FIGURE

Figure 3

ID

ZDB-FIG-211009-18

Publication

Pancholi et al., 2021 - RNA polymerase II clusters form in line with surface condensation on regulatory chromatin

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Figure 3

Large clusters of recruited Pol II are partially destabilized by hexanediol treatment, form stable patterns of non‐growing domains, and undergo repeated connection and splitting

Representative maximum‐intensity projection micrographs of nuclei in primary cell cultures of zebrafish embryos, treated for 5 min with control media or 3% 1,6‐hexanediol (HE), then fixed and labeled for Pol II Ser5P and Ser2P by immunofluorescence, images recorded by instant‐SIM, this panel shows only the Pol II Ser5P channel.

Quantification of changes in Pol II Ser5P cluster morphology upon hexanediol treatment. Area displayed as standard boxplots, solidity and the number of clusters per nucleus are mean±SEM. *** indicates P < 0.001 (two‐tailed permutation test for differences upon hexanediol treatment from control, N = 5, 3 independent samples; area: P < 0.0001 with n_Cluster = 1,435, 841 clusters for area; number of large clusters (area > 0.2 μm²) per nucleus: P < 0.0001 with n_Nuc = 212, 124 nuclei; solidity: P < 0.0001 with n_Large = 401, 135 large clusters).

Pol II Ser2P channel micrographs of the same nuclei shown in panel A.

Quantification of changes in Pol II Ser2P spots. n.s. indicates no statistically significant changes (area: P = 0.76 with n_Spots = 3,394, 1,937 spots; number of clusters per nucleus (no size cut‐off): P = 0.80 with n_Nuc = 212, 124 nuclei; solidity: P = 0.21 with n_Spots = 3,394, 1,937 spots).

Representative time‐lapse recording of Pol II Ser5P Fab in a live embryo (no hexanediol treatment). Similar results were observed in two independent experiments, each performed on three different embryos. Images are maximum‐intensity projections, and images recorded by instant‐SIM.

Representative close‐up time‐lapse showing transient merging and separation events within a Pol II Ser5P cluster. Single z‐sections, images were bleaching‐corrected and local background was subtracted (radius 3.3 μm). Images from same data set as panel (E). Contour plots are obtained by application of a manually adjusted threshold to assist interpretation, same threshold for all time points.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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