Author Summary Development of an embryo into different cell types relies on regulation of gene expression, whereby genes are coordinately turned on or off. CBP and the related p300 protein are central regulators of gene expression in animal cells. These co-activator proteins facilitate gene activation by a multitude of transcription factors, possibly through their histone acetyltransferase activity. Consequently, loss of CBP/p300 gene function disrupts development and is lethal in mice, worms, and flies. How CBP/p300 is targeted to regulatory DNA sequences is not understood. Here, we have compared genome occupancy of CBP with 40 different transcription factors in Drosophila embryos and find that master regulators of dorsal-ventral patterning, the transcription factors Dorsal and Medea, target CBP to the genome. CBP occupies mainly active genes in embryos, where histones become acetylated. Surprisingly, the presence of CBP at silent genes does not result in histone acetylation. We find that repressive chromatin prevents histone acetylation by CBP. Our results demon- strate that CBP preferentially associates with some gene regulatory networks and that CBP binds silent genes without causing histone acetylation. These data have implications for prediction of transcriptional regulatory sequences and for understanding gene regulation by one of the most widely used co-activators in animal cells. tissue. By contrast, a constitutively active form of Toll [15], Toll10B, results in high Dorsal concentration in all embryonic nuclei, generating embryos consisting entirely of presumptive mesoderm. In embryos derived from Tollrm9/rm10 mutant mothers [15], Dorsal enters all nuclei at an intermediate level corresponding to that found in the lateral, neuroectoderm region. Dorsal regulates gene expression in a concentration- dependent manner (reviewed by [16]). Target genes such as twist (twi) and snail (sna) with low-affinity bindning sites are turned on in ventral, presumptive mesodermal cells where Dorsal concen- tration is highest. When Dorsal sites are positioned in proximity to AT-rich binding sites, Dorsal is converted to a repressor that recruits the co-repressor Groucho and thereby prevents expres- sion of dorsal ectoderm targets such as zerknu¨llt (zen), tld, and dpp in lateral and ventral parts of the embryo [17–19]. This restricts Dpp-signaling to the dorsal ectoderm where it sub-divides this tissue by regulating gene expression in a concentration- dependent manner [8]. CBP can function as a Dorsal co-activator as they genetically interact and bind each other in vitro [7]. However, to what extent Dorsal relies on CBP for gene activation in vivo is not known. Here, we describe a high concordance in genome occupancy of CBP and Dorsal. We show that CBP occupancy differs in mutant embryos where Dorsal fails to enter the nucleus, and that this difference often correlates with changes in gene expression. Moreover, CBP occupancy in mutant embryos coincides with regions bound by the Smad protein Medea, a mediator of Dpp-signaling (reviewed in [20]). Thus, genome occupancy of CBP is most strongly associated with dorsal-ventral axis specification in Drosophila embryos, consistent with earlier studies on CBP mutant phenotypes. Although CBP associates with some Dorsal-target genes in tissues where they are not expressed, this does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chro- matin is present at the Dorsal-target genes, which does not preclude CBP binding, but restricts histone acetylation at these CBP-bound genomic sites. CBP Occupancy in Drosophila Embryos Results CBP-binding sites highly overlap Dorsal-bound genomic regions in early Drosophila embryos We compared the published CBP binding data in Drosophila 0– 4 hour embryos [5] to regions bound by 40 sequence-specific transcription factors mapped at a similar stage of embryo development [21,22]. There is a particularly strong correlation between genome occupancy of CBP and the key activator of dorsal-ventral patterning, the transcription factor Dorsal. Eighty- two percent of the CBP peaks overlap a Dorsal-bound region (Table S1). To normalize for different number of identified regions for the 40 factors, we used the 300 most strongly bound regions for each factor in comparison with all CBP peaks. This shows that the CBP peaks still overlap best with regions occupied by Dorsal (Table S1). To further investigate this overlap of CBP and Dorsal occupancy, we performed CBP ChIP-seq with chromatin extracts from 2–4 hour old wild-type and mutant embryos where Dorsal fails to enter the nucleus (gd7). The CBP serum is affinity-purified and CBP-specific, and quantitatively similar levels of CBP occupancy is found at several loci with another CBP antibody (Figure S1). To calculate peaks and bound regions, the 5% highest enrichment values in both wild-type and gd7 embryos were extracted, corresponding to a cut-off of 1.9 in wild-type and 1.9 in gd7. High-confidence peaks were then defined as regions of at least 200 bp with enrichment values of at least 1.9 (in log2 scale). We identified 3013 high-confidence peaks in wild-type and 1939 CBP peaks in gd7 embryos. These CBP peaks were compared to the occupancy of the previously mapped 40 transcription factors [21,22]. We divided the CBP peaks into bins of increasing cut-off, so that fewer but stronger CBP peaks are shown along the x-axis, and plotted the overlap with the 300 strongest regions for each factor. As shown in Figure 1A and Table S2, the CBP-peaks in wild-type embryos overlap most extensively with Dorsal. Furthermore, the stronger the CBP- peaks, the better the overlap with Dorsal. Fifty-two percent of the 174 strongest CBP-peaks overlap the top 300 Dorsal-bound regions (Figure 1A). By contrast, the CBP peaks do not overlap regions bound by transcriptional activators that pattern the anterior-posterior axis, such as Bicoid and Caudal, to more than 10–15 percent (Figure 1A). Similar results were obtained with unprocessed CBP data, showing that the high overlap with Dorsal is not due to the way we defined the CBP-bound regions (Table S3). We then determined how many of the 300 strongest regions for each factor overlap all CBP peaks in wild-type. As shown in Figure 1B, 95% of the 300 strongest Dorsal peaks overlap any of the 3013 regions bound by CBP in wild-type embryos. Thus, virtually all of the strong Dorsal-binding regions in the genome are also occupied by CBP. Since a lot of transcription factors bind to many of the same genomic sites [reviewed in 23], we expected other factors to overlap the CBP peaks to a similar degree as Dorsal. However, we find that other factors do not overlap CBP-bound regions to the same extent as Dorsal in wild-type embryos, although GAGA-factor (GAF) binding regions also overlap the CBP-peaks extensively (Figure 1A, 1B and Table S4). We conclude that Dorsal and GAF are associated with CBP to a larger extent than other factors in early embryos. To investigate if Dorsal is required for CBP’s association with the genome, we next compared CBP occupancy in wild-type and gd7 mutant embryos. As shown in Figure 1B, fewer of the Dorsal and GAF binding regions overlap CBP peaks in gd7 mutant embryos than CBP peaks in wild-type. By contrast, many other PLoS Genetics | www.plosgenetics.org 2 June 2012 | Volume 8 | Issue 6 | e1002769