genes located near Zld-bound regions (EASE analysis, p,0.05, except for the cellularization GO term, p,0.10). 2571 genes associated with significant Zld-bound regions were ranked accord- ing to the Ringo binding score into ten non-overlapping windows. Highest scores are in the 100-90 percentile. (B) Enrichment of GO terms for early embryonic biological processes among the genes closest to Zld-bound regions. The heights of the bars represent the log fold enrichment of the corresponding GO term associated with genes nearest to the bound regions with a 10% decrease of Zld binding scores, compared to all Drosophila genes. The fold enrichment was measured as in (A). (C) The distribution of binding scores for Zld-bound regions. The histogram shows the number of Zld-bound regions (Y-axis) vs. their binding scores (X-axis) calculated by the Ringo package in 50 intervals. The red dashed lines indicate the 50 and 90 percentile scores (10.97 and 32.55, respectively). Various Zld target genes are located on the graph according to their associated Zld binding scores. (PDF) Figure S4 Browser views of sex determination and proneural genes. RNA expression profiles are above the Zld binding scores/ profiles. All RNA peaks are on the same relative scale (maximum value is 15K) except for Sxl (B) with a maximum of 45K, and amos and ato (B) with a maximum of 7.5K. Ringo significance scores are shown as blue rectangles (maximum score is 130). TAG sites (limited to CAGGTAG, CAGGTAA, and TAGGTAG), CRMs, and the gene models are below the binding peaks. RNA models are collapsed except for Sxl to show the overlapping Sxl and CG14425 genes (not all Sxl transcripts are shown). Zld binds upstream, and often downstream, of sisA, os/sisC, all three genes in the ac/sc cluster (A), Sxl, dpn, amos, and ato (B), but not to other surrounding genes in the views, many of which appear to have high levels of maternal expression. One of the highest Zld-bound peaks is associated with amos (B, bottom). Most, but not all binding peaks are over TAGteam sites. Note the two clusters of TAGteam sites in the Sxl/CG14425 region, one just upstream of the early Sxl transcript, which correlates with the SxlPe enhancer [63], and a second even further upstream. All of these bound genes are down- regulated in zld2 except for CG14425 at 1–2 hrs. (PDF) Figure S5 Browser views of cellularization genes. Browser views are the same as described in Figure S4, with an RNA expression maximum of 45K. Zld binds upstream and in some cases downstream of slam (which has maternal and zygotic inputs), halo, btsz, bnk, nullo and a related gene CG14427, Sry-a, and frs/z600, but not to surrounding genes. The peak just upstream of nullo contains a CAGGCAA site (not shown), one of the new TAGteam sites found in the enrichment analysis that binds Zld in vitro. Bound genes are highly down-regulated in zld2. Note that Sry-a is down- regulated, but Sry-b (to the left) and Sry-d (to the right) are unaffected. (TIF) Figure S6 Browser views of DV genes. Browser views are the same as described in Figure S4 with an RNA expression maximum of 15K. Zld binds to regions of the dorsally expressed genes dpp and tsg, the Dpp-targets Race, egr, and C15, and the neuroectoder- mal genes vnd and ind. Expression of the Zld-bound genes is down- regulated in zld2 although to a lesser degree for the ventral ectodermal genes ind and vnd, which are also Dorsal targets. (TIF) Figure S7 Browser views of part of the Antp complex including ftz. Browser views are the same as described in Figure S4 with an RNA expression maximum of 15K. ftz shows greater expression Zelda Coordinates Early Gene Networks than the homeotic genes Dfd, Scr, and Antp (only 39 region of Antp is shown), which are not activated until mid-late nc 14 in specific segments. They all exhibit significant, but lower Zld binding scores. Interestingly, mir-10, located between Dfd and Scr (top panel) also appears to be under direct control of Zld. In addition, a transcription unit that remains unannotated but was described in Kuroiwa et al. [64] as gene ‘‘X’’ with no known function/ phenotype, is also controlled by Zld (bottom panel). (TIF) Figure S8 Target genes of the key patterning factors are more likely to be expressed if bound by Zld. (A) Bar graph showing the number of bound target genes of DV (DTS and Mad) and AP (Bcd, Cad and gap genes) [35,13,14] transcription factors (red) that are also bound by Zld (blue) divided into two groups: targets that are expressed (+) or not expressed (2) in blastoderm embryos (from MacArthur et al. [14], pol II ChIP-chip data). The analysis was restricted to genes within 2 kb of the factor-bound regions. (B– C) Heat maps showing the fraction of target genes that two factors have in common, including all target genes (B) or blastoderm- expressed targets (C). The number in each box represents the fraction of genes bound by a factor denoted in the row that are also bound by the factor denoted in the column. For example, 59% of all DTS targets are also bound by Zld (B). This number increases to 76% for those DTS targets that are expressed at 2– 4 hrs (C). (TIF) Figure S9 Summary of early gene circuitry coordinated by Zld. Genes are organized across the time line according to the nc in which transcripts were first detected in wild-type (A) or zld2 embryos (B) by in situ hybridization. Activation arrows and repression lines pointing to a box apply to all the genes in the box. Zld plays a widespread role in the timely activation of gene batteries involved in cellularization, sex determination, and DV and AP patterning. Zld functions in a complex network of coherent and incoherent feed forward loops that allow more comprehensive spatial and temporal regulation. Genes that are weakly expressed and/or shifted in zld2 are shown in gray (B). * denotes ectopic activation that occurs in zld2 embryos (B). (TIF) Table S1 Comparison of wild-type and zld2 expression profiles at 1–2 and 2–3 hrs of development. Expression data using Affymetrix gene expression arrays and NimbleGen tiling arrays is summarized as the number of genes (based on Drosophila melanogaster genome release 5.29) considered down- or up-regulated (fold change $2 with a p,0.05), or unchanged in zld2 compared to wild-type. Also listed is the number of genes that were not expressed (absent) and the total number of genes represented on the arrays. The NimbleGen down-regulated genes are further summarized as the number of genes that were also down-regulated in the Affymetrix gene arrays (overlap) or not, i.e., additional (new) down-regulated genes found using the tiling arrays. 77% (82/107) and 82% (275/337) of the 1–2 and 2–3 hr differentially expressed genes overlapped, respectively. BD% refers to the percentage of genes in each category that are associated with Zld-bound regions (within 2 kb) from the ChIP data. Down-regulated genes were more likely to be associated with Zld binding. Note the 10 fold difference between genes in the ‘‘down-regulated’’ and ‘‘absent’’ categories in both 1–2 and 2–3 hr datasets as well as the gene expression array and tiling array datasets. The difference between the ‘‘down-regulated’’ and ‘‘unchanged’’ genes is much lower, 2–3 fold, however many of the patterning genes that are mis-regulated in zld2 are included in the ‘‘unchanged’’ category because their overall expression levels did not change more than 2 fold. Many of PLoS Genetics | www.plosgenetics.org 14 October 2011 | Volume 7 | Issue 10 | e1002339