Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity
Fig 5
Overlapping activator and repressor binding sites are required for abdomen-specific RhoA activity.
(A) Sequences of tested RhoA variants. RhoA-RDM contains random nucleotides downstream of the Hox site. RhoA-SM contains mutations that decrease Sens binding, and RhoA-SM/SWT, RhoA-SM/SS and RhoA-SM/SM add either a low affinity (WT), high affinity (SS), or mutant (SM) Sens site downstream of the Hox site. (B-C) Quantification of β-gal immunostaining intensities in C1-SOPs in RhoBAD-LacZ versus RhoBAD-RDM-LacZ (B) or RhoBAD-SM-LacZ (C). Each box summarizes measurements from a single embryo. Two-tailed Welch’s T-test was used to compare RhoBAD-SM and RDM mutants to wildtype, n = 12 (WT) and 18 (RDM) in (B) and n = 12 (WT) and 15 (SM) in (C). (D) EMSAs comparing binding of purified Sens to RhoA probes (0, 57, 114, and 228 ng of Sens). (E) EMSAs assessing competition between purified Sens (114 or 228 ng) against purified AbdA (189 ng) and Exd/Hth (59.2 ng) on RhoA-SS and RhoA-SM-SS. (E’) Close-up view of Exd/Hth/Hox and Exd/Hth/Hox/Sens complexes on DNA probes. Schematics denote the formation of each transcription factor complex. (F-I) Lateral view of stage 11 RhoBAD-SM-lacZ (F), RhoBAD-SM/SWT-lacZ (G), RhoBAD-SM/SM-lacZ (H), and RhoBAD-SM/SS-lacZ (I) embryos immunostained for β-gal. Intensity of β-gal stain is represented by heat-map at left. “A1” indicates first abdominal segment. Note, no RhoBAD-SM/SS activity is detected in the PNS and the activity that is observed is in cells of the gut. (J) Quantification of β-gal intensities in thoracic and abdominal C1-SOPs of noted RhoBAD-lacZ embryos. Each box represents measurements from a single embryo. Statistical analysis was done using Kruskal-Wallis test followed by post-hoc pairwise Mann-Whitney U test with Bonferroni correction, n = 25 (SM), 23 (SM/SM), 22 (SM/SWT), and 24 (SM/SS). For all statistical comparisons, n.s. p ≥ 0.05; * p < 0.05, ** p < 0.001, *** p < 0.0001.