Figure 3. H3K9me2 is essential for histone H3 positioning at the nuclear periphery.

(A) Schematic illustrating C. elegans protein CEC-4 tethering H3K9me2-marked chromatin to the nuclear periphery; INM: inner nuclear membrane. (B) Localization of H3K9me2-marked chromatin (green) in wild-type (WT) and cec-4-null C. elegans embryo cells, counterstained with nuclear lamina marker Lamin 1 (red) and DAPI (blue); 3D reconstruction (top); immunofluorescent confocal images of C. elegans embryo cells (bottom). Scale bars: 3 μm (C) Dot plot of the proportion of total H3K9me2-marked chromatin at the nuclear lamina in WT, cec-4-null, and cec-4-rescued embryo cells (mean ± SD); n = 25 cells per condition. (D) Localization of indicated histone H3-GFP fusion proteins in NIH/3T3 cells; counterstained with H3K9me2 (green) and nuclear lamina marker Lamin B (red); spectral views (magnifications of top panels as indicated by dashed squares) illustrate H3-GFP signal intensity. Localization of the H3-GFP at the nuclear periphery (yellow arrowheads) or loss of peripheral localization (white arrowheads). Scale bars: 5 μm (top panels) and 1 μm (bottom panels). (E) Dot plot of the proportion of indicated H3-GFP fusion protein at the nuclear lamina (marked by Lamin B, top) or within the layer of peripheral heterochromatin (marked by H3K9me2, bottom), normalized to wt H3-GFP, calculated using Lamin B or H3K9me2 signal as a mask (mean ± SD); n = 30 cells per condition. (F) Line signal intensity profiles of corresponding images in panel D indicated by dashed lines. Statistical analyses performed using two-tailed student’s t-test for panel C and one-way ANOVA test for panel E; ****p<0.0001, **p=0.0024, ns: not significant; all comparisons relative to wild type (wt).

Figure 3—figure supplement 1. Localization of H3K9me2- and H3K9me3-marked chromatin in C. elegans wild-type (WT), cec-4-null, and cec-4-rescue embryo cells.

(A) Additional representative immunofluorescent confocal images of C. elegans embryo cells illustrate H3K9me2 (green) localized to the nuclear periphery as stained with Lamin1 (red) in WT and cec-4-null cells rescued with cec-4 transgene (CEC-4 mCherry), but not in cec-4-null cells; counterstained with DAPI (blue). (B) Representative immunofluorescent confocal images illustrate H3K9me3 (green) distribution, counterstained with Lamin 1 (red) and DAPI (blue); Scale bars: 3 μm.

Figure 3—figure supplement 2. Expression of histone H3-GFP fusion proteins.

Histone H3 immunoblot demonstrating expression of exogenous H3-GFP fusion proteins. (A) anti-histone H3 immunoblot; and (B) anti-GFP immunoblot.