Figure 6.

PRR11-mediated cyto-nucleoskeletal assembly regulates heterochromatin organization. a, immunofluorescence staining for H3K9me3 in cells transfected with Flag-tagged pVN173 or WT PRR11. The H3K9me3 localization of WT PRR11–expressing cells was diminished from the nucleoplasm. Representative images are shown. Bar, 20 μm. Zoomed images of boxed region are shown at the bottom-left corner (scale bars, 10 μm). b, quantification of cells with diminished H3K9me3 nucleoplasm distribution as normalized to total Flag-positive cells demonstrated in a. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. c, Western blot analysis for the indicated proteins. Cells were treated as in a and then were lysed and analyzed for expression of the indicated proteins by Western blotting. d–f, PRR11 silencing altered the localization of H3K9me3. d, immunofluorescence staining for H3K9me3 in cells treated with siNC or siRNA directed against PRR11. Representative images are shown. Bars, 20 μm. Zoomed images of boxed region are shown at the bottom-left corner (scale bars, 10 μm). e, qualification of cells with H3K9me3 nuclear peripheral distribution as normalized to total cells demonstrated in d. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. f, Western blot analysis for the indicated proteins. Cells were treated as in d and then were lysed and analyzed for expression of the indicated proteins by Western blotting. g and h, CK-666 restored H3K9me3 nucleoplasm distribution in WT PRR11-overexpression cells. g, H1299 cells were transfected with WT PRR11 with or without CK-666 (84 μm) treatment, and then cells were fixed and stained for Flag and H3K9me3. Representative images are shown. Bar, 10 μm. h, quantification of cells with diminished H3K9me3 nucleoplasm distribution as normalized to total Flag-positive cells. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. i and j, effects of PRR11-truncated mutations on the H3K9me3 redistribution. i, H1299 cells were overexpressed with Flag-tagged Δ1–100 or Δ290–360, and then cells were fixed 24 h after transfection and stained for Flag and H3K9me3. Representative images are shown. Bar, 10 μm. j, quantitative analysis of cells with diminished H3K9me3 nucleoplasm distribution. Ratio indicated that the number of cells with aberrant localization of H3K9me3 is relative to that of total Flag-positive cells. Results are shown as the mean ± S.E. (n = 3). Greater than 20 cells were counted per condition in every repeat. Cell nuclei were stained with DAPI (blue). ***, p < 0.001, or N.S., not significant (p > 0.05). k, proposed working model. PRR11 associates with and recruits Arp2/3 complex to facilitate F-actin assembly and rearrangement via the N and C termini (red regions) and thereby to regulate nuclear lamina integrity, which might be mediated by the LINC complex coupling both nuclear lamina and actin cytoskeleton. Nuclear laminae bind directly to lamina-associated domains and/or heterochromatin-associated proteins, such as H3K9me2/3 and H3K27me3, and this is important for chromatin organization and gene expression repression. Dysfunction of PRR11 disrupts cytoskeleton–nucleoskeleton, chromatin organization, and gene expression to promote lung tumorigenesis.