BAL1 and its partner E3 ligase, BBAP, link Poly(ADP-ribose) activation, ubiquitylation, and double-strand DNA repair independent of ATM, MDC1, and RNF8

Abstract

The BAL1 macrodomain-containing protein and its partner E3 ligase, BBAP, are overexpressed in chemotherapy-resistant lymphomas. BBAP selectively ubiquitylates histone H4 and indirectly promotes early 53BP1 recruitment to DNA damage sites. However, neither BBAP nor BAL1 has been directly associated with a DNA damage response (DDR), and the function of BAL1 remains undefined. Herein, we describe a direct link between rapid and short-lived poly(ADP-ribose) (PAR) polymerase 1 (PARP1) activation and PARylation at DNA damage sites, PAR-dependent recruitment of the BAL1 macrodomain-containing protein and its partner E3 ligase, local BBAP-mediated ubiquitylation, and subsequent recruitment of the checkpoint mediators 53BP1 and BRCA1. The PARP1-dependent localization of BAL1-BBAP functionally limits both early and delayed DNA damage and enhances cellular viability independent of ATM, MDC1, and RNF8. These data establish that BAL1 and BBAP are bona fide members of a DNA damage response pathway and are directly associated with PARP1 activation, BRCA1 recruitment, and double-strand break repair.

Fig 1

BAL1 and BBAP are recruited to DNA damage sites. GFP-BAL1 (A) and GFP-BBAP (B) recruitment to sites of laser microirradiation. HeLa cells were transfected with GFP-BAL1 or BBAP, laser microirradiated, and subsequently analyzed at serial time points for GFP fluorescence in the DNA damage site (left panels, representative photos; right panels, kinetics of recruitment). Variations in fluorescence intensity (I) were plotted as a function of time (t). GFP levels at each time point were determined by averaging values from 10 cells (±the standard error) from a representative experiment. (C) Kinetics of endogenous BAL1 recruitment to laser-induced DNA breaks. Images were obtained at baseline (0) and at serial time points (0.25 to 60 min) following laser microirradiation. (D) Endogenous BBAP localization to laser-induced DNA breaks (0, 1, and 4 min). (E) Kinetics of BAL1 focus formation following γ-irradiation. HeLa cells were treated with low-dose (100 cGy) irradiation and analyzed for BAL1 foci at baseline and at serial time points (0.5 to 60 min) thereafter.

Fig 2

BAL1 macrodomain 2 is required for recruitment to DNA damage sites. Top, BAL1 protein functional domains, including macrodomains 1 and 2 (orange and red), BBAP binding domain (BBD) (light blue in BAL1) (2), and a region with partial sequence homology to the PARP catalytic domain (dark blue) (domain sizes and amino acids below). GFP-BAL1 constructs are labeled and represented below the BAL1 diagram. Mutations in macrodomain 1 (D126A) and macrodomain 2 (IE 326,327AA [IE-AA]) are shown in black. Representative images of GFP-BAL1 (293T cell) transfectants at baseline and 2 min following laser microirradiation are shown on the right, and the percentages of cells with GFP-BAL1 recruitment to laser-induced DNA breaks are shown on the far right.

Fig 3

BAL1 and BBAP colocalize with PARP1 and PAR and physically associate with PARylated proteins following DNA damage. Colocalization of PARP1, PAR, and BAL1 (A) or PARP1, PAR, and BBAP (B) at laser-induced DNA breaks (2 min following laser microirradiation). (C) Colocalization of BAL1 and PAR foci in γ-irradiated cells. HeLa cells were treated with low-dose irradiation (100 cGy) and analyzed for BAL1 and PAR foci at baseline and at serial time points (0.5 to 60 min) thereafter. (D) Coimmunoprecipitation of PARylated proteins and BAL1. HeLa cells were untreated or treated with low-dose Dox (50 ng) for 10 min with or without PJ-34 pretreatment. Cell lysates were immunoprecipitated (IP) with anti-PARP1, anti-BAL1, or control IgG and immunoblotted with anti-PARP1, -PAR, -BAL1, or -BBAP antibodies. Input whole-cell lysates (left) were analyzed similarly and immunoblotted for actin as a loading control. Molecular weight markers are shown on the right. (E) Recruitment of GFP-PARP1, BAL1, and BBAP to laser-induced breaks in control cells or cells pretreated with the PARP inhibitor PJ-34 (2 min following laser microirradiation). (F) Kinetics of GFP-BAL1 recruitment to laser-induced DNA breaks in control or PJ-34-pretreated cells. GFP-BAL1 levels at each time point were determined by averaging values from 10 cells (±the standard error) in a representative experiment.

Fig 4

Recruitment of endogenous PARP1, PAR, BAL1, and BBAP to DNA damage sites. (A) Recruitment of endogenous PARP1, PAR, BAL1, and BBAP to laser-induced breaks in control or PJ-34-pretreated cells (2 min following laser microirradiation). (B) Depletion of PARP1, BAL1, and BBAP in HeLa cells. (C) Recruitment of BAL1, PARP1, BBAP, and PAR to laser-induced breaks in control cells or cells depleted of PARP1, BAL1, or BBAP (2 min following laser microirradiation).

Fig 5

BAL1 limits the cellular response to DNA-damaging agents. (A) BAL1 depletion augments the cellular response to DNA-damaging agents. HeLa cells were transfected with control or BAL1 siRNAs (siRNAs 1 and 2), treated with Dox at 50 ng/ml or 200 ng/ml, or left untreated for 1 to 96 h and subsequently evaluated by an MTS assay. The consequences of BAL1 depletion were most striking in cells treated with low-dose Dox (50 ng/ml) (P < 0.001, two-way ANOVA). After 72 h of treatment with low-dose Dox (50 ng/ml), cellular proliferation (as assessed by an MTS assay) was ≈70 to 80% lower in BAL1-depleted cells than in control RNAi or parental cells. OD, optical density. (B) Cellular apoptosis following BAL1 depletion and Dox treatment. Parental, control, and BAL1 siRNA-transfected HeLa cells were untreated or treated with Dox at 50 ng/ml and 200 ng/ml for 24 h and analyzed for apoptosis with anti-annexin V and PI staining. Error bars in panels A and B represent the standard deviations (SD) of the mean for three replicates in a representative experiment. (C) Recruitment of GFP-control, GFP-BAL1, or GFP-BAL1-DM to laser-induced breaks in (5′ UTR-specific) BAL1 siRNA knockdown cells. (a to c) GFP-control, GFP-BAL1, and GFP-BAL1-DM; (d to f) PARP-1 in these cells; (g to i) merged images. (D) Apoptosis of BAL1-depleted HeLa cells repleted with GFP-control, GFP-BAL1, or GFP-BAL1-DM and subsequently treated with doxorubicin (50 ng/ml) or left untreated. Apoptosis was assessed with annexin V and PI staining. Error bars represent the SD of the mean for 3 replicates in a representative experiment.

Fig 6

PARP-dependent recruitment of BBAP to DNA damage sites is required for early ubiquitin chain formation. (A) Ubiquitylation (conjugated ubiquitin, FK2 immunostaining) and PARP1 recruitment at serial time points following laser microirradiation (baseline [0] and 5 to 60 min) in control or PJ-34-treated cells. (B) BBAP and PARP1 recruitment in laser-microirradiated control or PJ-34-treated cells. (C) Ubiquitylation (FK2 immunostaining) and PARP1 recruitment in laser-microirradiated control cells or cells depleted of BAL1. (D) BBAP and PARP1 recruitment in laser-microirradiated control cells or cells depleted of BAL1. (E) Ubiquitylation (FK2 immunostaining) and PARP1 recruitment in laser-microirradiated control cells or cells depleted of BBAP. (F) Ubiquitylation (FK2 immunostaining) and BAL1 recruitment in BAL1-depleted cells replete with GFP-control or GFP-BAL1. Images were obtained 5 min after laser microirradiation. (G) RNF8 and PARP1 recruitment at serial time points following laser microirradiation in control or PJ-34-treated cells. (H) RNF8 and PARP1 recruitment in laser-microirradiated control cells or cells depleted of BAL1.

Fig 7

Functional analyses of PARP1-BAL1-BBAP- and MDC1-RNF8-associated DDRs by comet assay. Comet assays of HeLa cells pretreated with PJ-34 or vehicle alone (A), control, BAL1, MDC1, or BAL1 and MDC1 siRNAs (B), or control, BBAP, RNF8, or BBAP and RNF8 siRNAs (D), treated with low-dose irradiation (200 cGy) and analyzed under alkaline conditions 15 min, 60 min, or 24 h thereafter. Un Rx, untreated. (C) Depletion of BBAP or RNF8 following siRNA. HeLa cells treated with a scrambled control (SC) or BBAP or RNF8 siRNA were lysed, size fractionated, and immunoblotted with the respective antibodies and actin (as a loading control). The comet tail moment (% DNA in tail × tail length) was determined for 50 to 100 cells/condition using TriTek CometScore software. (A and B) Bar graphs (means ± SD); (C and D) representative photographs. Data are from one of three similar experiments.

Fig 8

Early 53BP1 recruitment to DNA damage sites requires PARP1, BAL1, and BBAP. (A) Recruitment of PARP1 and 53BP1 to laser-induced breaks in control or PJ-34-treated cells. Images were obtained at baseline (0) and 10 to 30 min following laser microirradiation. (B) PARP1 and 53BP1 recruitment to laser-induced breaks in control cells or cells depleted of PARP1, BAL1, or BBAP (via siRNA) (20 min following laser microirradiation). (C to F) Kinetics of 53BP1 and γH2AX focus formation following γ-irradiation of control or PJ-34-treated cells (C and D) or control siRNA or BAL1 siRNA-treated cells (E and F). Cells were treated with PJ-34 or vehicle alone (C and D) or control siRNA or BAL1 siRNA (E and F), subjected to low-dose (100-cGy) irradiation, and analyzed for 53BP1 and γH2AX foci at baseline and 1 to 60 min thereafter (C and E). Shown are the percentages of cells with ≥10 foci per nucleus at each time point and condition. Error bars represent the SD of the means for 3 independently stained slides for each time point and condition. At the earliest time points following irradiation (0 to 4 min), the development of repair foci (percentage of cells with ≥10 foci per nucleus) was compared in control and PJ-34-treated cells and control siRNA and BAL siRNA-treated cells with an ANOVA.

Fig 9

Early recruitment of RAP80-BRCA1 to DNA damage sites requires PARP1, BAL1, and BBAP. PARP1 and RAP80 (A) and BRCA1 (B) recruitment to laser-induced breaks in control or PJ-34-treated cells (0 to 60 min following laser microirradiation). (C) PARP1, RAP80, and BRCA1 recruitment to laser-induced breaks in control cells (a through f) or cells depleted of PARP1 (g to l), BAL1 (m to r), or BBAP (s to x) (via siRNA) (10 min following laser microirradiation).

Fig 10

DNA damage-induced ubiquitylation and recruitment of 53BP1 and RAP80-BRCA1 occurs via an early PARP1-, BAL1-, and BBAP-dependent pathway and a later phosphorylation-dependent ATM-MDC1-RNF8-associated route.