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

Figure 3. Acetylation within the 7K-patch enhances ChAM nucleosome association.

(A) In vivo acetylated lysine residues in chromatin-associated ChAM, detected by tandem MS analysis, are highlighted by arrows in the upper part (MS). Synthetic biotin-ChAM peptides non-acetylated (non-ac) or acetylated at single lysine residues 436 (436-ac), 437 (437-ac), and 438 (438-ac), or all three lysine residues (All 3-ac) are shown in the lower part. The position of the 7K-patch is highlighted in red. The position of the K438 residue, the endogenous target of KAT2A/2B within the ChAM (shown in Figure 1A), is highlighted in a box. (B) In vitro nucleosome binding assays for the synthetic biotin-ChAM peptides. After incubation with purified HeLa polynucleosomes in the presence of 5 µg salmon sperm DNA, biotin-ChAM peptides were pulled-down using streptavidin beads (Strep pulldown), and associated nucleosomes were detected by anti-histone H3 western blot. (C-D) FRAP assay of FE-PALB2 expressed in U2OS-shPALB2 cells, treated either with siRNA targeting KAT2A and KAT2B (siKAT2A/siKAT2B) or with negative control siRNA (siCntl). Representative images of live cells before bleaching (pre-bleaching) and during a 37.5 s recovery period (post-bleaching) are shown in (C), where dashed circles indicate bleached areas. FRAP data are quantified and plotted in (D). Dots represent values of half-recovery time (t1/2) for individual cells and bars mean values ± SD (siCntl, n=59; siKAT2A/2B, n=51). Statistical analyses were performed using GraphPad Prism 7.02 and p-values are for the unpaired Student’s t-test (*** p<0.0006). (E-F) As in C-D, except cells were treated with a cocktail of lysine deacetylase inhibitors (KDACi, 5 mM sodium butyrate, 5 µM trichostatin A, and 5 mM nicotinamide) or DMSO as a control. Representative images of live cells before bleaching (pre-bleaching) and during a 37.5 s recovery period (post-bleaching) are shown in (E), where dashed circles indicate bleached areas. FRAP data are quantified and plotted in (F). Dots represent values of half-recovery time (t1/2) for individual cells and bars mean values ± SD (DMSO, n=61; KDAC, n=94). Statistical analyses were performed using GraphPad Prism 7.02 and p-values are for the unpaired Student’s t-test (**** p<0.0001). Representative real-time cell images are shown in Figure 3—video 1 (siCntl), Figure 3—video 1 (siKAT2A/siKAT2B), Figure 3—video 1 (DMSO) and Figure 3—video 4 (KDACi).

Figure 3—source data 1. Original images for Figure 3B.
Figure 3—source data 2. Numerical data for Figure 3D and Figure 3F.

Figure 3.

Figure 3—figure supplement 1. PALB2 mobility increases upon deacetylation.

Figure 3—figure supplement 1.

(A) Depiction of cell lines used for FRAP assays of full-length PALB2. U2OS Flp-In T-REx, harbouring an inducible shRNA targeting the 3’-UTR of endogenous PALB2 (U2OS-shPALB2) was used as the parental cell line. FE-PALB2 under the tetO promoter, integrated at a defined FRT site, was induced by doxycycline exposure. (B) Diagram of experimental procedure, assessing the impact of KAT2A/2B using FRAP against FE-PALB2. KAT2A protein levels were measured by western blot analysis of U2OS-shPALB2 FE-PALB2 cells treated with siRNA targeting KAT2A and KAT2B (siKAT2A/siKAT2B) or scramble small interfering RNA (siCntl) (negative control) for 48 or 72 hr. Tubulin was used as a loading control. (C) Time plots of normalised mean fluorescence intensity of FE-PALB2 within the bleached area relative to that of the whole-cell in siCntl- or siKAT2A/2B-treated cells; related to the FRAP analysis shown in Figure 3C and D. Data represent mean values ± SD (siCntl, n=59; siKAT2A/2B, n=51). (D) Diagram of experimental procedure, assessing the impact of KDAC inhibition using FRAP against FE-PALB2. Levels of pan-acetylated histones in U2OS-shPALB2 FE-PALB2 cells treated with DMSO or deacetylases inhibitors (KDACi) are shown. Ponceau S staining of proteins after transfer onto nitrocellulose membrane was used as a loading control. (E) Time plots of normalised mean fluorescence intensity of FE-PALB2 within the bleached area relative to that of the whole-cell in DMSO- or KDACi-treated cells; related to FRAP analysis shown in Figure 3E and F. Data represent mean values ± SD (DMSO, n=61; KDAC, n=94).
Figure 3—figure supplement 1—source data 1. Original images for Figure 3—figure supplement 1B (left) and Figure 3—figure supplement 1D (right).
Figure 3—figure supplement 1—source data 2. Numerical data for Figure 3—figure supplement 1C and E.
Figure 3—video 1. Representative FRAP image of FE-PALB2 expressed in U2OS cells treated with control siRNA (siCntl), related to Figure 3C and D.
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Figure 3—video 2. Representative FRAP image of FE-PALB2 expressed in U2OS cell treated with siRNA targeting KAT2A and KAT2B (siKAT2A/siKAT2B), related to Figure 3C and D.
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Figure 3—video 3. Representative FRAP image of FE-PALB2 expressed in U2OS cell treated with DMSO, related to Figure 3E and F.
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Figure 3—video 4. Representative FRAP image of FE-PALB2 expressed in U2OS cell treated with a cocktail of lysine deacetylase inhibitors (KDACi), related to Figure 3E and F.
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