Supplementary Figure 4: Different genomic labeling approaches report on similar chromatin dynamics and transcription kinetics.
From: Dynamic interplay between enhancer–promoter topology and gene activity
a, Three methods of labeling genomic loci. b, The measured blue-green (MS2-parS) distances are not sensitive to labeling approach. The box plot shows the distributions of the instantaneous distance between spot pairs in the same nuclei. Distances shown are after chromatic aberration corrections. For all three genomic settings, the MS2-parS (blue-green) distances showed no significant differences (one-way Kruskal–Wallis test on individual embryo mean distances, n = 34, 9 and 6 embryos for sets A, B and C, respectively). This was observed regardless of the absence (Red-OFF, P = 0.17, χ2 = 4.3, d.f.=50) or presence (Red-ON, P = 0.60, χ2 = 1.04, d.f. = 49) of PP7 activity. Center values, medians; boxes, interquantile ranges (25–75% quantiles); whiskers, 1.5 times the interquantile range. The 0 kb control is the hbP2-MS2PP7-kni embryo described in Supplementary Fig. 3. c, The distances between spot pairs reflect their genomic arrangement. Distributions of the instantaneous distance between spot pairs are plotted. Box-and-whisker plots are as described in b. Distances shown are after chromatic aberration corrections. Note that the parS-PP7 (green-red) distance is significantly shorter when the parS tag is located at the 3′ side of the PP7 reporter (P = 4.5 × 10–6, two-tailed Wilcoxon rank-sum test). d, Mean square displacement (MSD) plots for set A and set B. Each MSD trace is a result of the population ensemble of all nuclei in a single embryo (embryo-averaged MSD; Methods). Results from the two genomic settings display subdiffusive characteristics with a scaling power of ~0.24, and their anomalous diffusion coefficients show no significant difference (two-tailed Student’s t test, P = 0.87, t = –0.1534, d.f. = 90; linear fits with mean ± s.d. across embryos). e–g, Transcriptional activation of eve-PP7 is not affected by labeling approach. The fraction of eve-MS2-expressing nuclei that also contain active PP7 (mean ± SE) is plotted as a function of time for three genomic settings. It seems that neither the presence nor the location of the parS tag interferes with either enhancer action or transcriptional activation. This is consistent with the hypothesis that the ParB-DNA complex is formed from specific ParB-parS nucleation sites followed by stochastic binding and trapping.
