Zelda Coordinates Early Gene Networks Figure 1. Zld protein expression in early embryos. (A) Time line of the first three hours of Drosophila development representing nc 1 through 14 [61]. Cycle length gradually increases after nc 10 from ten min to one hr at nc 14. Zygotic gene activation begins at 1–2 hrs followed by a larger wave of gene expression at 2–3 hrs. (B) Western analysis of Zld protein in 0–1 hr, 1–2 hr, and 2–3 hr embryos. 40 mg of protein was loaded in each lane and the blot was incubated with anti-Zld antibodies. A protein close to the predicted size of Zld (,180 kD) increased in concentration after 1 hr of development. (C–F) Zld antibodies detected nuclear protein in wild-type (C–E) but not zld2 embryos (F, nc 14). Note that Zld protein can be detected as early as nc 2 (C, arrows), and appears to accumulate to higher levels in blastoderm embryos (D, nc 10; E, nc 14). doi:10.1371/journal.pgen.1002339.g001 containing a TATCGAT site do not bind Zld in vitro (Figure 2D). The remaining two enriched sequences are simple repeats, CTCTCTC and C/GTCACAC (Figure S1B–S1D), and were not further analyzed. However, we noticed that all four enriched sequence types are similar to the motifs found over-represented in hotspots [5]. To examine the relationship between Zld binding and hotspots, we first calculated the percentage of hotspots that contained a Zld-bound region. 48%–64% of hotspots were bound by Zld depending on the transcription factor complexity (8–13 factors bound), which is striking considering that hotspots were defined using the binding profiles of 41 transcription factors, some of which were from late-staged embryos [5]. We next calculated the average Zld ChIP/input ratio in hotspot windows, which were ranked according to their transcription factor complexity scores [5]. The average Zld ChIP/input ratio increased with increasing complexity of the hotspots, ranging from 4.8 in the highest complexity window to 2.7 in the lowest (Figure 2E, blue line). In contrast, the background (see Materials and Methods) was close to 1 (p,0.0001; Figure 2E, green line). Comparison of ChIP and expression profiles reveals direct Zld targets To evaluate the regulatory role of Zld binding, we used the genomic tiling arrays to compare expression profiles of wild-type and zld2 embryos (see Materials and Methods). We reasoned that by 1) interrogating all transcripts with a greater number of probes per gene on the tiling arrays (to increase statistical power), 2) using competitive array hybridization to detect more subtle expression differences (by the ability to normalize data on the same array), and 3) extending the profiling to include a later time point (2–3 hrs) with both tiling and gene expression arrays, we would capture more Zld target genes. In addition, the tiling arrays allowed direct visual comparison between the Zld ChIP data and transcription profiles, as well as profiles from published datasets such as the hotspots [5]. Figure 3 shows browser views of the sc/sisB, zen, and sna genomic regions. RNA expression of sc/sisB and zen was greatly reduced in zld2 at both time points (Figure 3, top), while sna was only slightly reduced, consistent with our previous observation that sna expression was delayed but recovered in zld2 [3]. Zld-bound peaks (ChIP/input) were seen over well-defined enhancers (Figure 3, red boxes; REDfly [16]), which for sc/sisB and zen contain clusters of TAGteam sites (purple lines) known to be necessary for enhancer-driven expression [2]. At the sna locus Zld binding is associated with the primary and shadow enhancers [17], both of which are required for robust expression [17]. Note the significant overlap of Zld-bound peaks and hotspots (Figure 3 and Figure S1F). Comparison of the tiling and gene expression array datasets indicated that the tiling arrays are more sensitive, as has been previously noted [18], yielding three times more down-regulated genes (summarized in Table S1). 77% (1–2 hrs) and 82% (2–3 hrs) of the genes in the gene-array dataset were included in the tiling dataset, and importantly, two-thirds of these genes were associated with Zld-bound regions (Table S1). Many genes that were considered unaffected in our previous analysis [3] are now observed to be down-regulated in zld2, such as the gap gene gt (see Table S2 for a comparison of the different array datasets at each time point for a subset of pre-blastoderm genes). Comparing the two time points, many more genes (531) came to be expressed in 2–3 hr wild-type embryos, consistent with the burst in transcriptional activity known to occur at this time [4,7]. About half of these genes (44%) were down-regulated in zld2 and 19% of these were bound by Zld, indicating that Zld activates many of the newly transcribed genes, both directly and indirectly. PLoS Genetics | www.plosgenetics.org 3 October 2011 | Volume 7 | Issue 10 | e1002339