Topological chromatin density mapping of functionally relevant markers RNAP II, H3K4me3 and H3K27me3. (A) Mid z-sections through a C2C12 and an RPE-1 nucleus show abundant RNAP II foci preferentially at the boundary of chromatin and IC (insets 1 and 3). RNAP II signals are largely excluded from the Barr body in C2C12 (inset 2), while RPE-1 cells retain some sites of active transcription in the Barr body interior (inset 4, arrowheads; Barr bodies verified by Xist RNA, not shown here). Scale bars: 2 μm, insets 1 μm. (B) Over/underrepresentation of RNAP II in DAPI intensity classes of C2C12 (n = 7) and RPE-1 (n = 7) nuclei relative to the intensity class sizes as shown in Figure 1D and Additional file 1. Average RNAP II foci numbers and densities are indicated with standard deviations (P <0.001). (C) Clear separation of H3K4me3- and H3K27me3-marked chromatin shown in a mid (left) and apical z-section (right) of a C2C12 nucleus (arrow delineates the Barr body). H3K4me3 is located mainly at the decondensed periphery of CDCs, whereas H3K27me3 is enriched within compacted CDCs (insets 1 and 2). In the apical z-section H3K4me3-enriched chromatin is largely restricted to the vicinity of nuclear pores, whereas H3K27me3 is also found at more distant areas. Scale bars: 2 μm, insets 0.5 μm. (D) Comparative mapping of H3K27me3 (green) and H3K4me3 (red) signals on DAPI intensity classes in C2C12 nuclei (n = 10, distribution differences on classes P <0.001 for all markers). (E) Minimal distance distributions (nearest neighbor distances) for H3K27me3 and H3K4me3 signals displayed as box plots (median, Q1, Q3) with whiskers indicating the 1.5 IQR. Average minimal distances indicated with standard deviation (>100,000 distances determined from 20 cells; see Additional file 4 for all minimal distance distributions determined in this study). 1.5 IQR, 1.5 × interquartile range; CDC, chromatin domain cluster; DAPI, 4',6-diamidino-2-phenylindole; H3K27me3, trimethylated histone H3 lysine 27; H3K4me3, trimethylated histone H3 lysine 4; IC, interchromatin compartment; RNAP II, RNA polymerase II.