Department of Chemical and Biological Engineering andLewis-Sigler Institute for Integrative Genomics, PrincetonUniversity, Princeton, NJ 08544, USASummaryZygotic genome activation (ZGA) is a major genome pro-gramming event whereby the cells of the embryo begin toadopt specified fates. Experiments in Drosophila and zebra-fishhaverevealedthatZGAdependsontranscriptionfactorsthatprovidelarge-scalecontrolofgeneexpressionbydirectand specific binding to gene regulatory sequences [1–5].Zelda(Zld)playssucharoleintheDrosophilaembryo,whereit has been shown to control the action of patterning signals[1, 2]; however, the mechanisms underlying this effectremain largely unclear. A recent model proposed that Zldbinding sitesactasquantitative regulators ofthespatiotem-poral expression of genes activated by Dorsal (Dl), themorphogen that patterns the dorsoventral axis [6]. Here wetestedthismodelexperimentally,usingenhancersofbrinker(brk) and short gastrulation (sog), both of which are directlyactivated by Dl, but at different concentration thresholds[7–9]. In agreement with the model, we show that there is aclearpositivecorrelationbetweenthenumberofZldbindingsites and the spatial domain of enhancer activity. Likewise,the timing of expression could be advanced or delayed. Wepresent evidence that Zld facilitates binding of Dl to regula-tory DNA, and that this is associated with increased chro-matin accessibility. Importantly, the change in chromatinaccessibility is strongly correlated with the change in Zldbinding, but not Dl. We propose that the ability of genomeactivators to facilitate readout of transcriptional input iskey to widespread transcriptional induction during ZGA.Results and DiscussionInblastodermembryos,brinker(brk)isactivatedinaneight-toten-cell-widedomainthatdevelopsintotheventralneurogenicectoderm (NE), whereas short gastrulation (sog) is expressedin a broader band of 16–18 cells encompassing the entire NE(see Figures 1A and 1I). Both genes have the same ventralexpression boundary due to repression by Snail (Sna) in thepresumptive mesoderm [11–15]. The dorsal borders of theirdomains lie in regions of the Dorsal (Dl) gradient whereamounts are low and change little, raising the question ofhow their enhancers can interpret small differences in Dlconcentrations.sog and brk each have two reported cis-regulatory modules(enhancers) that are active in early embryos [10, 16–20]. Thesog intronic lateral stripe enhancer (LSE) [16] is less wellconserved and drives a slightly narrower stripe of expressionrelative to the sog shadow enhancer [17], also known as theneurogenic ectoderm enhancer (NEE), which recapitulatesthe broad endogenous sog pattern [18]. The brk 5