Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

Abstract

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1-TRF2 complex reveals that this interaction is mediated by the MCPH1 ₃₃₀YRLSP₃₃₄ motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

Fig. 1. Structure of the human TRF2^TRFH–MCPH1^TBM complex.

a Schematic representation of human TRF2 and MCPH1 domains, showing the interaction domains. b Comparison of MCPH1^TBM sequence with those of known TRF2-interacting protein. The conserved amino acid Y/H-X-L-X-P consensus sequence is highlighted. c Dimeric TRF2–MCPH1 structure is shown in a ribbon representation (TRF2, green/cyan; MCPH1, magenta/yellow). d TRF2 and MCPH1 are depicted in green and yellow, respectively, and the residues involved in their interaction are shown. Hydrogen bonding: magenta dashed lines. e MCPH1^TBM (in yellow) is buried inside a hydrophobic pocket formed by TRF2 helices α2 and α3 (in green). f ITC measurement of the interactions between TRF2^TRFH and different MCPH1^TBM mutant peptides. S333phos is a phosphorylated S333 peptide synthesized using a phosphorylated serine as starting material. Equilibrium dissociation constant (K_D) values derived from ITC data are shown in Table 2.

Fig. 2. MCPH1^S333 phosphorylation modulates MCPH1–TRF2 interaction.

a Schematic representation of the human MCPH1 constructs generated. The TBM sequence for each construct is shown and amino acid substitutions are depicted in red. b Co-IP with anti-Myc antibody-conjugated agarose beads from lysates of 293T cells expressing Myc-tagged TRF2 and either FLAG-tagged WT MCPH1, FLAG-MCPH1^TBM mutants or FLAG-MCPH1^ΔBRCT. γ-tubulin was used as a loading control. The blot shown is representative of four independent experiments. c Immunostaining-PNA FISH in HeLa cells overexpressing either empty vector or one of the FLAG-tagged WT MCPH1, MCPH1^AA, MCPH1^S333A, MCPH1^S333D, MCPH1^ΔBRCT constructs and either empty vector or HA-TPP1^ΔRD. MCPH1 proteins were detected using an anti-FLAG antibody (green) while telomeres were detected with a Cy3-OO-(CCCTAA)₄ PNA probe (red). 4,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei (blue). Representative images from three independent experiments are shown. White arrowheads indicate MCPH1 foci co-localizing with the telomere signals. Scale bar: 5 μm. d Quantification of the percentage of cells with >5 MCPH1-positive foci at telomeres from c. Data represent the mean ± standard deviation (SD) from three independent experiments. At least 200 cells were scored for each sample. Significance was determined with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. P values are shown. e Comparison of MCPH1^TBM amino acidic sequence across several mammalian species. The conserved residues are highlighted in yellow, while the residues in red differ from the canonical Y/H-X-L-X-P amino acid sequence. f Immunostaining-PNA FISH in MEFs overexpressing either Myc-WT MCPH1 or Myc-MCPH1^ΔBRCT together with either empty vector or FLAG-TIN2^A110R. Myc-MCPH1 proteins were detected with a Myc antibody (green), while telomeres were detected with either a telomeric PNA probe or a FLAG antibody that recognizes FLAG-TIN2^A110R (in red). Nuclei were stained with DAPI (blue). Representative images from three independent experiments. Scale bar: 5 μm. g Quantification of the percentage of cells with >5 MCPH1-positive foci at telomeres from f. Data are representative of the mean of three independent experiments ± SD. A minimum of 200 cells for each sample were scored. Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test.

Fig. 3. 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^Δ/Δ 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^Δ/Δ B2 + vector, TRF2^ΔBΔM or TPP1^ΔRD) or two (MCPH1^Δ/Δ A5 and samples with TRF2^ΔBΔM + TPP1^ΔRD) 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^Δ/Δ B2 + vector, TRF2^ΔBΔM and TPP1^ΔRD; n = 2 for MCPH1^Δ/Δ A5 and samples with TRF2^ΔBΔM + TPP1^ΔRD. A minimum of 200 cells were scored for each sample. c BARD1 TIF analysis in WT and MCPH1^Δ/Δ cells reconstituted with either empty vector, WT MCPH1, MCPH1^S333A or MCPH1^S333D and overexpressing either empty vector or FLAG-TPP1^ΔRD. 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^Δ/Δ cells reconstituted with the indicated constructs and overexpressing either empty vector or FLAG-TPP1^ΔRD (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^ΔRD-expressing U2OS cells treated with either scrambled or MCPH1 shRNA and reconstituted with either empty vector, WT MCPH1, MCPH1^S333A or MCPH1^S333D. 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.

Fig. 4. MCPH1–TRF2 interaction is required to promote HDR at telomeres lacking POT1-TPP1.

a Telomeric PNA-FISH staining of metaphase spreads from MCPH1^+/+, MCPH1^Δ/Δ B2, and MCPH1^Δ/Δ A5 HCT116 cells overexpressing either empty vector, TRF2^ΔBΔM, TPP1^ΔRD, or both TRF2^ΔBΔM and TPP1^ΔRD. Telomeres were detected using a PNA probe (red) and DAPI was used to stain the chromosomes (blue). Representative images from either three (MCPH1^+/+ and MCPH1^Δ/Δ B2 + vector, TRF2^ΔBΔM, or TPP1^ΔRD) or two (MCPH1^Δ/Δ A5 and samples with TRF2^ΔBΔM + TPP1^ΔRD) independent experiments. White and green arrowheads indicate chromosome and chromatid fusions, respectively. Scale bar: 5 μm. b, c Quantification of the percentage of chromosome (b) and chromatid (c) fusions observed in metaphase spreads shown in (a). Data represent the mean values ± SD. n = 3 for MCPH1^+/+ and MCPH1^Δ/Δ B2 + vector, TRF2^ΔBΔM, and TPP1^ΔRD; n = 2 for MCPH1^Δ/Δ A5 and samples with TRF2^ΔBΔM + TPP1^ΔRD. A minimum of 30 metaphases for each sample were examined per experiment. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. d Representative images of telomere sister chromatid exchanges (T-SCEs) (arrowheads) from three independent CO-FISH experiments on metaphase spreads of U2OS cells expressing TPP1^ΔRD and either scrambled or MCPH1 shRNA. Sister chromatid telomeres were labeled with a FAM-OO-(TTAGGG)₄ PNA probe (green) and with a Cy3-OO-(CCCTAA)₄ PNA probe (red) to detect telomeres generated by leading and lagging strand replication, respectively. Scale bar: 5 μm. e Quantification of the percentage of T-SCEs observed on metaphase spreads of U2OS cells expressing either empty vector or TPP1^ΔRD after MCPH1 depletion and reconstitution with the indicated constructs. Data are representative of the mean of three independent experiments ± SD. A minimum of 40 metaphases were analyzed per experiment. The indicated p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test.

Fig. 5. MCPH1 interacts with TRF2 in response to replication stress to promote replication fork progression at telomeres.

a Representative images from three independent experiments of telomeric PNA-FISH on chromosome spreads of WT and MCPH1^Δ/Δ HCT116 cells to detect multiple telomeric signals (MTS) (arrowheads). Scale bar: 5 μm. b Mean values ± SD of the percentage of MTS visualized by PNA-FISH in the indicated cell lines treated with either DMSO or 0.25 μM aphidicolin (APH). n = three independent experiments. At least 50 metaphases were scored. One-way ANOVA followed by Tukey’s multiple comparison test. c Co-IP with anti-TRF2 antibody from lysates of synchronized U2OS cells harvested at the indicated time points. Cyclin A was used as a control for cell cycle progression. Representative blots from two independent experiments. No Ab: no antibody control; *non-specific band. See also Supplementary Fig. 7c. d Percentage of cells showing ≥4 MCPH1/FLAG-TRF1 co-localizations in IMR-90 and HeLa cells expressing CDT1 (G₁) and Geminin (S/G₂). See also Supplementary Fig. 7d. Mean values ± SD from two independent experiments are shown. At least 200 nuclei were scored. Two-sided Student’s t test. e SMARD analysis of telomeric DNA fibers in U2OS treated with either Scrambled or MCPH1 shRNAs. Top: scheme of the CldU (green) and IdU (red) pulse label timing. Middle: representative images of telomeric fibers (telomeric DNA depicted in blue) from two independent experiments. Bottom: quantification of either CldU- or IdU-positive telomeric fibers. Scale bar: 10 μm. f Quantification of the length of CldU and IdU tracks from a representative experiment. Blue line: median. Two-sided Mann–Whitney test. g SMARD analysis of telomeric replication forks restart in U2OS cells treated with either Scrambled or MCPH1 shRNA and reconstituted with the indicated constructs. Top: pulse labeling timing scheme with hydroxyurea (HU)-induced replication block. Bottom: Representative images from two independent experiments. The dashed line separates the CldU-labeled portion (regular replication) and the IdU-labeled portion (replication restart). Scale bar: 10 μm. h Quantification of the IdU/CldU length ratio for the fibers labeled with both halogenated nucleotides in one representative experiment. Black line: median. Kruskal–Wallis test with Dunn’s multiple comparison test.

Fig. 6. MCPH1 is required for recruitment of HDR factors to stalled replication forks to promote fork restart.

a MCPH1 (red) and γ-H2AX (green) immunostaining in the indicated cells lines treated with either DMSO or 0.25 μM APH. Representative images from either three (MCPH1^+/+ and MCPH1^Δ/Δ + vector cells) or two (MCPH1^Δ/Δ + WT MCPH1 cells) independent experiments. b Mean values ± SD of the percentage of cells with >5 MCPH1/γ-H2AX co-localizations from (a). n = 3 for MCPH1^+/+ and MCPH1^Δ/Δ + vector cells, n = 2 for MCPH1^Δ/Δ + WT MCPH1 cells. At least 200 nuclei were scored. One-way ANOVA followed by Tukey’s multiple comparison test. c MCPH1 (red) and SMARCAL1 (green) immunostaining in U2OS cells treated with either DMSO or 0.25 μM APH. Representative images from three independent experiments. d Mean values ± SD of the percentage of cells with >5 co-localizing MCPH1/SMARCAL1 foci from (c). n = 3., at least 200 cells scored. Two-sided Student’s t test. e Percentage of cells with >5 BARD1, RAD51 or p-RPA32 (S4/S8) foci co-localizing with γ-H2AX in the indicated cell lines treated with either DMSO or 0.25 μM APH. Representative images are shown in Supplementary Fig. 8a. Data represent mean values ± SD, n = 2. At least 200 nuclei were examined. One-way ANOVA followed by Tukey’s multiple comparison test. f Representative images from two independent time course experiments to assess γ-H2AX foci (green) resolution in the indicated cell lines after release of HU block. Cells were incubated with 2 mM HU for 24 h before replacing the media and then fixed at the indicated time points. Untreated cells were used as control. g Number of γ-H2AX foci observed in 50–100 cells from each sample shown in (f). Data from one representative experiment. Red line: median. Kruskal–Wallis test followed by Dunn’s multiple comparison test. h, i Cell viability assay of MCPH1^+/+ and MCPH1^Δ/Δ cells treated with increasing amount of HU (h) or Olaparib (i), with or without concomitant BRCA1 depletion. Data from one of three independent experiments. Statistical significance is shown in Supplementary Table 1 (HU) and 2 (Olaparib). Scale bars for a, c, f: 5 μm.

Fig. 7. Schematic depicting MCPH1’s role at telomeres.

MCPH1 interacts with TRF2 when S333 is de-phosphorylated, while this interaction is disrupted upon S333 phosphorylation. In unperturbed conditions, MCPH1 exists in equilibrium between the phosphorylated and the de-phosphorylated forms and its telomeric localization increases in S phase. Removal of POT1 through the overexpression of TPP1^ΔRD increases MCPH1 localization to telomeres that is dependent upon the interaction with both TRF2 and γ-H2AX. MCPH1 binding to TRF2 promotes the recruitment of HDR and end resection factors to initiate HDR at telomeres lacking POT1-TPP1. Cells lacking MCPH1 or expressing MCPH1^S333D display reduced recruitment of HDR factors and reduced T-SCEs, suggestive of HDR defects. Similarly, induction of replication stress at telomeres increases MCPH1 telomeric localization, promoted by its interaction with TRF2. In the absence of MCPH1 or upon overexpression of the phosphomimetic mutant MCPH1^S333D, replication forks stalling at telomeres increases and telomere replication is impaired, resulting in telomere fragility. These observations suggest that telomeric localization of MCPH1 is required for proper telomere replication by promoting stalled replication fork restart.