expression was weaker and/or sporadic. Further analysis quanti- fying the number of nuclei within the rho and sog domains that contained nascent transcripts showed on average 40% of nuclei lacked detectable signal in zld2 embryos (Figure 4J, 4L, 4M). In contrast, the wild-type expression domains are more uniform with less than 10% inactive nuclei (Figure 4I, 4K, 4M). Interestingly, Zld binds to both primary and shadow enhancers of sog (Figure 4O) and brk (Figure S1F). The presence of a shadow enhancer is thought to increase the potential for reproducible and robust transcriptional activation [17]. Zld orchestrates timing within the segmentation gene network In the GO analysis, the pair-rule gene category was associated with one of the highest enrichment scores (Figure S3A, S3B). Zld- bound peaks were distributed across several of the known ‘‘stripe elements’’ as well as near the TSS of the primary pair-rule genes (Figure 5I–5K and Figure S7). Since their expression levels were only mildly reduced in zld2 (Table S2), we looked closely at the expression patterns. In wild-type embryos, eve, ftz, hairy, and runt were initially expressed in broad domains as early as nc 10, which refine into the respective seven-stripe patterns by the end of nc 14 (Figure 5A, 5C, 5E, 5G) [28]. In zld2, not only was activation delayed by two nuclear cycles, but the stripe patterns were dramatically altered in nc 14 (Figure 5B, 5D, 5F, 5H). Since pair- rule stripes are formed by localized gap repressors acting on stripe enhancers [29,30], we next examined gap gene expression. In wild-type embryos, gt (Figure 6A) and tll (Figure 6G) transcripts were detected at nc 10, while kni, Kr and hb transcripts were not observed until nc 11–12 (Figure 6, data not shown for hb). This varied activation foreshadows the appearance of the gap protein gradients [31]. In zld2 embryos initial transcription of all five gap genes was delayed by 1–2 nc (Figure 6, data not shown for hb). In addition, their patterns were significantly disrupted, which can be explained in part by miscued gap gene interactions. For example, in wild- type embryos, mutual repression between Gt and Kr is known to establish their complementary domains [32,33]. The overlap of gt and Kr transcripts in the head region of early zld2 mutants, which is never seen in wild-type, may be a consequence of delayed Gt repression (Figure 6B, top), but as Gt accumulates, anterior Kr expression disappears (Figure 6F, bottom). Another example involves Hb repressor function. Hb and Gt set the anterior Kr border, while Hb and Kr establish the anterior border of the posterior gt domain [32]. In zld2, the hb domain was reduced in size (Figure 6J), possibly due to lack of activation in regions of low- level Bcd, and consequently the anterior border of the Kr central domain (Figure 6F, bottom) and the gt and kni posterior domains shift anteriorly (Figure 6B, 6D, bottom). The shift in the posterior border of the Kr domain (Figure 6F, bottom) is likely due to expanded gt (Figure 6B). Tll is a strong repressor of gap genes [34], hence the ectopic expression of kni can likewise be explained by the delay in tll expression in zld2 (Figure 6H). The posterior tll domain, which expands anteriorly along the ventral surface (Figure 6H), could cause ventral repression of Kr (Figure 6F), as well as the changes observed in the posterior domains of gt, kni, hb, (Figure 6B, 6D, 6J, bottom) and the pair-rule genes (Figure 5). In summary, although many of the observed defects in the gap and pair-rule patterns in zld2 are due to delayed and mis-localized gap repressor activity, Zld binding to the gap and pair-rule enhancers (Figure 5, Figure 6, and Figure S7) points to a direct role for Zld in activating these genes. Zelda Coordinates Early Gene Networks Zld binds target genes of the key patterning factors destined to be expressed in the blastoderm embryo The ubiquitous nature of Zld binding to patterning genes prompted us to search for overlap between Zld-bound regions and regions bound by AP [13,14] and DV [35] transcription factors. 62% of the Bcd-bound regions, and 70% of the Dl-bound regions, which overlap extensively with Twi- and Sna-bound regions (referred to as DTS, [35]), are bound by Zld (data not shown). In contrast, only about 30% of the regions bound by Bcd are bound by DTS, and vice versa. We next looked at the genes bound by Zld and the other factors, and whether those genes were expressed in early embryos. Zld binds to 72% of the Bcd targets, 70% of the Cad targets, and 80% of the Tll targets (Figure 7 and Figure S8). Less but considerable overlap (about 50%) was observed between Zld targets and gap gene (Hb, Gt, Kr, Kni) targets (Figure S8). We further distinguished target genes by whether they were expressed at 2–4 hrs (defined as bound by RNA polymerase II (pol II) by MacArthur et al. [14]). About half of the AP factor-bound target genes were expressed. For each factor, expressed target genes were more likely to be bound by Zld than the non-expressed targets (Figure 7A and Figure S8A). Similarly, Zld binds 59% of the DTS target genes (351 genes; Figure 7B), but this increases to 76% for the expressed targets, and decreases to 35% for non-expressed targets (Figure 7A, 7C). Thus, the AP and DV target genes that are also bound by Zld are more likely to be expressed in the blastoderm embryo, indicating that Zld binding may promote transcriptional activity. Discussion Our combined approach of Zld ChIP-chip profiling, expression profiling, and genetic analysis revealed a wide-ranging regulatory role for Zld, and provides new insights into how essential embryonic processes are coordinated during early development. Our results demonstrate that Zld is required for timely and robust target-gene responses. The observed increase in Zld protein levels in the second hour of development raises the possibility that a ‘‘temporal gradient’’ of ubiquitously distributed Zld functions together with the spatial gradients of the patterning morphogens to define spatiotemporal specificity of zygotic gene expression in the early embryo. DNA sequences associated with Zld-bound regions are similar to motifs over-represented in hotspots Our Zld binding analyses indicate that there are at least eight TAGteam sites. CAGGTAG and CAGGTAA were the most over-represented and the most highly conserved in the Zld-bound regions (Figure 2A and Figure S1A). About half of Zld binding is TAGteam site dependent, and all of the sex determination, cellularization, and patterning genes we studied have TAGteam sites in their enhancers and in many cases near the TSS. Curiously, within the CAGGTAG site is CAGGTA, a motif found strongly enriched in hotspots [5]. Likewise, our TATCGAT, CT- repeat, and CAC-related sites (Figure S1B–S1D) are similar to additional motifs found in hotspots: GTATCGAT, CTCT- CTCTCT, and CTCACACG, respectively, which were proposed by modENCODE to be ‘‘candidate drivers’’ of hotspot formation [5]. TATCGAT is contained within the DRE (DNA replication related element) octamer site, TATCGATA, which is found near the TSS of genes involved in DNA replication [36]. Additionally, TATCGATA is similar to the BEAF-32 insulator site [37]. The CT-repeat site is also associated with an insulator sequence, the Trl/GAF motif [37]. It is unclear how Zld interacts with the non- TAGteam sequences since TATCGAT, for example, does not PLoS Genetics | www.plosgenetics.org 7 October 2011 | Volume 7 | Issue 10 | e1002339