VGLL3 modulates chemosensitivity through promoting DNA double-strand break repair

Abstract

Transcription cofactor vestigial-like 3 (VGLL3), as a master regulator of female-biased autoimmunity, also functions in tumor development, while the underlying mechanisms remain largely elusive. Here, we report that VGLL3 plays an important role in DNA damage response (DDR). VGLL3 can be recruited to damage sites in a PARylation-dependent manner. VGLL3 depletion impairs the accumulation of RNF8 and RAD51 at sites of DNA damage, leading to reduced homologous recombination efficiency and increased cellular sensitivity to chemotherapeutic drugs. Mechanistically, VGLL3 can prevent CtIP from KLHL15-mediated ubiquitination and degradation through competitive binding with KLHL15 and, meanwhile, stabilize MDC1 by limiting TRIP12-MDC1 but promoting USP7-MDC1 associations for optimal RNF8 signaling initiation. Consistently, VGLL3 depletion delays tumor development and sensitizes the xenografts to etoposide treatment. Overall, our results reveal an unexpected role of VGLL3 in DDR, which is distinct from its transcriptional cofactor function and not conserved among VGLL family members.

Fig. 1.. VGLL3 is recruited to DSBs depending on PARylation and CHD4.

(A and B) U2OS cells stably expressing GFP-VGLL3 were microirradiated. (A) Representative images. Scale bars, 2 μm. (B) Left: Representative live cell images. Right: The intensity of fluorescence at damage sites was quantified and presented as means ± SD from 10 cells. (C) U2OS cells expressing GFP-VGLL3 were pretreated with the indicated inhibitors before microirradiation. (D) Proportion of cells with VGLL3 accumulation was quantified. Data were derived from at least 20 cells per replicate and presented as means ± SEM from three experiments. (E) Immunoblotting (IB) validates inhibitor efficiencies. (F) Chromatin fractions of 293T cells expressing Flag-VGLL3 and GFP-CHD4 were harvested for immunoprecipitation (IP). WCL, whole-cell lysis. (G) Chromatin fractions (Triton X-100-insoluble fractions) of 293T cells were harvested for IP with anti-VGLL3 antibody. (H) CHD4-depleted U2OS cells were transfected with GFP-VGLL3 followed by microirradiation. Proportion of cells with VGLL3 accumulation was quantified. Data were derived from analysis of at least 25 cells per replicate and presented as means ± SEM from three experiments. (I) U2OS-DR-GFP or U2OS-DR-GFP cells expressing Flag-VGLL3 were transfected with siCHD1L. Chromatin immunoprecipitation (ChIP)–quantitative polymerase chain reaction (qPCR) at DSBs was performed using anti-Flag or anti-CHD4 antibodies. A representative result from at least three individual experiments is shown. Data are presented as means ± SEM from at least three replicates per experiment. (J) 293T cells were transfected with hemagglutinin (HA)–CHD4 and GFP-VGLL3 deletions for co-IP. (K) U2OS DR-GFP cells expressing Flag-VGLL3 wild type (WT) or ∆2 mutant were harvested for ChIP-PCR. Enrichment of VGLL3 at DSBs was measured, and a representative result from at least two individual experiments is shown (bottom left). Data are presented as means ± SEM from at least three replicates per experiment, unpaired t test. Expression of proteins was detected (right). ns, nonsignificant. ***P < 0.001 and ****P < 0.0001. bp, base pair; eGFP, enhanced GFP.

Fig. 2.. Loss of VGLL3 impairs HR and sensitizes cells to DNA damage agents.

(A) Percentage of VGLL3-depleted U2OS (left) or MDA-MB-231 (right) cells containing one or more micronuclei after treatment with bleomycin (4 μg/ml) for 2 hours. Data are presented as means ± SEM from three experiments, unpaired t test. (B) VGLL3-KO and GFP-VGLL3 complemented U2OS cells were treated with 20 μM ETO for 2 hours followed by recovery for 0.5, 3, and 8 hours. The levels of γH2AX were analyzed by IB. (C and D) Cells bearing HR (C) or NHEJ (D) reporter were transfected with siVGLL3s. The efficiencies of HR or NHEJ were analyzed by flow cytometry. A representative result from at least three individual experiments is shown (left). Data are presented as means ± SEM from at least two replicates per experiment, unpaired t test. VGLL3 expression was examined by IB (right). (E) Cells bearing HR reporter were transfected with siVGLL3s and complemented with Flag-VGLL3. The HR efficiencies were analyzed. A representative result from two individual experiments is shown (left). Data are presented as above. VGLL3 expression was examined by IB (right). (F to I) Sensitivity assays of VGLL3 KO or depleted MDA-MB-231 (F), MCF7 (H), SUM149PT (I) cells, or GFP-VGLL3 complemented MDA-MB-231 cells (G). Cells were treated with indicated doses of EPI or ETO for 24 hours, Olaparib for 72 hours. CCK8 assay was performed to detect the survival rate of cells. A representative result from three individual experiments is shown. Data are presented as means ± SEM from three replicates per experiment, unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.. VGLL3 regulates CtIP protein level.

(A to C) VGLL3-KO-1 U2OS cells stably expressing GFP-VGLL3 were treated with ETO (20 μM) for 1 hour and recovered for 3 hours before immunofluorescence (IF) for RAD51, BRCA1, and RPA. Representative images are shown (left). Scale bar, 10 μm. Percentages of cells with >10 foci were quantified (more than 200 cells per replicate) (right). Data are presented as means ± SEM from three experiments, unpaired t test. (D) Diagram of the DNA end resection detection system (left). U2OS-ER-AsiSI cells transfected with siVGLL3 were treated with 4-OHT for 6 hours followed by qPCR with the indicated primers amplifying regions including the positions at 364–base pair downstream from the DSB. A representative result from two individual experiments is shown (right). Data are presented as means ± SEM from at least two replicates per experiment, unpaired t test. (E) U2OS cells transfected with siVGLL3 were labeled with 10 μM BrdU for 48 hours, followed by treatment with ETO. Representative images under native conditions are shown (left). Percentage of cells with >10 BrdU foci was quantified, with data representing means ± SEM from three experiments (top middle). Knockdown efficiencies are examined by IB (bottom middle). Representative images of BrdU staining after denaturation with 2 M HCl are shown (right). (F) U2OS cells stably expressing GFP-VGLL3 were transfected with siVGLL3 followed by IB. (G) CtIP protein expression was examined in VGLL3 KO U2OS or MDA-MB-231 cells. LE, long exposure; SE, short exposure. (H) U2OS DR-GFP cells infected with pNL-cytomegalovirus-CtIP lentivirus were depleted of VGLL3 followed by HR analysis. A representative result from two individual experiments is shown. Data are presented as means ± SEM from at least three replicates per experiment. Protein expression was examined by IB. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. VGLL3 prevents CtIP from KLHL15-mediated proteasomal degradation.

(A) VGLL3-depleted U2OS cells were treated with 10 μM MG132 (top) or chloroquine (bottom) for 8 hours. CtIP protein expression was detected by IB. (B) VGLL3-depleted 293T cells were transfected with K48-only HA-Ub and SFB-CtIP. Thirty-six hours later, cells were treated with MG132 followed by denaturing IP with anti-Flag M2 beads. (C) U2OS cells were treated with indicated siRNAs followed by IB. (D) 293T cells were transfected with indicated siRNAs. Twenty-four hours later, the cells were transfected with SFB-CtIP and K48-only HA-Ub constructs. Thirty-six hours later, cells were treated with MG132 followed by denaturing IP with anti-Flag M2 beads. (E) U2OS DR-GFP cells were transfected with indicated siRNAs. A representative HR result from two individual experiments is shown (left). Data are presented as means ± SEM from three replicates per experiment, unpaired t test. Knockdown efficiencies are examined (right). (F to G) 293T cells expressing Flag-VGLL3 were transfected with GFP-KLHL15 (F) or GFP-KLHL15 truncations (G) followed by IP using anti-GFP beads. (H) 293T cells transfected with GFP-KLHL15 and gradual increased amount of Flag-VGLL3 (0 to 5 μg) were harvested for IP using anti-GFP beads. (I) 293T cells cotransfected with GFP-KLHL15 and Flag-VGLL3 deletions were treated with MG132 followed by IP using anti-GFP beads. (J) VGLL3 KO MDA-MB-231 cells infected with Flag-VGLL3 WT or ∆2 lentivirus were harvested for IB. (K) 293T cells depleted of CHD4 were transfected with the indicated constructs, followed by IP with anti-GFP beads. (L) 293T cells transfected with siCHD4 were harvested for IB. ***P < 0.001 and ****P < 0.0001. KD, knockdown.

Fig. 5.. VGLL3 promotes MDC1 protein expression and RNF8-RNF168 signaling after damage treatment.

(A) KLHL15 was knocked down in VGLL3-KO U2OS cells. After 72 hours, cells were treated with ETO (20 μM) for 1 hour and recovered for 3 hours before IF for RAD51. Scale bar, 10 μm. (B) U2OS or U2OS stably expressing GFP-VGLL3 cells were transfected with siVGLL3. After 72 hours, cells were treated with ETO and recovered for 30 min before IF for RNF8. Scale bar, 10 μm. (C) VGLL3-depleted U2OS cells were treated with ETO and recovered for 1 hour before IF for MDC1. Representative images are shown (left). Scale bar, 10 μm. Percentages of cells with >10 foci were quantified (200 cells per replicate) (right). Data are presented as means ± SEM from three experiments, unpaired t test. (D) U2OS or U2OS stably expressing GFP-VGLL3 cells were transfected with siVGLL3 followed by IB analysis. (E) U2OS cells were transfected with the indicated siRNAs followed by IB. (F) VGLL3-depleted U2OS DR-GFP cells were transfected with siKLHL15, HA-MDC1, or their combination for HR analysis. A representative result from two individual experiments is shown. Data are presented as means ± SEM from three replicates per experiment, unpaired t test. (G) VGLL3-depleted U2OS cells were transfected with siKLHL15, HA-MDC1, or their combination. The cells were treated with ETO for 1 hour and recovered for 3 hours before IF for RAD51. Representative images are shown (left). Percentages of cells with >10 foci were quantified and presented as above. Scale bar, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.. VGLL3 stabilizes MDC1 through promoting MDC1-USP7 but limiting MDC1-TRIP12 associations.

(A) VGLL3-depleted MDA-MB-231 cells were treated with MG132. MDC1 protein and mRNA levels were analyzed by IB (top) and qPCR (bottom). Data are presented as means ± SEM from three experiments, unpaired t test. (B) VGLL3-depleted 293T cells were transfected with Myc-Ub and HA-MDC1. Thirty-six hours later, cells were treated with MG132 followed by denaturing IP using anti-HA beads. (C) MDA-MB-231 cells transfected with the indicated siRNAs were harvested for IB. (D) 293T cells transfected with Flag-VGLL3 (top) or Flag-VGLL3 and HA-MDC1 (bottom) were harvested for IP using anti-Flag beads. (E) 293T cells transfected with Flag-VGLL3 and GFP-VGLL3 were harvested for IP using anti-Flag beads. (F) VGLL3-depleted 293T cells were transfected with Flag-USP7 and HA-MDC1. Cells were harvested for IP using anti-Flag beads. (G) 293T cells transfected with HA-MDC1 and GFP-VGLL3 deletions were harvested for IP using anti-GFP beads. (H) 293T cells cotransfected with HA-MDC1 and WT or Δ1 or Δ6 GFP-VGLL3 were harvested for IP using anti-HA beads. (I) 293T cells expressing WT or Δ1 or Δ6 GFP-VGLL3 were transfected with Myc-Ub and HA-MDC1. After 36 hours, cells were treated with MG132 followed by denaturing IP with anti-HA beads. (J and K) MDA-MB-231 cells transfected with indicated siRNAs were harvested for IB. (L) 293T cells were treated with indicated siRNAs. Twenty-four hours later, cells were further transfected with SFB-CtIP and K48 HA-Ub. Thirty-six hours later, cells were treated with MG132 followed by denaturing IP with anti-HA beads.

Fig. 7.. The E-rich and H-rich motifs of VGLL3 are required for optimal HR.

(A to C) U2OS cells stably expressing Flag, Flag-VGLL3, Flag-VGLL3-Δ2, or Flag-VGLL3-Δ6 were transfected with siVGLL3 or siNC for 72 hours. Cells were treated with ETO and cultured for 3 hours (for RAD51 and RPA32 IF) or 1 hour (for MDC1 IF), followed by IF with antibodies against RAD51 (A), MDC1 (B), and RPA32 (C). For (A) and (B), ETO-treated siNCs were also costained with anti-γH2AX antibody. Representative images are shown. Scale bars, 10 μm. Percentages of cells with >10 foci were quantified (more than 200 cells per replicate) (A and B: right; C: bottom). Data are presented as means ± SEM from three experiments, unpaired t test. (D) U2OS-DR-GFP cells stably expressing Flag, Flag-VGLL3, Flag-VGLL3-Δ2, or Flag-VGLL3-Δ6 were transfected with siNC or siVGLL3. After 24 hours, the cells were infected with I-SceI lentivirus. Percentage of GFP-positive cells was quantitated by fluorescence-activated cell sorting. A representative result from three individual experiments is shown. Data are presented as means ± SEM from three replicates per experiment. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 8.. The E-rich and H-rich motifs of VGLL3 contribute to tumor development.

(A to D) MDA-MB-231 cells stably expressing Δ2 or Δ6 or WT VGLL3 were depleted of VGLL3 followed by xenograft assay (n = 5 per group). Tumor graphs (A), tumor growth (B), tumor weight (C), and tumor weight inhibition rate (D) are shown. The arrow in (B) indicates each ETO treatment. (E and F) Representative histology stainings of Ki67 (E) and γH2AX (F) for tumor xenografts with indicated treatment. Scale bars, 100 μm. Data are presented as means ± SEM, unpaired t test. (G) Model for VGLL3 in DDR and chemoresponse. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.