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, 11 (1), 819

PALB2 Chromatin Recruitment Restores Homologous Recombination in BRCA1-deficient Cells Depleted of 53BP1

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PALB2 Chromatin Recruitment Restores Homologous Recombination in BRCA1-deficient Cells Depleted of 53BP1

Rimma Belotserkovskaya et al. Nat Commun.

Abstract

Loss of functional BRCA1 protein leads to defects in DNA double-strand break (DSB) repair by homologous recombination (HR) and renders cells hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors used to treat BRCA1/2-deficient cancers. However, upon chronic treatment of BRCA1-mutant cells with PARP inhibitors, resistant clones can arise via several mechanisms, including loss of 53BP1 or its downstream co-factors. Defects in the 53BP1 axis partially restore the ability of a BRCA1-deficient cell to form RAD51 filaments at resected DSBs in a PALB2- and BRCA2-dependent manner, and thereby repair DSBs by HR. Here we show that depleting 53BP1 in BRCA1-null cells restores PALB2 accrual at resected DSBs. Moreover, we demonstrate that PALB2 DSB recruitment in BRCA1/53BP1-deficient cells is mediated by an interaction between PALB2's chromatin associated motif (ChAM) and the nucleosome acidic patch region, which in 53BP1-expressing cells is bound by 53BP1's ubiquitin-directed recruitment (UDR) domain.

Conflict of interest statement

S.P.J. declares that he is founder and shareholder of Mission Therapeutics Ltd, Adrestia Therapeutics Ltd, Ahren Innovation Capital LLP, and Carrick Therapeutics Ltd. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. 53BP1 loss corrects HR in BRCA1- but not in PALB2- or BRCA2-deficient cells.
a HR reporter assay in U2OS-TLR WT cells siRNA-depleted for indicated proteins or treated with a control siRNA (siCTRL). The bars represent mean ± st.dev.; unpaired t test analyses were conducted to determine if differences between samples were statistically significant; n ≥ 3 experiments. Individual data points are plotted over bars. b Immunoblot of extracts of U2OS-TLR cells depleted for indicated proteins and used in HR assays shown in (a). Asterisks mark nonspecific bands. c, d Comparison of HR efficiencies measured by TLR assays performed in U2OS-TLR WT and 53BP1 KO cells siRNA-depleted for either BRCA1 (c) or PALB2 (d). Data representation and statistical analyses are as in (a); n ≥ 4 experiments. e Immunoblot of extracts of U2OS-TLR WT and 53BP1 KO cells siRNA-depleted for BRCA1 and PALB2 and used in HR assays in (c, d). f Quantification of RAD51 IRIF in RPE1 cells siRNA-depleted for indicated proteins. Cells were treated with 6 Gy of IR, fixed at 4−8 h after irradiation, stained with antibodies specific to cyclin A and RAD51 proteins, imaged and quantified using OPERA Phoenix HT microscope; n = 4 repeats. Source data are provided as a Source Data file. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant (P ≥ 0.05).
Fig. 2
Fig. 2. 53BP1 depletion rescues PALB2 focus formation in BRCA1-deficient cells.
a Quantification of Venus-PALB2 IRIF in RPA focus-positive RPE1 cells. Two independently generated RPE1 Venus-PALB2 cell lines (#1 and #15) were siRNA-depleted for indicated proteins, exposed to 6 Gy of IR and 6 h later, fixed and stained with anti-GFP and anti-RPA2 antibodies. Imaging and IRIF quantifications were performed in three independent experiments, using OPERA Phoenix HT microscope. b Representative images, acquired on OPERA Phoenix HT microscope, of RPE1 cells with endogenously Venus-tagged PALB2 gene. The cells were stained with anti-GFP (to enhance the signal of the Venus tag) and anti-RPA2 antibodies. Scale bar, 50 µm. c Venus-PALB2 association with RPA filaments in cells depleted for 53BP1. RPE1 cells expressing endogenously tagged Venus-PALB2 were depleted for BRCA1 and/or 53BP1, irradiated with 6 Gy of IR and, 8 h later, processed for immunofluorescence analyses. Images were acquired using super-resolution 3D-SIM OMX microscope. Scale bar, 5 µm. Graphs to the right of the images represent distribution of relative frequencies of Venus-PALB2 foci numbers adjacent to each RPA focus. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. ChAM of PALB2 is important for PALB2 recruitment to DSB sites in BRCA1/53BP1-deficient cells.
a Domain structure of GFP-tagged derivatives of PALB2 stably integrated at the FRT site in Tet repressor-expressing RPE1 cells. b Representative images of RPE1 p53 KO cells siRNA-depleted of endogenous PALB2 and complemented with indicated GFP-PALB2 variants. Cells were exposed to 6 Gy of IR and 6 h later fixed and stained with anti-GFP (to detect PALB2) and anti-RPA antibodies. Scale bars, 10 µm. Additional images are in Supplementary Fig. 4a. c Violin plots represent quantification of the integrated density of GFP-PALB2 IRIF in RPA-positive cells following exposure to 6 Gy of IR. Numbers next to the names of the cell lines indicate numbers of the individual clones used in the experiment. d Representative images of RPE1 p53/BRCA1/53BP1 KO cells siRNA-depleted of endogenous PALB2 and complemented with indicated GFP-PALB2 variants. Cells were treated as in (a). Scale bars, 10 µm. Additional images are in Supplementary Fig. 4b. e Violin plots represent quantification of the integrated density of GFP-PALB2 IRIF in RPE1 p53/BRCA1/53BP1 cells following exposure to 6 Gy of IR. Numbers next to the names of the cell lines indicate numbers of the individual clones used in the experiment. f, g Clonogenic survivals of RPE1 p53 KO (f) or p53/BRCA1/53BP1 KO (g) cells complemented with either PALB2WT or PALB2∆ChAM in response to indicated doses of olaparib. Lower panels show area under curve (AUC); n ≥ 3. The bars represent mean ± s.e.m.; one-way ANOVA; *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant (P ≥ 0.05); n = 3 independent experiments. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. ChAM domain of PALB2 interacts with nucleosome acidic patch.
a In vitro pull-down (PD) assay using purified components. 6xHis-MBP-tagged PALB2 ChAM domain or 6xHis-MBP immobilised on Ni-NTA affinity beads, incubated with recombinant nucleosome variants. Acidic patch mutant (Ac-patch mut) comprises H2A E61/91/92A and H2B E105A; ΔH3 NT comprises H3 residues 25−135; ΔH4 NT comprises H4 residues 21−106. Anti-H3 and H4 antibodies showed increased reactivity towards histones with shorter N-terminal tails. b The nucleosome and its acidic patch. Left panel: electrostatic potential view of the protein histone surface; right panel: ribbon representation of the nucleosome core particle. c Sequence alignment of ChAM with alanine scanning mutation areas highlighted (blue line) and basic residues mutated in 4 M mutant highlighted (red arrows). d, e Clonogenic survivals of RPE1 p53 KO (e) or p53/BRCA1/53BP1 KO (f) cells complemented with PALB2WT, PALB2∆ChAM or PALB24M in response to indicated doses of olaparib. Lower panels show corresponding area under curve (AUC) bar graphs; n ≥ 3. The bars represent mean ± s.e.m.; one-way ANOVA; **P < 0.01, ***P < 0.001; ns, not significant (P ≥ 0.05); n = 3 independent experiments. Source data are provided as a Source Data file. f Pull-down assays comparing NCP association of 6xHis-MBP-PALB2 ChAM variants with charge removal and charge retaining ChAM mutations on Arg414 and Arg421. g Pull-down assays between NCPs and immobilised 6xHis-MBP-ChAM variants containing alanine mutations across the proposed nucleosome-interacting region.

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