ChAM, a novel motif that mediates PALB2 intrinsic chromatin binding and facilitates DNA repair

Abstract

The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the repair of DNA damage by homologous recombination. Here, we report that chromatin-association motif (ChAM), an evolutionarily conserved motif in PALB2, is necessary and sufficient to mediate its chromatin association in both unperturbed and damaged cells. ChAM is distinct from the previously described PALB2 DNA-binding regions. Deletion of ChAM decreases PALB2 and Rad51 accumulation at DNA damage sites and confers cellular hypersensitivity to the genotoxic drug mitomycin C. These results suggest that PALB2 chromatin association via ChAM facilitates PALB2 function in the cellular resistance to DNA damage.

Figure 1

Identification of an evolutionarily conserved region of PALB2. (A) Graph representing the percentage of sequence identity across human, mouse, chicken, opossum, zebrafish and frog PALB2 orthologues. Each data point represents the mean percentage sequence identity over 20 consecutive residues. For illustration purposes, only residues aligned with human PALB2 were implemented. Coloured boxes represent protein- and DNA-interaction regions of human PALB2. (B) Sequence alignment of ChAM. Black boxes indicate residues with >60% identity. (C) Composition of complexes containing EYFP–PALB2 (WT or ΔChAM) purified from EUFA1341 whole-cell extracts. Mock purifications from parental cells (−) and those expressing EYFP (vector) are shown as controls. Lamin A and H2A.X are markers for extraction of nuclear and chromatin-associated proteins, respectively. BRCA, breast cancer; ChAM, chromatin-association motif; Dr, zebrafish; EYFP, enhanced yellow fluorescent protein; Gg, chicken; Hs, human; IP, immunoprecipitation; Mm, mouse; Md, opossum; PALB2, partner and localizer of breast cancer 2 susceptibility protein; WT, wild type; Xt, frog.

Figure 2

ChAM deletion impairs PALB2 chromatin association. EUFA1341 cells expressing EYFP-PALB2 (WT or ΔChAM) were subjected to DNA-damaging treatment (A) or transfected with 6 × His-MRG15 expression vector (B) and subjected to cellular fractionation. (C) HT1080 cells expressing Flag–EGFP–PALB2 (WT or ΔChAM) were treated with control or MRG15-targeting siRNAs and subjected to cellular fractionation. Tagged PALB2 and other components of the BRCA complex were detected by western blotting. Lamin A and histones H2A.X or H3 are markers for extraction of nuclear and chromatin-associated proteins, respectively. γH2A.X is a marker of the DDR. Asterisks indicate nonspecific bands. γH2A.X, Ser 139 phosphorylated histone H2A.X; BRCA, breast cancer; ChAM, chromatin-association motif; DDR, DNA damage response; EYFP, enhanced yellow fluorescent protein; PALB2, partner and localizer of breast cancer 2 susceptibility protein; siRNA, small interfering RNA; vec, empty vector; WT, wild type.

Figure 3

ChAM is sufficient to trigger chromatin association. (A) EMSA performed with ssDNA and recombinant ChAM. (B) Competition EMSAs performed with D-loop, dsDNA, ssDNA and P2T3 (lanes 1–6) or P2T3^ΔChAM (lanes 7–12). (C) Competition EMSAs performed as above with PALB2 (lanes 1–6) or PALB2^ΔChAM (lanes 7–12). (D) Fluorescence microscopy images of GFP (pDEST53 vector) and GFP–ChAM (pDEST53-ChAM vector) expressed in HEK293T cells. (E) Cellular fractionation of HEK293T cells expressing either GFP or GFP–ChAM. (F) Co-purification of GFP–ChAM with core histones after mild Benzonase DNA digestion. Asterisks indicate nonspecific bands. (G) Analysis of DNA fragment sizes after Benzonase DNA digestion. Total DNA from samples analysed in (F) was recovered with a QIAquick column after RNaseA and Proteinase K treatments. The symbols 1n–4n indicate the nucleosome positions. pDEST53 is a mammalian vector for N-terminal GFP-fusion protein expression. ChAM, chromatin-association motif; cyto., cytoplasmic; chrom., chromatin enriched; DAPI, 4′,6-diamidino-2-phenylindole; dsDNA; double-stranded DNA; EMSA, electromobility shift assay; GFP, green fluorescent protein; IP, immunoprecipitation; nucl., nuclear soluble; ssDNA, single-stranded DNA; WCE, whole cell extract.

Figure 4

ChAM is required for normal PALB2 function in vivo. HT1080 cells were challenged with IR or MMC and scored for the percentage of cells with ⩾10 Flag–EGFP–PALB2 (WT or ΔChAM) foci (A), γH2A.X foci (B) and colocalizing γH2A.X and Flag–EGFP–PALB2 foci (C). HT1080 cells were treated with PALB2 3′-UTR siRNA, challenged with MMC and scored for the percentage of cells with ⩾10 Rad51 foci (D). HT1080 cells were treated with BRCA1 siRNA, challenged with MMC and scored for the percentage of cells with ⩾10 Flag–EGFP–PALB2 (WT or ΔChAM) foci (E). Survival curves after MMC (F) or olaparib (G) treatment of EUFA1341 and derivative cell lines. All data are means (±s.d.) of ⩾3 independent experiments. More than 200 cells were scored for each individual sample. P-values are from the two-tailed Student's t-test, and asterisks indicate P-values <0.05 (^*), <0.01 (^**) or <0.001 (^***). γH2A.X, histone H2A.X phosphorylation; BRCA, breast cancer; ChAM, chromatin-association motif; IR, ionizing radiation; MMC, mitomycin C; PALB2, partner and localizer of breast cancer 2 susceptibility protein; siControl, negative control siRNA; siRNA, small interfering RNA; UTR, untranslated region; Untr., untreated; WT, wild type.

Figure 5

Model for the role of ChAM-mediated PALB2 chromatin association during HR. See main text for detailed description. BRCA, breast cancer; ChAM, chromatin-association motif; HR, homologous recombination; PALB2, partner and localizer of breast cancer 2 susceptibility protein.