Fig 2: Characterization of the MCPH1–CDH1 interaction. A) Schematic diagram of MCPH1 fragments to be used in CDH1 pull-down studies. The sites of potential D-Box and KEN-Box degron sequences are indicated. B) GFP or GFP–MCPH1 variants were transfected into HEK293 cells for 24 h before purification and incubation with FLAG–CDH1 cell extracts (from transfected HEK293 cells). The ability of CDH1 to bind MCPH1-L was investigated by using Western blotting. C) The KEN-Box sequence was mutated to “AAA” in GFP–MCPH1-L and GFP–MCPH1-S before performing the CDH1 pull-down experiment as described in B. D) Schematic diagram of CDH1 fragments to be used in pull-down studies with GFP–MCPH1-L. E) FLAG–CDH1 (or CDH1 fragment) pull-down was performed as in B.

Fig 3: D-Box4 phosphorylation is not involved in CDH1-mediated regulation of MCPH1. A) Schematic diagram of MCPH1-L showing the positions of D-Box4 and the novel phosphorylation site in or near this degron sequence. B) GFP or GFP–MCPH1 variants were transfected into HEK293 cells for 24 h before purification and incubation with FLAG–CDH1 cell extracts (from transfected HEK293 cells). The ability of CDH1 to bind MCPH1 was investigated by using Western blotting. C) HeLa Tet-On cells were cotransfected with GFP–MCPH1-L (or the indicated phosphomutants/phosphomimetics) and either FLAG empty vector or FLAG–CDH1. GFP–MCPH1-L expression was induced by the addition of tetracycline 10 ng/ml. After 24 h, cells were lysed and samples analyzed by using Western blotting.

Fig 4: Reduction of neuroprogenitors in Mcph1-?e8 embryonic cortex. (A) Immunofluorescence staining of sagittal sections of the E15.5 brain with Sox2 and Tbr2 antibodies. (B) Quantification of Sox2+ and Tbr2+ cells. N: the number of mice analyzed. The total numbers of cells of control (Con) and mutant (Mcph1-?e8) scored are summarized under the respective graphs. Unpaired Student’s t-test was used for statistical analysis. *, p < 0.05; n.s, not significant.

Fig 5: MCPH1 promotes the recruitment of DDR factors at dysfunctional telomeres lacking POT1-TPP1 in a TRF2-dependent manner.a Immunostaining for MCPH1 telomeric localization in MCPH1+/+ HCT116 and two CRISPR/Cas9 MCPH1?/? HCT116 clones (B2 and A5) overexpressing the indicated constructs. MCPH1 localization at telomeres was assessed using an anti-MCPH1 antibody (green) and telomere were detected through PNA-FISH (red). Representative images from either three (MCPH1+/+ and MCPH1?/? B2 + vector, TRF2?B?M or TPP1?RD) or two (MCPH1?/? A5 and samples with TRF2?B?M + TPP1?RD) independent experiments. b Percentage of cells with >5 MCPH1-positive foci at telomeres from (a). Data represent mean values ± SD. n = 3 for MCPH1+/+ and MCPH1?/? B2 + vector, TRF2?B?M and TPP1?RD; n = 2 for MCPH1?/? A5 and samples with TRF2?B?M + TPP1?RD. A minimum of 200 cells were scored for each sample. c BARD1 TIF analysis in WT and MCPH1?/? cells reconstituted with either empty vector, WT MCPH1, MCPH1S333A or MCPH1S333D and overexpressing either empty vector or FLAG-TPP1?RD. Representative images from two independent experiments. d Percentage of cells with >5 BARD1-positive TIFs from (c). The means from two independent experiments ± SD are shown. At least 200 cells were scored for each sample. e–g Quantification of the percentage of cells with >5 p-RPA32 (S33) (e) and CTIP (f) TIFs and with >3 EXOI (g) TIFs in WT and MCPH1?/? cells reconstituted with the indicated constructs and overexpressing either empty vector or FLAG-TPP1?RD (see also Supplementary Fig. 6f–h). Data represent the mean ± SD from two independent experiments. At least 200 cells were scored for each sample. h RAD51 TIF analysis in TPP1?RD-expressing U2OS cells treated with either scrambled or MCPH1 shRNA and reconstituted with either empty vector, WT MCPH1, MCPH1S333A or MCPH1S333D. Representative images from three independent experiments. i Quantification of the percentage of cells with >5 RAD51-positive TIFs shown in (h). Data represent the mean values ± SD, n = 3. At least 200 cells were scored for each sample. The statistical analysis for b, d–g and i was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Scale bars for a, c, h: 5 µm.