Figure 5. ChAM 7K-patch mediates PALB2 global chromatin association.

(A) Western blot analysis of U2OS-shPALB2 cells constitutively expressing FLAG (EV) or FLAG-PALB2 variants. Where indicated, cells were treated with 2 µg/mL doxycycline (+Dox) to deplete endogenous PALB2. Lamin A was used as a loading control. (B-C) Subcellular distribution of FLAG-PALB2 variants in cytoplasmic (Cyt), nuclear soluble (Nuc) and chromatin-enriched (Chr) fractions upon doxycycline-induced PALB2 depletion. Histone H3 was used as a control for cellular fractionation. Protein levels, detected by western blotting, were quantified using ImageJ. PALB2 levels in the chromatin fraction were normalised against PALB2 levels in whole-cell extracts shown in (A) and expressed as a percentage of the WT in (C). Data indicate mean values ± SD from three independent experiments (n=3). Statistical analyses were performed using GraphPad Prism 7.02, and p-values are from the unpaired Student’s t-test for pairwise comparisons (ns, not significant; **** p<0.0001). (D-E) FRAP analysis of FE-PALB2 WT, 7R and 7Q in U2OS-shPALB2 cells in which endogenous PALB2 was depleted via doxycycline exposure. Representative images before bleaching (pre-bleaching) and during the recovery period (post-bleaching) are shown in (D), where dashed circles indicate bleached areas. FRAP data are quantified and plotted in (E). Dots represent values of half-recovery time (t_1/2) for individual cells and bars mean values ± SD (WT, n=33; 7R, n=31; 7Q, n=26). Statistical analyses were performed using GraphPad Prism 7.02 and p-values are from the unpaired Student’s t-test for pairwise comparisons (ns, not significant; **** p<0.0001). Representative real-time cell images are shown in Figure 5—video 1 (WT), Figure 5—video 2 (7R) and Figure 5—video 3 (7Q).

Figure 5—figure supplement 1. PALB2 ChAM limits its mobility.

(A) Depiction of the cell lines used in this study. The U2OS-shPALB2 cell line was used as the parental cell line. FE-PALB2 variants under the tetO promoter or FLAG-PALB2 variants under the CMV promoter were integrated at a defined FRT site. (B-D) FRAP analysis of FE-PALB2 wild-type (WT) or harbouring an internal deletion of the ChAM (ΔChAM) conditionally induced in U2OS-shPALB2. Images of FE-PALB2 in live cells before bleaching (pre-bleaching) and during the recovery time after bleaching are shown in (B). FRAP data are quantified and plotted in (C). Dots represent values of half-recovery time (t_1/2) for individual cells and bars mean values ± SD (WT, n=18; ΔChAM, n=17). Statistical analyses were performed using GraphPad Prism 7.02 and p-values are for the unpaired Student’s t-test (**** p<0.0001). Time plots of normalised mean fluorescence intensity of FE-PALB2 WT or ΔChAM within the bleached area relative to that of the whole-cell are shown in (D). Data represent mean values of relative florescence intensity ± SD. Representative real-time cell images are shown in Figure 5—video 4 (WT) and Figure 5—video 5 (ΔChAM).

Figure 5—video 1. Representative FRAP image of FE-PALB2 WT expressed in U2OS cell, related to Figure 5D and E.

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Figure 5—video 2. Representative FRAP image of FE-PALB2 7R variant expressed in U2OS cell, related to Figure 5D and E.

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Figure 5—video 3. Representative FRAP image of FE-PALB2 7Q variant expressed in U2OS cell, related to Figure 5D and E.

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Figure 5—video 4. Representative FRAP image of FE-PALB2 WT expressed in U2OS cell, related to Figure 5—figure supplement 1B and C.

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Figure 5—video 5. Representative FRAP image of FE-PALB2 with an internal deletion of the ChAM (ΔChAM) expressed in U2OS cell, related to Figure 5—figure supplement 1B and C.

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