Figure 4

Xist RNA topography within the Barr body of somatic cells. Gallery of consecutive 3D-SIM z-sections (125 nm z-distance) through the Barr body of (A) a C2C12 and (B) a RPE-1 nucleus stained with DAPI (grey) after Xist RNA-FISH (green). Xist RNA penetrates the entire Barr body (with exception of the chromocenter region of C2C12 cells; arrowheads in z 1 and z 2). Scale bars: 1 μm. Higher magnifications (z 3 and z 6, respectively) illustrate the preferential albeit not exclusive localization of Xist RNA along lower intensity DAPI regions. (C) 3D surface renderings of Xist RNA foci of the entire Barr bodies shown in (A) and (B). (D) Boxplots with number and volume distribution of 3D-SIM discernible Xist RNA foci in single Barr bodies of C2C12 (n = 10) and RPE-1 (n = 22) nuclei. Median numbers determined for C2C12 and RPE-1 cells were 95 and 54, median volumes 0.0195 and 0.0198 μm³, respectively. (E) C2C12 nucleus after induced HCC, resulting in similar chromatin density between the Barr body and surrounding chromatin. Note widening of IC channels in the Xist RNA decorated Barr body and accumulation of Xist RNA foci at their border. Scale bars: 2 μm, inset 1 μm. (F) Relative fraction (representation) of Xist RNA signals (green) in Barr bodies of C2C12 (n = 9), RPE-1 (n = 13) and HCC-induced C2C12 cells (n = 14) mapped to each DAPI intensity class (grey) reveal a shift of Xist signals toward lower intensity classes, most prominent after HCC treatment. Distribution differences of Xist on classes P <0.001 for all cell types. 3D-SIM, three-dimensional structured illumination microscopy; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; HCC, hypercondensed chromatin; IC, interchromatin compartment; Xist, X inactive specific transcript.