kinases to impose cell cycle checkpoints (Bartek and Lukas, 2003). We tested whether the localization of cMcph1 to centrosomes was dependent on ATM or Brca1 by examining EGFP-cMcph1 localization in Atm^-/- and Brca1^-/- DT40 cells. As shown in Figure 3a, we observed EGFP-cMcph1 at the centrosome in both knockout lines, demonstrating that its centrosomal localization is independent of ATM and Brca1. We also observed EGFP-cMcph1 at the centrosome in Chk1^-/- DT40 cells (unpublished data), but the further definition of the localization was impaired by an apparent reduction in H2AX phosphorylation in Chk1-deficient cells. Centrosomal localization of cMcph1 was unaffected by ionizing radiation, was independent of cell cycle stage, and occurred at multiple centrosomes that were induced by DNA damage (unpublished data), suggesting that cMcph1 is a constitutive component of the centrosome, similar to the products of the three other identified genes that are mutated in primary microcephaly, CDK5RAP2 (MCPH3), ASPM (MCPH5) and CENPJ (MCPH6; Bond et al., 2005).

Figure 3.

Genetic dependencies of cMcph1 IRIF formation. (a) Localization of EGFP-cMcph1 in ATM^-/- and Brca1^-/- DT40 cells. Micrographs show colocalization of EGFP-tagged cMcph1 (green) with -tubulin (blue) and -H2AX foci (red) induced by 10 Gy IR. (b) Micrograph showing colocalization of EGFP-tagged cMcph1 (green) with Rad51 foci (red) induced by 10 Gy IR. DNA is shown in blue. (c) Genetic dependencies of cMcph1 localization to damage foci. Wild-type, ATM^-/- and Brca1^-/- DT40 cells were transfected with pEGFP-cMcph1. Twelve hours post transfection, cells were subjected to 5 Gy IR, then fixed at different times and stained for -H2AX and -tubulin. Transfected cells were identified by centrosomal EGFP-cMcph1 signals and -H2AX foci were quantitated by eye in the transfected cells. Results shown are the meanss.d. of three separate experiments in which at least 300 cells were counted at each time point. (d) Localization of EGFP-cMcph1 in H2AX mutant DT40 cells. Micrographs show colocalization of EGFP-tagged cMcph1 (green) with -tubulin (blue) and -H2AX foci (red) induced by 10 Gy IR. Scale bars, 5 m.

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Next, we tested for the formation of IRIF by cMcph1. Myc-tagged cMcph1 colocalized with EGFP-labelled cMcph1 (Figure 1c), which in turn localized to -H2AX foci (Figure 3a). The initial, rapid induction of -H2AX foci after irradiation (IR) is followed by a reduction in the number of foci over several hours, presumably reflecting the loss of a DNA damage signal following repair (Paull et al., 2000). cMcph1 also localized to homologous recombinational repair foci formed by the Rad51 recombinase after IR (Figure 3b; Paull et al., 2000), showing that cMcph1 persists at sites of DNA damage beyond the initial recognition phase. The kinetics of EGFP-cMcph1 focus formation and disappearance paralleled the behaviour of -H2AX in wild-type and in Atm^-/- and Brca1^-/- DT40 cells (Figure 3c), consistent with an upstream role in the DNA damage response for Mcph1 (Rai et al., 2006). Notably, the delay in the disappearance of IRIF in Brca1^-/- cells that reflects the repair deficiency in this cell line (Vandenberg et al., 2003) was mirrored by a delay in the resolution of Mcph1 foci. These data demonstrate that cMcph1 foci are controlled in a DNA damage responsive manner similar to radiation-induced -H2AX foci.

We then tested whether the formation of cMcph1 IRIF is dependent on H2AX phosphorylation. As shown in Figure 3d, H2AX^-/– DT40 cells showed no cMcph1 IRIF, 2 h after 10 Gy IR. While transgenic expression of wild-type H2AX protein restored cMcph1 IRIF formation, expression of H2AX in which the conserved Ser-139 was mutated to Ala (S139A) did not support IRIF formation by cMcph1 (Figure 3d). These data show clearly that -H2AX is required for cMcph1 IRIF formation.

We next investigated the functions of the different domains of cMcph1 in responding to DNA damage by transfection of a deletion series of EGFP-tagged cMcph1 expression constructs (Figures 4a and b). As shown in Figure 4c, the N-terminal BRCT1 domain was required for centrosomal localization of cMcph1 in irradiated cells, but not IRIF formation. Interestingly, the loss of the BRCT1 domain still allowed centrosomal localization in unirradiated cells. This may suggest that the tandem BRCT2, 3 repeat has a limited affinity for the centrosome that is weaker than the IRIF interaction. The constitutive centrosome localization suggests the recognition of a centrosome component by the BRCT1 domain, although the extent to which single BRCT domains can interact with phosphoproteins is unclear (Manke et al., 2003; Yu et al., 2003).

Figure 4.

Analysis of deletion mutants of cMcph1. (a) Diagrammatic representation of cMcph1 protein showing BRCT domains. (b) Table summarizing the expression constructs analysed and localization patterns 2 h after 10 Gy irradiation (IR) determined for the recombinant proteins indicated. (c) Micrographs showing localization of EGFP-tagged cMcph1 deletion mutants (green), -H2AX (red) and -tubulin (blue) in DT40 cells 2 h after 10 Gy IR. Scale bars, 5 m. (d) DT40 cells transiently transfected with EGFP-tagged full-length cMcph1 or cMcph1BRCT3 were subjected to 10 Gy IR and analysed by immunofluorescence microscopy for cleaved caspase-3 at the indicated time points post treatment. Transfectants were identified by EGFP signal. Data points are the means.d. of six separate experiments in which 300 cells were counted per time point. (e) Immunoblot of expression levels of the EGFP-cMcph1 transgenes used for the experiment in (d). 'Control', 'WT', '3', untransfected, EGFP-tagged full-length cMcph1 and cMcph1BRCT3, respectively.

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The C-terminal BRCT3 domain was necessary for the formation of cMcph1 IRIF, but not for centrosomal targeting (Figure 4c). Loss of part of the region between BRCT1 and BRCT2 did not abrogate IRIF formation or centrosome localization, demonstrating that the BRCT domains are the key determinants directing cMcph1 localization. Importantly, we saw a similar localization pattern with both the N- and C-terminally tagged forms of cMcph1, consistent with the observed localization being independent of tagging. Expression of each of the EGFP-tagged cMcph1 proteins was confirmed by immunoblot analysis (unpublished data). Tandem C-terminal BRCT domains, which can act as phosphoserine/phosphothreonine-binding modules, are common motifs in IRIF-forming proteins (Manke et al., 2003; Yu et al., 2003). The C-terminal BRCT domains of MDC1/NFBD1 bind directly to -H2AX to form MDC1/NFBD1 IRIF (Stucki et al., 2005). The C-terminal BRCT domains of PTIP (Manke et al., 2003) and BRCA1 (Au and Henderson, 2005) are required for IRIF formation, and H2AX is also necessary for IRIF formation by 53BP1 and BRCA1 (Celeste et al., 2002, 2003). However, pull-down experiments in HeLa nuclear extracts with a H2AX phosphopeptide found MDC1/NFBD1 to be the principal -H2AX-binding protein in human cells (Stucki et al., 2005), so that any other potential BRCT–-H2AX interactions, such as Mcph1–-H2AX, in IRIF are likely to be indirect.

To determine whether the inability to form cMcph1 IRIF impeded the cellular response to DNA damage, we quantitated the induction of apoptosis in wild-type and cMcph1-overexpressing DT40 cells following IR treatment. As shown in Figures 4d and e, the same level of apoptosis was induced by IR in cells that express EGFP-cMcph1 as in wild-type DT40 cells. However, expression of EGFP-tagged cMcph1BRCT3 caused a higher level of apoptosis after IR, suggesting an elevated radiosensitivity resulting from an inability to form cMcph1 IRIF.