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.

Figure 1. Mutations in TRAIP cause primordial dwarfism

(a) Mutations identified in TRAIP. Top, schematic of TRAIP gene structure; middle, TRAIP protein structure; bottom, sequence electropherograms demonstrating (middle, right panels) homozygous nonsense mutations in Patient 1 (P1) and Patient 2 (P2) and (left) a homozygous missense mutation, Arg18Cys in Patient 3 (P3). (b) A physiochemically similar residue is present at codon 18 in all vertebrates. Sequence alignments of Homo sapiens, Pan troglodytes, Mus musculus, Gallus gallus, Xenopus tropicalis and Danio rerio using Clustal W. (c) Patients with TRAIP mutations have prenatal onset severe growth failure with disproportionate microcephaly. Birth weight (BWGT), current height (HGT) and current head circumference (OFC) plotted as z-scores (standard deviations from population mean for age and sex). 97.5% of general population will lie above the dashed line at −2 S.D. Black bars indicate mean values. (d) Cerebral cortical size is markedly reduced with simplification of gyral folding. MRI T2-weighted sagittal and axial images of P3 (age 3 years) compared with control scans of a healthy child (age 3 years 1 month). Scale bar, 2 cm. (e) Photographs of affected individuals with TRAIP mutations demonstrating facial similarities, including an elongated narrow face and micrognathia. Informed consent to publish photographs was obtained from families.

Figure 2. TRAIP mutations result in reduced cellular levels of TRAIP protein

(a) The Arg185* mutation severely reduces TRAIP transcript levels in P1 and P2 patient cell lines. RT-PCR using primers in 5′ and 3′ UTR to amplify TRAIP transcripts in primary fibroblasts and lymphoblastoid cell lines (LCLs) demonstrates marked decrease in full length TRAIP transcript, consistent with nonsense-mediated decay. Additional low intensity PCR products are evident, that represent alternatively spliced transcripts, confirmed by subcloning and Sanger sequencing (lower panels). These include transcripts, which through omission of exon 7, or exons 6, 7 and 8, retain an open reading frame and result in protein products with small internal deletions. Loading control, GAPDH. (b) TRAIP protein levels are reduced in patients with Arg185* and the Arg18Cys mutations. Immunoblotting with an affinity purified rabbit anti-TRAIP antibody raised against recombinant TRAIP protein demonstrates reduced levels of the 53 kDa TRAIP protein in P3, and marked depletion in P1 and P2 where protein is only detectable on prolonged exposure (Supplementary Fig. 2). Loading controls, actin and vinculin.

Figure 3. TRAIP localizes to sites of UV-induced DNA damage

(a) TRAIP localizes to DNA damage sites induced by UV laser microirradiation both in the absence and presence of pre-treatment with BrdU as a damage sensitizer. Representative images, before and after UV laser microirradiation. Scale bar, 5 μm. (b) GFP-TRAIP colocalizes with γH2AX and with RFP-PCNA at sites of UV laser-induced damage. Representative images of UV laser-irradiated GFP-TRAIP expressing cells immunostained for γH2AX (pre-sensitized with BrdU) or co-expressing RFP-PCNA (no BrdU pre-treatment) as indicated. Scale bar, 5 μm. (c, d) GFP-TRAIP is detected by a Proximity Ligation Assay (PLA) in close proximity to PCNA, an association enhanced after UV-induced damage. (c) Representative images of PLA signals/nucleus in doxycycline-inducible GFP-TRAIP HeLa cells before and after damage with 25 J/m² UV-C. Scale bar, 5 μm. (d) Quantification of PLA signals/nucleus. Box plots, center line denote mean values, box 25/75 %, whiskers 5/95 %, data pooled from n=2 independent experiments, n>65 data points per condition per experiment; Mann Whitney rank sum test: *** p<0.001. (e) TRAIP accumulates at sites of localized UV damage, colocalising with RPA and γH2AX. Representative immunofluorescence images of MRC5 cells transfected with GFP-TRAIP or GFP alone after UV-C irradiation through 3 μm microfilters. Scale bar, 5 μm.

Figure 4. TRAIP is required for UV-induced RPA2 and H2AX phosphorylation during S-phase

(a) Phosphorylation of downstream ATR substrates is unaffected by TRAIP depletion. 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 Western blot as indicated. (b, c) TRAIP loss reduces RPA2 phosphorylation and histone H2AX phosphorylation (γH2AX) in response to UV. HeLa cells transfected with TRAIP and control siRNAs (b) or primary patient fibroblasts (c) were UV-C treated, harvested and immunoblotted as indicated. (d, e) Quantification of pS4/S8-RPA2 (d) and γH2AX (e) in primary fibroblasts after UV. Chemiluminescence from Western blots quantified using ImageQuant. Mean ± SEM, n=3 experiments; values normalized to total RPA2 signal; two-way ANOVA across all time points: *** p<0.001, ** p<0.01. (f) Retroviral complementation with wild-type TRAIP rescues impaired phosphorylation of RPA2 and H2AX after UV-C irradiation in TRAIP^Arg185* cells. Fibroblasts derived from P2^Arg185* were immortalized with hTERT, denoted P2^TERT; and reconstituted with pMSCV-TRAIP, P2^TERT+TRAIP. (g) TRAIP is required for optimal RPA2 and H2AX phosphorylation in S-phase. P2^TERT and P2^TERT+TRAIP cells were irradiated with 15 J/m² UV-C, labeled with EdU for 4 h, pre-extracted, fixed and co-stained for pS4/S8-RPA2 or γH2AX, EdU and DAPI. Representative images of immunofluorescence staining of pS4/S8-RPA2 (left), quantification of pS4/S8-RPA2 (middle) and γH2AX (right) signal integrated density in EdU-positive and EdU-negative cells. Mean ± SEM for n=3 experiments, values normalized to P2^TERT+TRAIP; Student’s t-test: *p<0.05; ***p<0.001. Scale bar, 10 μm.

Figure 5. Impaired growth and cell cycle progression in TRAIP deficient cells

(a) TRAIP mutations impair cell proliferation. Cell numbers of passage-matched primary fibroblast cell lines derived from patients or controls were determined over 17 days to establish growth rates. Mean ± SEM for n=3 experiments; fold growth relative to day 0. (b) Complementation with wild-type TRAIP rescues the slow growth phenotype of TRAIP^Arg185* cells. P2^TERT fibroblasts were reconstituted with pMSCV-vector only or pMSCV-TRAIP. Growth rates of passage-matched cell lines were analyzed over 18 days. Mean ± SEM for n=3 experiments; fold growth relative to day 0. (c) Doubling times of fibroblast cells from (a) and (b). Mean ± SEM for n=3 experiments; Student’s t-test: *p<0.05; **p<0.01. (d) TRAIP-depleted cells exhibit delayed S/G2 phase progression. HeLa cells were transfected with RNAi against TRAIP or luciferase (Luc) control. 72h later cells were pulse 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. Left, Western blots of RNAi for TRAIP or control Luc demonstrate effective TRAIP depletion. Middle, representative images of gating used in analysis of flow cytometry data. Right, quantification of the relative number of cells with 4n content; fold change relative to 0h. Mean ± SEM, n=4 experiments. Fold change relative to 0h; two-way ANOVA across all time points: ** p<0.01.

Figure 6. Replication fork stalling is increased following UV-induced DNA damage in TRAIP patient cells

(a) First label termination events (representing elevated replication fork stalling) are increased after UV irradiation in TRAIP patient fibroblasts. Top left panel, schematic of the experimental plan. Bottom left panel, representative images of ongoing or stalled replication forks and new origin firing from DNA fiber spreads of primary fibroblasts. Right panel, quantification of ongoing forks, 2^nd label only (new origin firing) and 1^st label termination (fork stalling) structures in fibroblast cells. Mean ± SD, n=3 independent experiments, >400 structures per cell line per experiment quantified. Student’s t-test: *p<0.05; **p<0.01; ns, not significant. (b, c) Substantial fork asymmetry is seen in UV-treated patient cells. (b) Representative images of DNA fibers from controls (Con1, Con2) and patient fibroblasts (P2, P3) after 30 J/m² UV treatment. (c) Quantification of replication fork asymmetry. 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 points pooled from n=2 independent experiments, >50 structures per cell line per experiment quantified.