Microcephalin/MCPH1 associates with the Condensin II complex to function in homologous recombination repair

Abstract

Microcephalin/MCPH1 is one of the causative genes responsible for the autosomal recessive disorder primary microcephaly. Patients with this disease present with mental retardation and dramatic reduction in head size, and cells derived from these patients contain abnormally condensed chromosomes. MCPH1 contains an N-terminal BRCT and tandem C-terminal BRCT domains. More recently, MCPH1 has been implicated in the cellular response to DNA damage; however, the exact mechanism remains unclear. Here, we report the identification Condensin II as a major MCPH1-interacting protein. MCPH1 and Condensin II interact in vivo, mediated by the CAPG2 subunit of Condensin II binding to a middle domain (residues 376-485) of MCPH1. Interestingly, while Condensin II is not required for the IR-induced G2/M checkpoint, Condensin II-depleted cells have a defect in HR repair, which is also present in MCPH1(-/-)MEFs. Moreover, the Condensin II binding region of MCPH1 is also required for HR function. Collectively, we have identified a novel function of MCPH1 to modulate HR repair through Condensin II, and thereby maintain genome integrity.

FIGURE 1.

Isolation of Condensin II as a MCPH1-associated protein. A, tandem affinity purification of MCPH1-containing complexes. 293T cells stably expressing MCPH1 underwent two rounds of affinity purification, and the final elution was run on SDS-PAGE and proteins visualized by silver stain. The pooled elution was sent for MALDI-TOF. B, list of peptides obtained from MS analysis. C, MCPH1 associates with Condensin II in vivo. Co-immunoprecipitation experiments were performed using the indicated antibodies.

FIGURE 2.

A middle region of MCPH1 specifically mediates its interaction with Condensin II. A, CAP-G2 subunit of Condensin II associates with MCPH1. Immunoprecipitation reactions were performed using S beads and blotted with anti-Myc or anti-FLAG antibodies. Arrows show input bands for the Condensin II subunits. The asterisk denotes the nonspecific band. B, Condensin II binds to a middle region (residues 376-485) of MCPH1. A schematic representation of MCPH1 deletions used to map Condensin II binding region on MCPH1 is shown. Wild-type and deletion mutants of MCPH1 were ectopically expressed in 293T cells and immunoprecipitated with anti-FLAG antibodies and blotted with the indicated antibodies specifically for Condensin II subunits. C, Δ2-2 region contains a 20-amino acid conserved region across species. The ClustalW alignment of the conserved region among higher eukaryotes within the Condensin II binding domain of MCPH1 is presented.

FIGURE 3.

MCPH1^-/- MEFs display prematurely condensed chromosomes. A, MCPH1 MEFs have abnormal condensed DAPI staining. Examples of DAPI staining of MCPH1^+/+ (top) and MCPH1^-/- (bottom) MEFs were presented. The top right panel shows number of mitotic cells in each cell line, MCPH1^+/+ and MCPH1^-/- cells were collected and analyzed by FACS for phospho-Histone H3 as a mitotic marker. An average of three experiments is presented. The bottom right panel displays quantification of percentage of cells with abnormal condensed DNA measured by DAPI staining. On average, three independent experiments were performed, and 500 cells were counted per experiment. Bars represent the S.D. B, MCPH1^-/- MEFs have condensed chromosomes. Examples of metaphase spreads of MCPH1^+/+ (top) and MCPH1^-/- (bottom) cells were included. Three independent experiments were performed, and at least 100 metaphase spreads were counted per experiments. The average of percentages of cells with condensed chromosomes is shown. Bars indicate the S.D.

FIGURE 4.

Condensin II binding is not required to rescue the PCC defect in MCPH1^-/- MEFs. A, diagrams of wild-type and deletion mutants of MCPH1 used in this study. B, N terminus of MCPH1 is important for the rescue of PCC in MCPH1-deficient cells. MCPH1^-/- reconstituted cells were treated with 10 Gy, fixed, and stained 6 h later with the indicated antibodies. C, quantification of data presented in B. 500 cells were counted, the results of three independent experiments is shown. Bars represent S.D. D, loss of the N terminus but not the Condensin II binding region causes PCC. Chromosome spreads from MCPH1^-/--reconstituted cells were counted. Shown on the left are examples of metaphase spreads seen in each cell line; on the right is quantification of condensed chromosomes in each cell line tested. Bars indicate the S.D.

FIGURE 5.

Condensin II is not required for G2/M checkpoint control, but plays a role in HR repair. A, G2/M checkpoint is active in Condensin II-depleted cells. HeLa cells were transfected with control or CAP-D3 siRNA. Cells were irradiated and harvested 1 h later, and mitotic cells were counted based on p-H3 staining. Average of three independent experiments with S.D. is shown. B, both Condensin II and MCPH1 are required for efficient HR repair. Left panel, U2OS-DRGFP was transfected with control or CAP-D3 siRNA, allowed to recover, and then transfected with 16 μg of pCBASce. 24 h later, cells were collected and analyzed for GFP expression by FACS. Right panel, MCPH1^+/+ cells, MCPH1^-/- cells, and MCPH1^-/- cells reconstituted with indicated wild-type and deletion mutant of MCPH1 were assayed for HR capability by co-transfection of DR-GFP reporter plasmid with pCBASce. Cells were allowed to recover and analyzed 48 h later for GFP expression.