CBP Occupancy in Drosophila Embryos Figure 2. CBP binds to Dpp-target genes in gd7 mutant embryos. A) Percent overlap of CBP peaks in 2–4 hour gd7 mutant embryos with the 300 strongest binding regions of transcription factors in wild-type embryos. Seven out of 40 factors are shown. The stronger CBP peaks overlap regions bound by the Smad4 protein Medea, a transducer of Dpp-signaling, more than any other factor. B) Dorsal fails to enter the nucleus in gd7 mutant embryos, resulting in conversion of the entire embryo into dorsal ectoderm tissue where signaling by the TGF-ß molecule Dpp occurs. Consequently, expression of dpp is expanded (ii), expression of the Dpp-inhibitor sog absent (iv), and expression of the Dpp-target gene ush expanded (vi) in gd7 mutant embryos compared to wild-type (i, iii, v). Two to four hour old embryos were hybridized with digoxigenin-labeled probes and are oriented with anterior to the left. Lateral views with dorsal up are shown in (i) and (ii), ventro-lateral views in (iii) and (iv), and dorsal views are shown in (v) and (vi). C) CBP and Medea occupancy overlap at the ush locus, a Dpp-target gene. CBP ChIP-seq peaks (as defined in Materials &Methods) in wild-type (wt) and gd7 mutant embryos, as well as Medea ChIP-chip peaks in wt are shown. Occupancy is plotted as log2-fold enrichment over input. D) ChIP-qPCR confirms that CBP occupancy is elevated at the ush locus in gd7 embryos (T-test p = 0.045). CBP occupancy is plotted as fold enrichment relative the Mi-2 locus. Background occupancy levels are detected at two intergenic loci, the average of which is plotted (IG). Mean fold enrichment and standard deviations from 6 (wt) or 5 (gd7) independent biological replicates are shown. doi:10.1371/journal.pgen.1002769.g002 As illustrated in Figure 2C, CBP occupancy of the Dpp-target gene ush is hardly detectable in wild-type embryos, but highly evident in gd7 embryos. Similar results were obtained for other Dpp-target genes, including Race, tail-up, GATAc, and pannier (Figure S2). As expected, the CBP peaks are found at Med- binding regions at Dpp-target genes (Figure 2C and Figure S2). We confirmed that CBP occupies Dpp-target genes to a larger extent in gd7 embryos than in wild-type by ChIP-qPCR (Figure 2D and Figure S2, T-test of CBP at ush in gd7 vs wild- type, p = 0.045, at pnr p = 0.012). Our results indicate that CBP becomes recruited to Dpp-target genes upon signaling, consis- tent with previous observations that Dpp-signaling is impaired in CBP mutant embryos [8,10,11]. We conclude that whereas Dorsal and CBP binding strongly coincide genome-wide in wild- type embryos, CBP associates with Smad binding sites genome- wide upon increased TGF-ß signaling. This shows that the gene regulatory networks controlled by the two key morphogens in dorsal-ventral patterning, Toll/Dorsal and Dpp/Medea, are to a larger extent than others associated with CBP in early embryos. In embryos lacking nuclear Dorsal, CBP occupancy is reduced at regions where Dorsal, but few other factors, binds in wild-type embryos We next separated the CBP-peaks into those that remain unchanged between wild-type and gd7, those where CBP binding increases at least 2-fold in gd7 versus wild-type (gd7 Up), those where CBP binding decreases at least 2-fold in gd7 versus wild-type (gd7 Down), and those that are completely lost in gd7. We first looked at how many other factors that occupy these CBP-bound regions, measured in their HOTness. High occupancy target (HOT) regions are defined as genomic sites binding at least one of the 40 transcription factors [22]. The minimum HOTness value is 1 and increases with the number of factors and the number of sites for each factor found within the genomic region. Very HOT regions, or hotspots, are found across the Drosophila, C. elegans, and human genomes, and are associated with open chromatin, but their function is not understood [22]. As shown in Figure 3A, most CBP-peaks overlap a HOT region. CBP-peaks that increase in gd7 have a high HOTness, whereas unchanged, down and lost in gd7 are decreasingly HOT. This is also illustrated in Figure 3B, where PLoS Genetics | www.plosgenetics.org 4 June 2012 | Volume 8 | Issue 6 | e1002769