Figure 1.
UVB irradiation induces NR4A nuclear foci formation.
(A–B) MM96L cells transiently transfected with EYFP-NR4A1 (A) or EYFP-NR4A2 (B) were mock (No UV), or UVR (+UV) treated, and fixed 4 hours post treatment. Localisation of the NR4A fusion proteins was determined by fluorescence imaging, representative cells are shown. (C–D) Relative quantification of UV induced NR4A nuclear foci was performed by blind scoring images as diffuse or nuclear foci positive for NR4A1 (C) or NR4A2 (D). Data represents the pooled relative percentages from at least three independent experiments. An unpaired student t-test was used to determine the statistical differences between control and UV treated cells (**p<0.01, ***p<0.001). (E) A schematic representation of the NR4A2 WT, NTD alone and sequential N-terminal deletion mutants of NR4A2 used to identify the region responsible for translocation to foci. In all cases the EYFP fluorophore is fused at to the N-terminal end of the protein. (NTD, N-terminal domain; DBD, DNA binding domain; LBD, ligand binding domain). (F) Representative images of nuclear localisation of EYFP-WT and ΔNT and NTD NR4A2 proteins following mock or UVR treatment, as indicated. (G) Localisation of EYFP-NR4A2 WT, Δ84, Δ91 and ΔNT proteins to nuclear foci in response to mock (white boxes) or UV irradiation (black boxes) was scored as foci positive or negative in blind analysis. Data represents the mean +/− SEM of three independent experiments. Statistical analysis was performed using ANOVA with a Tukeys post-test (***, p<0.001; **, p<0.01, ns = not significant, nd = 0 foci positive nuclei detected). (H) Schematic representation of an NR4A2 N-terminal domain- PPARγ dominant negative-LBD chimera termed N2-DS. Lower panels show the post UVR localisation of EYFP tagged PPARγ and N2-DS chimera in the nucleus of transfected cells.
Figure 2.
p38 signalling mediates UV induced localisation of NR4A proteins to sites of DNA damage repair.
(A) Fluorescent images of EYFP-NR4A protein localisation in nuclei following UVR treatment in the presence of inhibitors of p38 (SB203580; SB), PKA (H89), PKC (GF109203X; GF) or MEK1/2 (PD98059; PD) or DMSO (Vehicle). Representative images from 4 independent experiments are shown. (B) Nuclear images were scored as diffuse or foci positive from vehicle and p38 inhibitor treatments described above in mock and UVR treated cells. Bar graph shows the mean percentage of foci positive cells (+/− SEM). (C) Schematic representations of NR4A2 protein showing position of potential of Alanine substitutions at p38 phosphorylation sites in the N-terminal domain at amino acids S126 and S181. Clustal alignment below shows sequences flanking the serine residues (arrow head) and phospho sites (underlined). (D) Scoring of foci containing nuclei in cells expression WT and S126A, S181A and S126A/S181A mutant proteins as indicated. Bar graph represents the mean +/− SEM of results from 4 independent experiments. Statistical analysis in panels (B) and D was performed using ANOVA with a Tukey’s post-test (***, p<0.0001; **, p<0.001; *, p<0.01; ns, not significant).
Figure 3.
NR4A2 co-localises with DNA repair proteins following UVR treatment.
MM96L cells transfected with pEYFP-NR4A2 were irradiated with 25 mJ/cm2 UV, fixed at 4 hours post-UV treatment and probed with primary antibodies against endogenous (A) γH2A.X or (B) DDB2 or (C) XPC proteins as indicated. Left panels, pEYFP-Nurr1 fluorescence (green); middle panel, immunofluorescence (red); centre right, merged images; right, DAPI staining. These data are representative images from 4 independent experiments.
Figure 4.
Mutations in the NR4A2 LBD result in mis-localisation of the receptor with DNA repair proteins.
(A) Schematic representation of the WT and ΔH12 mutant forms of NR4A2 indicating the position of the C-terminal helix 12. MM96L cells transfected with WT or ΔH12 forms of NR4A2 were treated with 25 mJ/cm2 UV, fixed at 4 hours post-UV treatment. Immunofluorescence was performed to detect (B) γH2A.X; (C) DDB2; and (D) XPC proteins. Left panels (green fluorescence), pEYFP-NR4A-WT (upper) or pEYFP-NR4A2-ΔH12 (lower); Middle panels, γH2A.X, DDB2 or XPC immunofluorescence (red); Right panels, merged images; Representative images from 4 independent experiments is shown.
Figure 5.
Trichostatin A (TSA) treatment restores co-localisation of NR4A2-ΔH12 and DNA repair proteins.
The co-localisation of the NR4A2-ΔH12 mutant (green) and (A) γH2A.X, (B) DDB2 or (C) XPC shown in red was performed in the presence of vehicle (upper panels) or TSA (lower panels). These data are representative images from 4 independent experiments. Left panels (green fluorescence), pEYFP-NR4A2-ΔH12; Middle panels, γH2A.X, DB2 or XPC immunofluorescence (red); Right panels, merged images; Representative images from 4 independent experiments is shown.
Figure 6.
NR4A2 shows increased UV induced interaction with PARP1.
(A) Fluorescence images of EYFP-NR4A2 protein localisation in nuclei following UVR treatment in the presence of inhibitor of PARP (PJ34; PJ) or Vehicle (DMSO). Representative images from 4 independent experiments are shown. (B) Co-immunoprecipitation of COS-1 cells transfected with MYC-NR4A2 and Flag-PARP1, as indicated, and mock or UV irradiated. Immunoprecipitation was performed with an anti-MYC antibody and precipitates probed with anti-FLAG antibody. A representative image taken from three independent experiments is shown. (C) MM96L cells co-transfected with WT or ΔH12 forms of NR4A2 and Flag-PARP1 (fl-PARP1) and treated with 25 mJ/cm2 UV, fixed at 4 hours post-UV treatment. Immunofluorescence was performed to detect FLAG-PARP1 protein. Left panels (green fluorescence), pEYFP-NR4A-WT (upper) or pEYFP-NR4A2-ΔH12 (lower); Middle panels, FLAG-PARP1 immunofluorescence (red); Right panels, merged images. (D) The co-localisation of the NR4A2-ΔH12 mutant (green) and FLAG-PARP1 shown in red was performed in the presence of vehicle (upper panels) or TSA (lower panels). Left panels (green fluorescence), pEYFP-NR4A2-ΔH12; Middle panels, FLAG-PARP1 immunofluorescence (red); Right panels, merged images.
Figure 7.
NR4A2 expression facilitates removal of UVR induced DNA lesions.
(A) A2058 melanoma cell lines engineered to stably express either GFP or NR4A2 were subjected to mock or UVR treatment after which the levels of CPD’s were determined by immunofluorescence 24 hours post irradiation. Representative images are shown. (B) Quantification of CPD fluorescence of multiple images from three independent experiments is shown with values normalised to the A2058-GFP mock treated cells. Data is expressed as the mean +/− SEM of 3 independent experiments. (C) A2058-GFP and A2058-NR4A2 cells were treated as described above and 6-4-PP levels determined by immunofluorescence at 4 hours post irradiation. Representative images are shown. (D) Quantification of 6-4-PP fluorescence was performed as described above for the mock (no UV) and 4 hour post UVR conditions. Data is expressed as the mean +/− SEM of 3 independent experiments. (E) South-western blot analysis of CPD levels in A2058 cells stably expressing GFP, NR4A2-WT or N2-DS proteins following UVR treatment. (F) South-western blot analysis of CPD levels in MM96L cells, transfected with negative or pooled NR4A2 and NR4A1 siRNAs as indicated, following UVR treatment. In panels E and F, total genomic DNA was harvested prior to irradiation (0 min) and 5 minutes and 24 hours post irradiation as indicated.