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. 2022 Oct 21;11:e57736. doi: 10.7554/eLife.57736

Figure 1. KAT2A/2B acetylate PALB2 within a 7K-patch in its ChAM domain.

(A) PALB2 lysine residues identified as acceptors of KAT2A/2B-dependent acetylation in vivo and in vitro by tandem MS analyses are shown in blue and black, respectively. An asterisk indicates residues that are only detected in endogenous PALB2, but not in PALB2 acetylated in vitro. The acetylated lysine residues within the ChAM are highlighted in red. Full-length PALB2 (FL,~131,3 kDa) and fragments 1–4 used for in vitro acetylation assays are also shown. 1: PALB2 1–320 (36.2 kDa), 2: 295–610 (35.6 kDa), 3: 580–900 (35.3 kDa) and 4: 867–1186 (35.3 kDa). (B) In vitro acetylation of PALB2 by KAT2A and catalytically inactive KAT2A-ED. Purified GST-fusions of PALB2 full-length (158.1 kDa) and fragments 1 (63 kDa), 2 (62.4 kDa), 3 (62.1 kDa), and 4 (62.1 kDa) (depicted in A) were incubated with either purified Flag-KAT2A or Flag-KAT2A-ED in the presence of acetyl-CoA, followed by SDS-PAGE. Total and acetylated proteins were visualized by Ponceau S and anti-acetyl lysine (pan-AcK) western blot, respectively. PALB2 fragment 2 acetylation by KAT2A is highlighted with a red dashed box. (C) A heat map of acetylated lysine residues, as identified by quantitative MS analysis of in vitro acetylated PALB2 FL or fragment 2 by KAT2B, KAT2A or a catalytically inactive KAT2A (KAT2A-ED). The abundance of each acetyl-lysine was evaluated as previously described (Fournier et al., 2016). The lysine residues which were detected as endogenous acetylation acceptor are shown to the left. (D) ChAM protein sequences from twelve PALB2 orthologues were aligned using MUSCLE (EMBL-EBI sequence analysis tool) and visualised using the ClustalW software with default colour-coding. Lysine residues acetylated by KAT2A and KAT2B in vitro are respectively highlighted by red and green dots. Hs (Homo sapiens, human), Pt (Pan troglodytes, chimpanzee), Pp (Pan paniscus, bonobo), Bt (Bos taurus, cow), Ml (Myotis lucifugus, little brown bat), Ss (Sus scrofa, wild boar), Ec (Equus caballus, horse), Cl (Canis lupus familiaris, dog), Am (Ailuropoda melanoleuca, giant panda), Oa (Ovis aries, sheep), Gg (Gallus gallus, red junglefowl), Tg (Taeniopygia guttata, zebra finch).

Figure 1—source data 1. Original images for Figure 1B.

Figure 1.

Figure 1—figure supplement 1. PALB2 is directly acetylated by KAT2B.

Figure 1—figure supplement 1.

(A) PALB2 FL and fragments 1–4 were acetylated in vitro by KAT2B with 14C-labelled acetyl-CoA, and following SDS-PAGE, acetylated proteins and total proteins were detected by 14C-autoradiography and Coomassie blue staining, respectively. RAD51 was not identified as a target of KAT2A/2B in vivo (Fournier et al., 2016) and was used as a negative control. (B) In vitro acetylation of PALB2 by KAT2B. Purified PALB2 full-length and fragments 1–4 (Figure 1A) were incubated with purified Flag-KAT2B in the presence of acetyl-CoA, followed by SDS-PAGE. Total and acetylated proteins were visualized by Ponceau S staining and anti-acetyl lysine (pan-AcK) western blot, respectively. PALB2 full-length and fragment 2 acetylated by KAT2B are highlighted by black arrows.
Figure 1—figure supplement 1—source data 1. Original images for Figure 1—figure supplement 1A.
Figure 1—figure supplement 1—source data 2. Original images for Figure 1—figure supplement 1B.