Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay

Abstract

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. We performed a CRISPR-Cas9 knockout screen focused on ubiquitin E3 ligases and related proteins with the DNA topoisomerase I inhibitor camptothecin. This led us to establish that MAEA, a core subunit of the CTLH E3 ligase complex, is a critical regulator of homologous recombination and the replication stress response. In tandem, we identified eight patients with variants in MAEA who present with a neurodevelopmental disorder that we term DIADEM (Developmental delay and Intellectual disability Associated with DEfects in MAEA). Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart and protection as a likely cause of disease. Mechanistically, we find that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which we propose may contribute to the presentation of DIADEM by compromising genome integrity and cell division during development.

Keywords: DNA Repair; DNA Replication; Neurodevelopmental Disorder; Ubiquitin.

Figure 1. Loss of CTLH components confers hypersensitivity to seDSB-inducing agents.

(A) Overview of the E3 ubiquitin ligase CRISPR screen. (B) DrugZ analysis of (A). Black symbols denote positive control proteins, while red symbols denote CTLH complex members. Diamonds and circles denote significant and non-significant hits, respectively. (C) MAEA domain map displaying CRISPR-induced edits in MAEA KO and MAEA hypomorph U2OS cells. (D) Clonogenic survival assays in MAEA KO U2OS cells using camptothecin and olaparib. (E) Clonogenic survival assays in HAP1 cell lines using camptothecin and olaparib. (F) Clonogenic survival assays using etoposide in the indicated U2OS cell lines. (D–F) n = 3 independent experiments; error bars denote mean ± SEM. KO knockout, HM hypomorph. Source data are available online for this figure.

Figure 2. Clinical MAEA variants associated with neurodevelopmental defects in humans hypersensitize cells to seDSB-inducing agents.

(A) Cα trace representation of the MAEA/RMND5A/UBE2H complex crystal structure (PDBID: 8PJN). Altered residues are shown as CPK spheres colored by atom (C – cyan, O – red, N – blue, S – yellow). The environments of each mutant site are shown in the expansion insets. (B) Domain map of MAEA with C > S mutations and clinical variants labeled. Red text indicates cells were complemented with these MAEA variants and tested in subsequent assays. (C–F) Clonogenic survival assays in the indicated U2OS cell lines using camptothecin or olaparib, as indicated. Data in (C–F) are the combined results of three independent experiments. Bars denote mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. 1S = eGFP-MAEA^1S (C340S), 2S = eGFP-MAEA^2S (C314S, C317S), 3S = eGFP-MAEA^3S (C314S, C317S, and C340S). Source data are available online for this figure.

Figure 3. MAEA loss impairs RAD51 loading, but not DNA end resection.

(A) TLR assay in U2OS cells with the indicated siRNAs (n = 3). Statistics were generated by performing an ordinary one-way ANOVA comparing each siRNA to the siLuc control. siCtIP and siLuc data are from five independent experiments. siMAEA and siRMND5A data come from three independent experiments. (B, C) Immunofluorescence-based quantification (B) and representative images (C) of chromatinized RPA in S phase cells treated with DMSO (1 h) or camptothecin (1 μM, 1 h). (D, E) Quantification (D) and representative images (E) of BrdU in U2OS cells treated with DMSO (1 h) or camptothecin (1 μM 1 h). (F, G) Representative images (F) and quantification (G) of RAD51 foci in S phase U2OS cells treated with DMSO or camptothecin. All quantifications of foci are the combined results of three independent experiments. Extended fluorescent images from (C, E) can be found in Fig.EV3C,D. For (B–G), P values were generated using a two-tailed Kruskal–Wallis test. γ is a measure of effect size. Data represents three independent experiments. Bars denote mean ±95% CI. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; exact P values can be found in Appendix Table S1. Scale bars = 20μm. Source data are available online for this figure.

Figure 4. MAEA KO cells are hypersensitive to replication stress.

(A–C) Clonogenic survival assays in WT and MAEA KO cells with HU (A), aphidicolin (B), and ATRi (C). (D, E) Quantification of chromatin-associated γH2AX and RPA signal in WT and MAEA KO cells upon HU (D) or ATRi (E) treatment for the indicated times. (F) Quantification of chromatin-associated γH2AX and RPA signal upon ATRi treatment in MAEA KO U2OS cells complemented with the indicated eGFP expression constructs. Representative images are shown in Fig. EV4F. (G) DNA fiber assay measuring replication fork progression in eGFP Only (MAEA^−/−) and eGFP-MAEA^WT cells following treatment with camptothecin. Clonogenic data (A–C) are from three independent experiments. Statistics were generated using an ordinary two-way ANOVA. Bars represent the mean ± SEM. DNA fiber data are the combined result of three independent experiments. P values were generated by performing a two-tailed Kruskal–Wallis test. γ is a measure of effect size. Bars represent the mean ± 95% CI. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; exact P values can be found in Appendix Table S1. Scatter plots represent two independent experiments. HU hydroxyurea, ATRi ATR inhibitor (AZD3768). Source data are available online for this figure.

Figure 5. MAEA loss impairs replication fork stability, restart, and protection.

(A, B) Quantification of replication tract lengths (A) and spontaneous replication fork stalling (B) in MAEA KO U2OS cells complemented with the indicated eGFP expression constructs. (C, D) Quantification of HU-induced replication fork stalling (C) and fork restart after HU wash-out (D) in MAEA KO U2OS cells complemented with the indicated eGFP expression constructs. (E) Quantification of replication fork degradation after HU treatment in MAEA KO U2OS cells complemented with the indicated eGFP expression constructs. (F) Quantification of replication fork degradation in primary fibroblasts from Patients 1, 2, and 4 versus a WT control fibroblast cell line following treatment with HU. The data were the combined result of three independent experiments. P values were generated by performing a two-tailed Kruskal–Wallis test on scatter plots and an ordinary one-way ANOVA on histograms. γ is a measure of effect size. Bars represent the mean ± 95% CI. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; exact P values can be found in Appendix Table S1. HU hydroxyurea, KO knockout. Source data are available online for this figure.

Figure EV1. MAEA KO and HM cell line validation.

(A) Immunoblot for MAEA in the indicated U2OS cell lines; n = 2 independent experiments. (B) TIDE analysis of the sgRNA target site in MAEA KO and MAEA HM cells. (C) Sanger trace analysis of the sgRNA target site in MAEA KO and HM cells. (D) Cell cycle analysis of MAEA KO and HM cells. (E, F) Representative images of untreated WT and MAEA-deficient U2OS (E) and HAP1 (F) cells. (G) Immunoblot for MAEA in U2OS following siRNA transfection; n = 3 independent experiments. (H, I) Representative images (H) and colony counts (I) of U2OS cells with or without siRNA-mediated MAEA depletion. (J) The average colony size of siRNA-transfected U2OS cells at 500 cells per well. (K) Total area coverage of U2OS colonies following siRNA transfection. (I–K) Bars represent the mean ± SEM. KO knockout, HM hypomorph. Source data are available online for this figure.

Figure EV2. Clinical and C > S MAEA variants are evolutionarily conserved.

(A) Aminode analysis (Chang et al, 2018) of MAEA, with C > S mutations annotated. The red line represents conservation across species. Species compared in the analysis are listed in the box. (B) Immunoblot assessing expression of eGFP-MAEA constructs; n = 2 independent experiments. Source data are available online for this figure.

Figure EV3. MAEA loss compromises RAD51 foci formation.

(A) Immunoblot against the indicated proteins with the indicated siRNAs; n = 3 independent experiments. (B) Quantification of BrdU immunofluorescence, after camptothecin treatment in the presence or absence of siRNA-mediated CtIP depletion. (C, D) Representative images from Fig. 3C,E expanded to include γH2AX staining, indicative of the S phase cells. (E) Immunoblot against RAD51 in WT and MAEA KO U2OS cells. (F) Immunoblot for indicated proteins in U2OS WT or MAEA KO cells. (E, F) are each n = 3 independent experiments. P values were generated by performing a two-tailed Kruskal–Wallis test. γ is a measure of effect size. The data were the combined results of two independent experiments. Bars denote mean ± 95% CI. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; exact P values can be found in Appendix Table S1. Scale bars = 20 μm. NT non-treated, KO knockout. Source data are available online for this figure.

Figure EV4. RAD51 loading defects in MAEA-deficient SH-SY5Y cells.

(A) Immunoblot for MAEA depletion in polyclonal SH-SY5Y cells following CRISPR-Cas9 editing; n = 3 independent experiments. (B) Quantification and (C) representative images of BrdU in SH-SY5Y cells treated with DMSO (1 h) or camptothecin (1 μΜ, 1 h). (D) Quantification and (E) representative images of RAD51 foci in S-phase SH-SY5Y cells treated with DMSO or camptothecin. Bars in (B, D) represent median and interquartile range. P values were generated by performing a two-tailed Kruskal–Wallis test. γ is a measure of effect size. The data represent three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; exact P values can be found in Appendix Table S1. Scale bars = 10 μm. For (B, C), outliers were removed using ROUT analysis (Q = 1%). (F) Representative images of Fig. 5F. Source data are available online for this figure.