Fig. 1: Reporter cell system reliably monitors intranuclear position of the MyoD1 locus in vivo.
From: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers
a Schematic overview of experimental strategy for monitoring the 3D nuclear position of the MyoD1 locus in living myoblasts by inserting a 64x LacO array downstream of MyoD1 coupled with expression of GFP-LacR. b Representative 3D reconstructed z stack of images showing intranuclear 3D position of the MyoD1 locus in proliferating myoblasts (green dots). Scale bars are indicated on images. See also Supplementary Movie 1. c Immunofluorescence microscopy of proliferating and 4-days differentiated MyoD1 and Pax7 reporter myoblasts. MyoD1 and Pax7 loci are indicated by yellow arrowheads. Lamin A/C staining (red) marks nuclear border. Scale bars, 10 μm. d Schematic drawing depicting the analysis of the radial distance of gene loci to the nuclear periphery and normalization based on nuclear height. e Density plots of the normalized radial distance of loci to the periphery. Left: Distribution of MyoD1 in proliferating (red) and differentiating (day 4, green) C2C12 myoblasts. nprolif = 106, ndiff = 120, p = 1 × 10−4. Right: Distribution of MyoD1 and Pax7 loci in proliferating myoblasts. nMyoD1 = 208, nPax7 = 79, p = 6.3 × 10−4. p values for loci were calculated using two-sample, two-sided KS test and corrected for multiple testing using Hochberg method. Numeric values displayed on violin plots represent median values. f Normalized radial position distribution of data shown in (e) displayed in violin plots. H3K9me2-marked region (gray-shaded horizontal bar) represents peripheral heterochromatin layer as measured in (g). ****p = 1 × 10−4, ***p = 6.3 × 10−4 p values were calculated using two-sample, two-tailed KS test and corrected for multiple testing using Hochberg method. g H3K9me2 immunofluorescence staining of MyoD1 C2C12 reporter myoblasts. Width of H3K9me2-marked layer is measured in rendered images (middle panel, yellow dots and lines illustrate measurement method for heterochromatin layer), bar graph depicts normalized mean thickness ± SEM of heterochromatin layer measured at 100 different locations. Scale bars, 10 μm. Images in (c) were processed with Fiji and in (b) and (g) with Imaris. Source data are provided as a Source Data file.
