Author Summary Development of a fertilized egg into a multicellular organism comprises a series of precisely timed events initially controlled by factors deposited into the egg. Some of these factors are localized to specific regions of the embryo and instruct cells to adopt certain fates. In this way, these ‘‘morphogen’’ factors lend pattern to the body plan so that different appendages are formed in the right places. In contrast, other factors are evenly distributed throughout the embryo and regulate processes concern- ing all cells. For example, in Drosophila, Zld is a ubiquitous factor that collectively activates batteries of genes essential for further development. Here we show that Zld also functions alongside the spatial morphogens to ensure timely and robust activation of their target genes. In the absence of Zld, activation of these genes is delayed, which derails the proper order of gene interactions and ultimately disrupts gene expression patterns. Our results demonstrate the significance of a timing mechanism in coordinating regulatory gene networks during early development, and they bring a new perspective to classical concepts of how spatial regulation can be achieved. (TSS) of genes down-regulated in zld2 [3]. However, many genes with upstream TAGteam sites were unaffected in our profiling studies. They may not be expressed at 1–2 hrs, or they could have a maternal component masking the effect of Zld on their zygotic expression, or like sna, they may have gone undetected in the profiling analysis due to more subtle effects in zld2. Therefore, Zld may play a more extensive role in regulating early developmental genes than previously suggested. To further investigate Zld targets, and possible mechanisms of their coordinated expression, we analyzed Zld binding across the genome in pre-cellular blastoderm embryos. These results, combined with our expression profiling studies, uncovered many new Zld targets, and demonstrated that Zld is responsible for timing the activation of genes across all three patterning systems, DV, AP and terminal. Our expression assays further showed that proper transcriptional onset is critical for the cascade of cross- regulatory interactions among patterning genes, and that changes in timing can lead to profound changes in positional information throughout the blastoderm. We found a remarkable overlap between Zld-bound regions and HOT (high occupancy transcrip- tion factor binding) regions, or hotspots, reported by the modENCODE consortium [5]. The observation that Zld can be visualized in nuclei before other known transcription factors, and that the most over-represented motif in hotspots is the Zld binding site, hints at a role for Zld in marking, establishing, or maintaining hotspots. Results Zld binds proximal to over 2,000 transcription start sites in 1–2 hr embryos Zygotic gene activation begins with a subset of genes transcribed between 1–2 hrs after fertilization (see time line in Figure 1A) [6]. A second more dramatic wave of transcription occurs between 2– 3 hrs while the embryo undergoes cellularization in nc 14 [4,7]. To identify the genes directly bound by Zld, we performed chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). We first prepared polyclonal antibodies against the C- terminal region of Zld known to bind DNA [3]. This antibody Zelda Coordinates Early Gene Networks recognized a protein of ,180 kD, the predicted size of Zld (Figure 1B), and stained whole mount wild-type embryos (Figure 1C–1E) but not zld2 embryos (Figure 1F). Interestingly, Zld can be detected in nuclei as early as nc 2 (Figure 1C), in contrast to other maternal factors such as Bicoid (Bcd) and Dorsal (Dl), which do not appear in nuclei before nc 9 and 10, respectively [8–11]. Zld protein levels appear to increase substantially during the second hour of development (Figure 1B, 1D) [12], coincident with the activation of the zygotic genome [6]. We used the Zld antibody to immunoprecipitate chromatin from 1–2 hr embryos, and hybridized labeled DNA to high- resolution NimbleGen tiling arrays (see Materials and Methods). Our genome-wide binding data indicated that Zld behaves similarly to other transcription factors in early Drosophila embryos binding thousands of genomic regions [13,14], but showing a stronger tendency to bind close to the TSS. Specifically, Zld binds 2626 regions (p,0.05; see Materials and Methods), and 83% of these (2180) lie within 2 kilobases (kb) of their TSS. Binding near the TSS may be a distinguishing feature of Zld, and may provide a hint regarding its function. In comparison, only 43% of the Bcd- bound regions are within 2 kb of the TSS [13]. DNA motifs associated with Zld-bound regions We examined the enrichment and placement of TAGteam sites within Zld-bound regions (Figure 2A; see Materials and Methods). Of the five TAGteam sites defined initially by ten Bosch et al. [2], CAGGTAG was the most enriched site (9 fold enriched), followed by CAGGTAa (4.5 fold), tAGGTAG (3 fold), CAGGTAt (2 fold), and CAGGcAG (1.8 fold; Figure 2A). In addition, two derivatives (tAGGTAa and CAGGcAa), which are located upstream of the nullo gene (a likely Zld target [3]), were enriched 1.9 fold and 1.6 fold, respectively (Figure 2A). Note that the enriched sequences tend to be located in the center of the bound regions (Figure 2A), suggesting that they represent binding of Zld to TAGteam sites in vivo. We generated a position weight matrix (PWM) using all sites in the bound regions that corresponded to the seven TAGteam sequences above (Figure 2B; see Materials and Methods). To identify additional TAGteam sites that might bind Zld, we used the PWM to scan the bound regions (p#0.0003; see Materials and Methods). Only eight heptamers were recovered, including the seven aforementioned TAGteam sites, plus an eighth site, CAGGTAc. This site was found to be enriched 2.3 fold in the bound regions (Figure 2A). We emphasize that the PWM is derived from Zld ChIP data, and together with in vitro binding results for all eight sites (Figure 2D), these data define a Zld consensus site. In total, 1240 (47%) of the bound regions contain at least one of the eight sites. Of these, more than half (712) contain CAGGTAG or CAGGTAA, which are also highly conserved among the sequenced Drosophila genomes when located in bound regions versus non-bound regions, and also when compared to other sequences within the bound regions (Figure S1A). To address whether Zld might bind to a secondary site, as has been found for other transcription factors [15], we searched for enriched heptamer sequences in Zld-bound regions relative to the genome (see Materials and Methods). Four types of heptamers were recovered (Figure S1B–S1D). The top-ranked enriched site recovered was CAGGTAG (Figure S1B, S1C), validating this approach. Also enriched were TATCGAT and related sequences (TAT sites; see PWM in Figure 2C). 695 (26.5%) of the bound regions contain at least one of these sites, however, these regions have on average lower binding scores than CAGGTAG- containing regions (Figure S1E). The TATCGAT site is less conserved than CAGGTAG (Figure S1A), and oligonucleotides PLoS Genetics | www.plosgenetics.org 2 October 2011 | Volume 7 | Issue 10 | e1002339