Figure 4

Example view of a genomic region containing a gene with high Pol II Ser5P levels at the promoter (rnf19a).

Overview of Pol II Ser5P and H3K27ac ChIP‐seq levels for 50‐kb windows covering the entire genome, windows covering regions containing super‐enhancers, and symbols indicating regions covered by specific oligopaint probe sets.

Top row—representative optical section obtained by three‐color STED microscopy from a fixed zebrafish embryo, showing Pol II Ser5P and H3K27ac (immunofluorescence) and DNA (JF646‐Hoechst). Bottom row—Detail views as marked in the top row. The details views are 2.4 μm across.

Mean fluorescence intensities (normalized against median intensity of a given nucleus) within Pol II Ser5P clusters. Overall levels of H3K27ac and DNA inside clusters were determined (values for individual clusters in gray, median with 95% bootstrap confidence interval in black) as well as correlations between H3K27ac intensity and Pol II Ser5P intensity, cluster area, and cluster solidity (Pearson correlation coefficient, red line is a linear fit to guide the eye). n = 470 clusters and n = 130 large clusters (area > 0.08 μm², used for solidity analysis) were extracted from images of N = 29 nuclei obtained from five embryos.

Assessment of Pol II Ser5P and Pol II Ser2P immunofluorescence signal intensities at the location of super‐enhancer probe sets. Genomic target regions are sorted left‐to‐right by descending Pol II Ser5P signal. Intensities are normalized by the whole nucleus median level, shown is the median with 95% bootstrap confidence intervals. N = 2 embryos imaged per probe set, yielding n = 67, 66, 91, 63, 53, 70, 93, 63, 71, 78, 59, 58, 103, 55, 65, 127 oligopaint foci for the probe sets SE1, SE2, SE3, SE4, SE5, SE6, SE7, SE8, SE9, SE10, SE11, SE12, cdc25b, celf1, crsp7, rnf19a, respectively. Each probe set contained approximately 500 probe sequences, covering 50 kb sequence length, images acquired by instant‐SIM (genomic regions see Appendix Fig S8, probe sets see Materials and Methods).

Representative images of indicated oligopaint probe sets and immunofluorescence signal, two examples are shown for SE5 and crsp7 to illustrate positioning outside a Pol II Ser5P cluster and inclusion into a cluster. Shown are single z‐sections with segmentation outlines of the oligopaint signal.