Fig. 3: Depletion of lamin A/C and LBR induces reorganization of H3K9me3-marked heterochromatin and detachment of LADs from the nuclear periphery.
From: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers
a Immunostaining and 3D reconstruction of H3K9me3-marked heterochromatin in wildtype and lamin A/C-LBR double knockout C2C12 cells. Scale bars, 10 μm. b Graphs show quantification of mean area per intranuclear H3K9me3-positive spot (left), and number of intranuclear foci per cell (right). Bar graphs represent mean ± SD. nWt = 40, nKO = 31, nKO2 = 31 (single data points are displayed). Left graph: H(2) = 11.72, **pKO = 0.0096, **pKO2 = 0.0058; right graph: H(2) = 52.07, ****pKO = 9.8 × 10-8, ****pKO2 = 6.6 × 10−11 (Kruskal-Wallis test). c Nucleoplasmic over peripheral H3K9me3 signal ratio determined using line intensity profiles across the nucleus in wildtype and lamin A/C-LBR double knockout cells. Line graph on the right shows a representative H3K9me3 fluorescence intensity plot measured along the dashed lines in the images shown in (a). Bar graph displays mean values ± SD (nWt= 4, nKO = 5). t(7) = 8.31, ****p = 7.1 × 10−5 (two-tailed t-test). d Lamin B1 immunostaining of indicated cell lines. Scale bars, 10 μm. e Lamin B1 ChIP-qPCR analyses demonstrating loss of lamin B1 binding to LADs at the nuclear periphery of lamin A/C-LBR double knockout cells. Data represent mean values ± SEM of three biological replicates. Horizontal dashed line indicates the signal obtained using unspecific IgG antibodies in control ChIP. The genomic positions of tested LADs are indicated in the table. f Representative confocal images of fluorescence in-situ hybridization (FISH) signal using a GFP-labeled BAC probe for a LAD region (green dots). Dashed line indicates nuclear border. Scale bars, 10 μm. g Radial distance of LAD region to the nuclear periphery upon depletion of lamin A/C and LBR as detected by FISH. nWt = 909, nKO = 666, ****p = 7.6 × 10-4 (two-sample, two-tailed KS test). Images in (a) processed with Imaris, and in (d) and (f) with Fiji. Source data are provided as a Source Data file.
