TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

Abstract

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

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Figure 1: Mutations in TRAIP cause primordial dwarfism.
Figure 2: TRAIP mutations result in reduced cellular levels of TRAIP protein.
Figure 3: TRAIP localizes to sites of UV-induced DNA damage.
Figure 4: TRAIP is required for UV-induced RPA2 and H2AX phosphorylation during S-phase.
Figure 5: Impaired growth and cell cycle progression in TRAIP-deficient cells.
Figure 6: Replication fork stalling is increased following UV-induced DNA damage in patient cells.

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Acknowledgements

We thank the families and clinicians for their involvement and participation; N. Hastie, W. Bickmore, D. Fitzpatrick, J.-C. Acosta, M. Nowotny, V. Vitart and A. Lehmann for helpful discussions; R. Geahlen (Purdue University), S. Taylor (University of Manchester), D.-J. Kleinjan (University of Edinburgh), A. Lichawska and J. Mansfeld (Gurdon Institute) for their kind gifts of reagents; J. Ding, P. Hari and E. Milz for technical assistance; E. Freyer for assistance with FACS analysis; the IGMM core sequencing service; A. Wheeler and the IGMM imaging facility for assistance with microscopy, E. Maher and A. Pearce for performing SNP genotyping arrays. This work was supported by funding from the Medical Research Council and the European Research Council (ERC, 281847) (A.P.J.), the Lister Institute for Preventative Medicine (A.P.J. and G.S.S.), Medical Research Scotland (L.S.B.), German Federal Ministry of Education and Research (BMBF, 01GM1404) and E-RARE network EuroMicro (B.W.), Wellcome Trust (M.E. Hurles), CMMC (P.N.), Cancer Research UK (C17183/A13030) (G.S.S. and M.R.H.), Swiss National Science Foundation (P2ZHP3_158709) (O.M.), AIRC (12710) and EU FP7-PEOPLE (CIG_303806) (S.S.), Cancer Research UK (C6/A11224) and ERC/EU FP7 (HEALTH-F2-2010-259893) (A.N.B. and S.P.J.).

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Contributions

M.E. Hurles, L.S.B., M.H., G.Y., J.A., H.T., P.N., and B.W. performed exome sequencing and analysis. L.S.B., M.E. Harley, G.Y., S.M. and B.W. performed sequencing, genotyping, linkage analysis and other molecular genetics experiments. M.E. Harley, O.M., A.L., M.R.H., A.N.B., A.Z., K.R., M.A.M.R., A.F., C.-A.M., S.S., S.P.J. and G.S.S. designed and performed the cell biology experiments. N.H.E., L.G., L.C., M.M., M.S., M.B.B., K.J.M. and B.W. ascertained subjects, obtained samples and/or assisted with clinical studies. M.E. Harley and A.P.J. wrote the manuscript. The study was planned and supervised by B.W., G.S.S. and A.P.J.

Corresponding authors

Correspondence to Bernd Wollnik or Grant S Stewart or Andrew P Jackson.

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M.E. Hurles is a cofounder of, shareholder in and consultant to Congenica Ltd., a clinical diagnostics company.

Integrated supplementary information

Supplementary Figure 1 Patients P1 and P2 have distant common ancestry, sharing a common 4.3 Mb homozygous haplotype across the TRAIP locus.

(a) Schematic of high density SNP genotyping of the TRAIP locus in P1 and P2 (blue shading) demonstrates that both patients have 4.6 Mb and 8.4 Mb regions of homozygosity surround the TRAIP gene which lies at 49.9 Mb. An identical haplotype of 117 SNPs is evident in both patients within the shared region of homozygosity consistent with a shared common ancestor. Heterozygous SNP markers delineating regions of homozygosity are shown in red. 102 homozygous SNP markers within the 4.3 Mb region are omitted for clarity. (b) Inbreeding coefficients (F) estimates for P1 and P2 from SNP genotype data. FEstim analysis software that utilizes hidden Markov chain maximum likelihood estimation approach on the basis of observed genome-wide marker genotypesS3 was used to derive inbreeding coefficients for patient P1 and P2 from genome wide SNP genotyping data. An inbreeding coefficient of 0.003 was found for P2 that corresponds to an inbreeding coefficient seen in offspring of 3rd cousin parents. This estimate was confirmed by a complimentary approach that derives a statistic FROH from the percentage of the genome containing large regions of homozygosity, a measure that correlates well with inbreeding coefficients derived from pedigreesS4. Additionally, two point linkage analysis using the known family pedigree in P3 and a pedigree equating to the calculated inbreeding coefficient of 0.003 for P2, generates a significant combined LOD score of 4.42 at θ=0.

Supplementary Figure 2 TRAIP protein levels are markedly reduced in patient P2.

Expression of TRAIP protein is only detectable on prolonged exposure of immunoblots. Residual protein detected may include the isoform encoded by a transcript that skips exon 7. Deletion in this exon results in an in-frame deletion of 37 amino acids, which would be predicted to result in a 47 kDa protein that will be difficult to distinguish from the 53 kDa full-length TRAIP on immunoblotting. Immunoblotting performed on cell lysates using TRAIP and H2A (loading control) antibodies.

Supplementary Figure 3 A TRAIP polyclonal antibody raised against recombinant GST-TRAIP1-270 detects WT, Arg18Cys and Arg185*-truncated TRAIP proteins.

(a) N-terminal FLAG-tagged TRAIP proteins, either WT or with patient mutations, were generated by in vitro transcription/translation (IVT) and then immunoblotted with either anti-FLAG or the rabbit polyclonal anti-TRAIP antibody generated in this study. Empty vector (-) was used as a negative control. The TRAIP antibody recognized all forms of TRAIP as efficiently as the anti-FLAG antibody, including the missense Arg18Cys mutation from patient P3 and the truncated TRAIP protein corresponding to aa1-184 that might be translated from transcripts containing the truncating Arg185* mutation in patients P1 and P2. WT and Arg18Cys were present at the same level (anti-FLAG) and detected equally well with TRAIP antibody, whereas Arg185* was present at a lower level (anti-FLAG), but still detected well with TRAIP antibody. The additional lower molecular band in each lane on the anti-TRAIP antibody blot likely results from untagged TRAIP protein generated during in vitro translation through usage of an alternative methionine start site, present immediately after the FLAG tag. (b) Full immunoblots of patients P1 and P2 reveal no short truncated form of TRAIP protein, indicating that the mutated transcripts are most likely degraded via nonsense-mediated decay.

Supplementary Figure 4 TRAIP transcript levels in patient P3 primary fibroblasts are not depleted.

RT-PCR using primers in 5’ and 3’ UTR to amplify TRAIP mRNA in primary fibroblasts demonstrates similar transcript levels in patient P3 cells when compared to control fibroblast cell line. Loading control, GAPDH.

Supplementary Figure 5 TRAIP is degraded via the proteasome in response to UV damage.

(a) Immunoblots of control fibroblast cells (upper panel) and HeLa cells (lower panel) treated with UV-C, before harvesting at the indicated times. The proteasome inhibitor MG132 (10 µM) was added to cell media at t=0 h. Cell lysates were analyzed by immunoblotting using antibodies against TRAIP and actin or H2A (loading controls). (b) Reduction in TRAIP protein levels is not accounted for by an indirect effect on cell cycle, as TRAIP protein levels remain relatively constant through the cell cycle. HeLa cells synchronized at the G1/S phase boundary by double thymidine block; time course following release into fresh media. Samples harvested at the indicated times and cell lysates analyzed by immunoblotting using antibodies against TRAIP, CDK1, cyclin A, cyclin B1, pSer10-Histone H3 and Histone H3.

Supplementary Figure 6 TRAIP-deficient cells are generally proficient in ATM, DNA-PKcs and ATR signaling in response to UV-C.

(a) TRAIP-depleted HeLa cells are proficient in ATM and DNA-PKcs signaling. HeLa cells were transfected with RNAi against TRAIP or luciferase (control). After 72h, cells were UV-C treated, before harvesting at indicated times. Cell lysates were analyzed by immunoblotting as indicated. (b) Phosphorylation of Ser33-RPA2 is unaffected by TRAIP depletion. HeLa cells transfected with RNAi against TRAIP or control were UV-C treated and harvested at indicated times. Cell lysates prepared using NP-40-based lysis buffer were analyzed by immunoblotting using antibodies against TRAIP, pSer33-RPA2, RPA2 and Vinculin. (c) TRAIP protein expression is restored in hTERT-immortalized patient fibroblasts following retroviral complementation with pMSCV-TRAIP. Immunoblots of patient fibroblasts after hTERT immortalization and retroviral transduction of pMSCV-vector only or pMSCV-TRAIP. * denotes a non-specific band. (d) Characterization of ATM, DNA-PKcs and ATR activation in P2TERT and P2TERT+TRAIP fibroblasts after UV-C irradiation. P2TERT and P2TERT+TRAIP fibroblasts were UV-C treated and harvested at indicated times. Cell lysates were analyzed by immunoblotting as indicated. ATM and DNA-PKcs autophosphorylation and NBS1 and KAP1 phosphorylation were similar in both cell lines, while a small reduction in CHK1 phosphorylation was observed in P2TERT cells. (e) TRAIP-depleted HeLa cells are proficient in G2/M checkpoint assay. Mitotic index is unchanged in TRAIP-depleted HeLa cells (plotted relative to the mitotic index of untreated cells in the same experiment). Mean ± SEM; n=3 independent experiments.

Supplementary Figure 7 TRAIP promotes S-phase–specific RPA2 and H2AX phosphorylation induced by UV-C.

(a, b) Scatter plots represent pSer4/Ser8-RPA2 (a) or γH2AX (b) signal integrated densities (arbitrary units) of individual EdU-negative and EdU-positive P2TERT and P2TERT+TRAIP fibroblasts shown in Figure 4g. Red lines denote mean ± SEM for n=3 independent experiments. Pooled data plotted, n>50 EdU positive cells and n>150 EdU negative cells quantified per cell line per treatment per experiment.

Supplementary Figure 8 TRAIP mutations do not detectably alter the accumulation of DNA strand breaks following UV-C treatment.

Primary patient fibroblasts or controls were UV-C treated and harvested 4 h later for neutral comet assay. Graph represents quantification of mean olive tail moments ± SEM for n=4 independent experiments. >250 comets analyzed per cell line per treatment.

Supplementary Figure 9 TRAIP deficiency delays cell cycle progression in S/G2.

(a) Quantification of number of mitotic cells contributing to 4n cell number at 6 h in Figure 5d. The mitotic index of siTRAIP EdU positive cells 6 h after EdU pulse labeling is not significantly different from control siRNA treated cells. Mean ± SEM of n=3 independent experiments; >800 EdU positive cells quantified per experiment. (b, c) Complementation with TRAIP restores delayed S/G2 phase progression of P2TERT fibroblasts. P2TERT and P2TERT+TRAIP cells were labeled with 10 µM BrdU for 30 min before washing out and replacing with fresh media. At indicated times, cells were harvested, fixed and prepared for flow cytometry. (b) Representative images of gating used in analysis of flow cytometry data. (c) Quantification of BrdU positive cells at 0, 4 and 8 h after BrdU pulse labeling. Left, percentage of cells with 2n content; middle, percentage of cells with mid S phase content; right, percentage of cells with 4n content. Mean ± SEM of n=4 independent experiments; for 8 h time point, Student’s t-test: *p<0.05.

Supplementary Figure 10 Fork velocity, inter-origin distance and new origin synthesis rates are unaltered in TRAIP patient primary fibroblasts.

(a) Top panel, schematic indicating DNA labeling protocol. Bottom panel, quantification of IdU tract lengths in control and patient fibroblasts. Mean ± SEM of n=3 independent experiments; >100 fibers measured per cell line per experiment. (b) Quantification of inter-origin distances. Box plots, center lines indicate mean, box 25/75% and whiskers 5/95%. n=3 independent experiments; >30 structures measured per cell line per experiment. (c) Quantification of ongoing forks, 2nd label only (new origin firing) and 1st label termination (fork stalling) structures. Mean ± SD, n=3 independent experiments; > 300 fibers measured per cell line per experiment.

Supplementary Figure 11 Complementation with wild-type TRAIP rescues increased fork stalling and fork asymmetry in UV-irradiated TRAIPArg185* cells.

(a) Fork velocities in UV-treated TRAIP patient primary fibroblasts are similar to wild-type control cells. Mean ± SEM, n=2 independent experiments, >100 structures measured per cell line per experiment. (b) Quantification of ongoing forks, 2nd label only (new origin firing) and 1st label termination (fork stalling) structures, untreated or after 30 J/m2 UV-C irradiation. Mean ± SD, n=3 independent experiments. >400 structures measured per cell line per experiment. Student’s t-test: **p<0.01; ns, not significant. (c) Quantification of replication fork asymmetry in P2TERT and P2TERT+TRAIP fibroblasts after 30 J/m2 UV, 0.5 µM aphidicolin or 50 ng/ml mitomycin C treatment. Ratio of left/right fork length; mean ratio for each cell line is indicated in italics; Mann Whitney Rank sum test: ***p<0.001; ns, not significant. Data plotted pooled from n=2 independent experiments, with >50 structures quantitated per cell line per experiment. (d) Quantification of ongoing forks, 2nd label only (new origin firing) and 1st label termination (stalled fork) structures, untreated or after 50 ng/ml mitomycin C treatment. (e) Track lengths are not detectably altered in TRAIP-deficient fibroblasts compared with controls, after UV-C or low dose aphidicolin treatment. P2TERT and P2TERT+TRAIP fibroblasts untreated or following treatment with 30 J/m2 UV or 0.5 µM aphidicolin. Mean ± SEM, n=2 independent experiments. >100 tracts measured per cell line per experiment.

Supplementary Figure 12 Replication restart is not impaired in TRAIP patient cells after hydroxyurea-induced fork arrest

Top, schematic of the experiment. Bottom, quantification of ongoing forks, 2nd label only (new origin firing) and 1st label termination (fork stalling) structures. Data are mean ± SD for n=2 independent experiments. >300 structures quantified per cell line per experiment.

Supplementary Figure 13 Impaired DDR to replication-blocking lesions such as UV photodimers in TRAIP-depleted patient cells can reduce growth through reduction in cell number generated during development

Replication blocking lesions (red circles) arise in the genome during development, which require efficient DDR for their resolution and to ensure efficient cellular proliferation utilizing multiple repair pathways that include translesion synthesis/post-replication repair. In TRAIP patient cells, impaired DDR during replication results in delayed S/G2 phase progression. Increased cell cycle length and reduced numbers of cycling cells will consequently lead to reduction in overall cell proliferation, decreasing total cell number. Impaired spindle checkpoint function could also increase aneuploidy levels leading to increased cell deathS5. However, unlike mutations in BUBR1S6, an archetypal SAC protein, variegate aneuploidy is not observed with normal karyotypes in all TRAIP patients (Table 1). Decrease in cell number results in diminished growth potential, with reduction in both brain and body size.

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Supplementary Figures 1–13, Supplementary Table 1 and Supplementary Note (PDF 1778 kb)

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Harley, M., Murina, O., Leitch, A. et al. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism. Nat Genet 48, 36–43 (2016). https://doi.org/10.1038/ng.3451

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