Zelda Coordinates Early Gene Networks Figure 2. Conserved TAGteam motifs are differentially enriched in Zld-bound regions. (A) Enrichment indices (Y-axis) of TAGteam sequences were calculated in 100 bp non-overlapping windows across Zld-bound peaks (5 kb to either side of the center of the peak, marked as 0 on the X-axis). Background was estimated by averaging the enrichment indices of 20 randomly selected heptamers (orange line). (B) PWM derived from the enriched TAGteam sites (see Materials and Methods). (C) PWM derived from the enriched TAT sites (see Materials and Methods). (D) Gel shift assay of labeled oligonucleotides containing different TAGteam sites (lanes 1–16), a mutation of the TAGGTAG site (TGAATAG, lanes 17–18), and the TATCGAT site found in the genome-wide enrichment test (lanes 19–20) without or with recombinant Zld protein (alternating lanes). The eight TAGteam sites bind Zld with varying affinities, while the mutant and TATCGAT site do not bind. (E) Hotspots (3163 with at least eight factors bound) were divided into windows containing 100 hotspots each, and ranked from high to low according to hotspot scores [5] (X-axis, 1 = highest ranking score). The average Zld ChIP/input ratios were calculated for each window (Y-axis). Blue line represents Zld ChIP/input ratios in ranked hotspots; green line represents background (see Materials and Methods). doi:10.1371/journal.pgen.1002339.g002 To further explore the relationship between the position of Zld binding relative to the TSS and effect on gene expression, for each bound region we plotted its location relative to the TSS against the fold change in expression of that gene in wild-type versus zld2. Zld-bound regions showed a tendency to be close to the TSS of genes regardless of whether they were down-regulated in zld2 or not; in fact many unaffected genes were bound by Zld within 2 kb of the TSS (Figure S2A). However, we observed a correlation between the location of Zld binding and the level of wild-type expression. Genes considered expressed were more likely to be bound by Zld within 2 kb than genes considered not expressed (Figure S2B). Moreover, the higher the level of expression, the more likely Zld binds near the TSS (Figure S2B), suggesting that such binding is important for transcriptional activation by Zld (see Discussion). Zld ensures timely and robust transcriptional activation As a first step to gain insight into the regulatory networks established by Zld, we performed Gene Ontology (GO) analysis on the genes associated with Zld-bound regions. First, we ranked the PLoS Genetics | www.plosgenetics.org 4 October 2011 | Volume 7 | Issue 10 | e1002339