SET nuclear oncogene associates with microcephalin/MCPH1 and regulates chromosome condensation

Abstract

Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.

FIGURE 1.

MCPH1^S25X/S25X cells express a truncated form of MCPH1 lacking its N terminus. A, LCLs from patients with either heterozygous or homozygous S25X mutations were assayed for MCPH1 foci formation after ionizing radiation. After a 10 Gy ionizing radiation, cells were spun down onto coverslips and stained with the indicated antibodies. B, upper panel, the MCPH1 antibody specifically detects endogenous MCPH1 protein. Cell extracts prepared from WT cells and a primary microcephaly patient cell line (MCPH1^{Δex1–8/Δex1–8}) with a homozygous deletion of the promoter and exons 1–8, were immunoprecipitated and blotted with anti-MCPH1 antibodies. Lower panel, a truncated form of MCPH1 is detectable in patient cells with the S25X mutation. Cell extracts prepared from WT cells and cells from primary microcephaly patients were immunoprecipitated and blotted with anti-MCPH1 antibodies. Anti-actin immunoblotting of whole cell extract (WCE) was included as loading controls. C, cytogenetic analysis of peripheral blood lymphocytes from a primary microcephaly patient with the MCPH1^{V50G,I51V/V50G,I51V} mutation, illustrating the PCC phenotype. Left, low power view. Right, high power view of prophase-like cells with high condensed chromosomes. D, sequence electropherogram of a primary microcephaly patient homozygous for c.149T>G and c.151A>G mutations.

FIGURE 2.

SET is a novel binding partner of MCPH1. A, schematic representation of WT MCPH1, MCPH1 deletion mutants, and SET nuclear oncogene. B, left panel, TAP of complexes containing MCPH1-N terminus revealed SET as an MCPH1-associated protein. 293T cells stably expressing an MCPH1 N-terminal fragment were used for TAP. MCPH1-associated proteins were identified by LC-MS/MS performed at the Taplin Mass Spectrometry Facility at Harvard University. Right, SFB-tagged WT MCPH1 and MCPH1 N-BRCT co-precipitated with endogenous SET, but the N-terminal deletion (ΔN) mutant of MCPH1 did not. C, GST-fused WT MCPH1 and MCPH1 N-BRCT, bound to SET in vitro, but the GST-fused MCPH1 ΔN or GST alone did not. D, GST-SET, but not GST alone, pulled down the MCPH1 N-BRCT. Arrows indicate the expected migration for each recombinant protein. NLS, nuclear localization signal; NAP, nucleosome assembly protein.

FIGURE 3.

SET deficiency leads to aberrant chromosome condensation. A, Mcph1^−/− MEFs and mouse fibroblasts depleted for SET by siRNA, displayed substantial numbers of prophase-like cells with condensed chromosomes. B, knockdown of SET using two different siRNA (9 and 10) in WT MEFs increased the percentage of cells with condensed metaphases compared with MEFs transfected with control siRNA. C, Western blot analysis confirmed the knockdown of SET in MEFs. D, knockdown of SET using two different siRNA (5 and 6) in human H1299 cells increased the percentage of cells with condensed metaphases compared with H1299 cells transfected with control siRNA. Co-depletion of hCAP-D3 partially rescued the percentage of H1299 cells with condensed metaphases in SET knockdown cells. Bar graphs are presented as means ± S.E. *, p < 0.05; relative to control, si-Con, Mann-Whitney U test.

FIGURE 4.

MCPH1 V50G/I51V mutant protein has reduced affinity for SET and fails to fully rescue the chromosome condensation defect. A, MCPH1 V50G/I51V mutations greatly reduced the binding of MCPH1 with SET. Lysates were prepared from 293T cells transfected with plasmids encoding SFB-tagged WT MCPH1 or MCPH1 V50G/I51V mutant. Precipitation was performed using streptavidin beads, and immunoblotting was conducted using antibodies as indicated. B, beads coated with GST or GST-SET were incubated with 293T cell lysates containing SFB-tagged WT MCPH1 or MCPH1 V50G/I51V mutant. After extensive washing, associated proteins were eluted and immunoblotted with anti-FLAG antibodies to detect epitope-tagged MCPH1. Coomassie staining was included to indicate the equal amounts of GST and GST-SET used in these experiments. C, reciprocal GST pulldown assay shows reduced in vitro binding between SET and MCPH1 V50G/I51V mutant. Arrow indicates the expected migration for these recombinant proteins. D, a MCPH1 V50G/I51V mutant reconstitution did not fully rescue the abnormal chromosome condensation phenotype in Mcph1^−/− MEFs. E, a MCPH1 V50G/I51V did not rescue the PCC phenotype in Mcph1^−/− MEFs. *, p < 0.05 with respect to Mcph1^+/+ cells; Mann-Whitney U test.