WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

Here, Caron et al. show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. Their findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

(H) Same as in G, except that phleomycin (Phleo) and neocarsinostatin (NCS) were used to induce DSBs. Blots were probed for Ub(K48), GFP and gH2AX. Tubulin is a loading control.
(I) Pulldown of ubiquitylated proteins by the TUBE approach in U2OS cells stably expressing GFP-RPB1. Cells were treated with the indicated siRNAs and with proteasome inhibitor (MG-132) 25 minutes before treatment with DNA damaging agents. Blots were probed for Ub(K48), GFP, WWP2 and gH2AX. Tubulin is a loading control. Figure S4. DNA-PK and WWP2 co-operate to promote DNA damageinduced RPB1 ubiquitylation (A) Pulldowns of ubiquitylated proteins in U2OS using the TUBE approach. Cells were treated with a broad-spectrum inhibitor of de-ubiquitylating enzymes (PR169) and the indicated siRNAs before phleomycin (Phleo) treatment. Blots were probed for p-RPB1 (S2), gH2AX and H2A.

Supplemental
(B) Western blot analysis of the indicated proteins in U2OS cells from A. Tubulin is a loading control.
(D) As in C, except that cells were treated with siRNAs against Luciferase (siLuc) and WWP2 and that blots were probed for WWP2 instead of Ku80, LigIV and DNA-PKcs.
(E) Immunofluorescence images (upper panel) of Ub(K48) accumulation at DNA damage tracks generated by multiphoton laser micro-irradiation in U2OS cells that were left untreated or treated with DNA-PK inhibitor (DNA-PKi). gH2AX is a DNA damage marker. Western blot analysis (lower panel) of DNA-PK activation 1 hour after 10 Gy of irradiation radiation (IR).
(F) Same as in E, except that cells were transfected with the indicated siRNA (upper panel).
Western blot analysis (lower panel) of WWP2 expression levels. Blots were probed for WWP2. Tubulin is a loading control.
(G) Quantification of upper panels in E (left) and F (right). The mean ±S.E.M from 3 independent experiments is shown. Statistical significance was calculated using the Student's t-test (p<0,05 *, p<0,01 **, p<0,001 ***). Figure S5. DNA-PK regulates RPB1 levels at broken genes (A) ChIP-qPCR against gH2AX in DMSO-treated (control) and DNA-PKi-treated U2OS HA-ER-I-PpoI cells at the indicated time points after (4-OHT) treatment and at the indicated positions at DAB1 and SLCO5a1. The mean ±SD from qPCR replicates of a representative experiment is shown.
(C) Western blot analysis of WWP2 expression in cells from A. Blots were probed for WWP2. Tubulin is a loading control.
(D) Western blot analysis of the indicated proteins in soluble and chromatin fractions from untreated and Phleomycin (Phleo-)treated U2OS cells. Tubulin and H4 are loading controls.
gH2AX is a DNA damage marker. A representative experiment is shown.
(F) Immunofluorescence images of gH2AX foci formation 1 hour after 10 Gy of IR in U2OS cells transfected with the indicated siRNAs.
(G) Quantification of F. The mean ±S.E.M from 3 independent experiments is shown.
(H) Western blot analysis of WWP2 expression in cells from F. Blots were probed for WWP2. Tubulin is a loading control.