Fig. S1. Specificity of antibodies recognizing covalent histone modifications. (A) Western blots of NIH-3T3 cell acid extracts were divided and probed with anti-H3K27-Me₃, anti-H3K4-Me₃, or anti-H3K36-Me₃ in the presence of blocking peptides, as indicated. (B) E14 ES cells were immunostained with the indicated antibodies and blocking peptides. Widefield images were taken with the same exposure time, set at identical contrast levels, and size-scaled the same. Bottom panels are DAPI stain of same cells in panels directly above, where antibody signals were completely blocked. H3K27-Me₃ and H3K4-Me₃ signals varied in intensity across cell populations, and were more pronounced for H3K27-Me₃. Erosion measurements of the strong versus weak staining cells showed similar distributions and numbers of foci. Therefore, data from all cells were combined for analyses shown in Figs. 1 and 4. Scale bar: 5 m.

Fig. S2. Relative co-localization of H3K4-Me₃, H3K36-Me₃, and H3K27-Me₃ with RNA Pol II-Ser2-P. (A-C) Mouse ESCs were immunostained with anti- H3K4-Me₃ antibody (A, red; B, white) and anti-Pol II-Ser2-P (A, green; C, white). (D-F) Mouse ESCs were immunostained with anti- H3K36-Me₃ antibody (D, red; E, white) and anti-Pol II-Ser2-P (D, green; F, white). (G-I) Mouse ESCs were sequentially immunostained with anti-H3K27-Me₃ (G, red; H, white) followed by anti-Pol II-Ser2-P antibodies (G, green; I, white). Arrows indicate domains of Pol II-Ser2-P between areas of H3K27-Me₃ at the nuclear periphery. Scale bar: 1 m. Widefield images are shown.

Fig. S3. Comparison of nuclear morphology and expression of stem cell markers in ESCs and NPCs. (A, B) X-Z confocal sections of feeder independent E14 ESCs grown on glass coverslips, fixed, stained with anti-Lamin B1 antibody (green) and DAPI (blue) and mounted on (A) a conventional glass slide or (B) a depression slide, preventing any compression of the cell. Cells in A and B show similar morphology and height, indicating lack of compression in normal mounting protocol. (C,D) Confocal X-Z sections of (C) strongly adherent, elipsoidal NIH-3T3 fibroblast and (D) weakly adherent, spherical NPC stained with anti-lamin B1 antibody (green) and DAPI (blue) and mounted on conventional glass slide as in A. (E) Progenitor B lymphoma cell (kind gift of Dr Kevin Mills, The Jackson Laboratory), processed for FISH and mounted as in A, C, and D, also maintains spherical morphology. In A-E, nuclear surfaces closest to coverslips are at bottom. Scale bars: 1 m. (F) Immunostaining of E14 ESCs showed that these cultures express both SOX2 (green) and SSEA1 (red). Scale bar: 5 m. (G) Immunostaining of NPC cultures indicated a subset of cells expressing NESTIN (green). These cells all expressed SOX2 (red). Anti-NESTIN was used to mark NPCs for histone mapping experiments (Fig. 4). Scale bar: 1 m. Widefield images are shown in F and G.

Fig. S4. Relative shell volumes generated from Erosion analysis of (A) ESCs or (B) NPC and NIH-3T3 cells immunostained to detect the indicated histone H3 modifications or Pol II-Ser2P. Shell volumes were normalized relative to the total nuclear volume and scaled for comparison to Erosion measurements of fluorescent signals in Figs. 1-4. Errors are s.e.m.