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Figure 5 | Epigenetics & Chromatin

Figure 5

From: Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Figure 5

Spatial association of Xist RNA with the nuclear matrix protein SAF-A. Immuno-RNA-FISH detection of Xist RNA (green) and SAF-A (red) in C2C12 cells, counterstained with DAPI (grey). (A) Immunolabeled endogenous SAF-A is abundant in the IC and at low DAPI intensity sites of random autosomal nuclear regions (inset 1), but scarce within the Xi (insets 2 and 3, representing different z-sections of the nucleus). SAF-A and Xist RNA foci are in close spatial proximity within the narrowed ANC of the Xi (arrowheads). (B) C2C12 nucleus with HCC. SAF-A localizes within the widened IC channels (inset 1) and is largely restricted to the periphery of the Xi (inset 2; four consecutive z-sections with step size 0.125 μm). Arrowheads point to Xist RNA foci closely associated to SAF-A signals. (C) C2C12 nucleus with transiently expressed hrGFP-tagged murine isoform of SAF-A detected with an antibody against hrGFP (red) together with Xist RNA (green). In comparison to autosomal regions (inset 1) SAF-A:hrGFP is enriched within the Barr body and shows a high degree of association to Xist RNA foci (inset 2). Scale bars (A,B,C): 2 μm, insets 0.5 μm. (D) Nearest neighbor distances (minimal distance distribution) with a median of approximately 120 nm for segmented red and green Xist RNA signals to SAF-A (n = 11 nuclei; >400 distances) and mSAF-A:hrGFP (n = 11 nuclei; 270 distances) are displayed as box plots (median, Q1, Q3) with whiskers indicating the 1.5 IQR. Mean values with standard deviations are indicated. 1.5 IQR, 1.5 × interquartile range; ANC, active nuclear compartment; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; HCC, hypercondensed chromatin; IC, interchromatin compartment; SAF-A, scaffold attachment factor-A; Xi, inactive X chromosome; Xist, X inactive specific transcript.

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