FIG 6.

Recruitment of Arkadia to Polycomb bodies is directed by its SUMO-binding domain and M domain. (A and B) Nuclear localization of Arkadia mutants (as indicated) combining M domain deletions, C-terminal truncations, and point mutations in the SUMO-binding domain. Flag-tagged Arkadia constructs (green) were coexpressed with Myc-tagged CBX4 (red) in U2OS cells and visualized through immunofluorescence microscopy. Nuclear DNA (blue) was stained with Hoechst 33342. (C) Polycomb body localization of ArkN865 in the presence of a nuclear-diffused, C-box mutant form (Cmut) of CBX4 (red). Diagram at bottom illustrates the point mutations in the C-box of CBX4 that replaced three residues with Ala. (D) Quantitative measure of the degree of colocalization between Arkadia and CBX4 proteins. Each dot in the plot corresponds to the Pearson's correlation coefficient (Pearson's r) between the red and green channels from a cropped image (200 by 200 pixels, as presented in this figure and in Fig. 5), calculated using the respective pixel intensities of all 20,000 pixels. Horizontal lines mark the average of Pearson's r from multiple images in each experimental group (as indicated).