Figure 9. 7K-patch is important for normal cell survival.

(A) Diagrams of experimental procedures, assessing survival of U2OS-shPALB2 stably expressing FLAG (EV) or FLAG-PALB2 variants upon doxycycline (Dox)-induced endogenous PALB2 depletion by WST-1 assay. (B) Cellular survival normalised against those without doxycycline exposure. Data represent mean values ± SD from three independent experiments (n=3). Statistical analyses were performed using GraphPad Prism 9 and p-values are the unpaired one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001). (C) Diagrams of experimental procedures, assessing the FLAG-PALB2 fusion enrichment at previously reported PALB2-bound loci by ChIP-qPCR. (D) ChIP-qPCR analysis of FLAG-PALB2 WT, 7Q and 7R enrichment at known PALB2-bound loci (Bleuyard et al., 2017a), namely the coding regions of the ACTB, TCOF1, and WEE1 genes, in cells untreated or treated with 4 Gy IR. Data are expressed as fold enrichment over IgG and represent mean values ± SD from three independent experiments (n=3) with triplicate qPCR reactions. Statistical analyses were performed using GraphPad Prism 8 and p-values are from two-way ANOVA with Tukey’s multiple comparisons test (*p<0.05; **p<0.01). (E) Model for PALB2 acetylation function in the maintenance of genome stability. MRG15 and KAT2A/2B-mediated ChAM acetylation, which occurs locally at active genes, jointly promote PALB2 enrichment at undamaged transcriptionally active chromatin. DNA damage triggers ChAM deacetylation at these loci and temporarily releases PALB2 from chromatin. This allows PALB2 to interact with damage sensors, such as BRCA1, which in turn recruits the entire HR complex to sites of DNA damage. ChAM binding to naked DNA through the deacetylated 7K-patch may promote RAD51 loading and HR repair. The constitutively highly mobile PALB2 variant (7Q) is efficiently recruited to sites of DNA damage but, unlike WT PALB2, unable to promote efficient RAD51 foci formation. The randomly chromatin-bound and less capably mobilised PALB2 variant (7R) is inefficiently recruited to sites of DNA damage and hence fails to promote HR.

Figure 9—figure supplement 1. Summary of ChAM properties and post-translational modifications.

Residues targeted for phosphorylation and acetylation within ChAM and its vicinity are indicated below the depiction of the ChAM domain. Previously identified point mutations which impaired ChAM interaction with nucleosomes are shown above the depiction of the ChAM domain.