This indicates that Polycomb-mediated repression is involved in keeping twi and sna off in the neuroectoderm and dorsal ectoderm. By contrast, H3K9me3, a mark for HP1-mediated repression, is absent on the twi and sna promoters in all three tissues (Figure 5E and 5J). Interestingly, the high levels of H3K27me3 over the sna promoter in gd7 embryos does not prevent CBP binding, indicating that Polycomb-repressed H3K27me3 chromatin is compatible with CBP binding. We therefore conclude that whereas CBP binding is not prevented by H3K27me3-repressed chromatin, histone acetylation is restricted. This conclusion is reinforced by our results from Tollrm9/rm10 mutants, where sna is also repressed and H3K27me3 present, and CBP binding not significantly different from that in other genotypes. Relative Toll10B mutants, histone acetylation remains low in Tollrm9/rm10 embryos (T-test of H3K9ac, H3K27ac, plus H4ac in Tollrm9/rm10 vs Toll10B, p = 0.041), which is likely explained by the higher amounts of H3K27me3 in Tollrm9/rm10 as compared to Toll10B embryos (Figure 5I and 5J, T-test of H3K27me3 in Tollrm9/rm10 vs Toll10B, p = 0.0025). We note that H3K18ac levels in mutant embryos correlate with changes in CBP amount to a better extent than other histone acetylations, indicating that H3K18 may be a major in vivo target for CBP’s HAT activity. The dorsal ectoderm targets tld and zen are repressed by Dorsal, and therefore more highly expressed in gd7 mutants that lack nuclear Dorsal, but completely repressed in Tollrm9/rm10 and Toll10B mutants, except at the embryonic poles (Figure 6A and 6F). In gd7 embryos, where these genes are expressed in more cells than in wild-type, binding of CBP is higher according to ChIP-seq (although not statistically significant by ChIP-qPCR) and acety- lation of histones increases (Figure 6B–6D and 6G–6I, T-test of histone acetylations at tld in wild-type vs gd7, p = 0.00046, at zen in wild-type vs gd7, p = 0.0094). This is consistent with the genome- wide correlation of CBP occupancy and gene expression (Figure 4). In both Tollrm9/rm10 and Toll10B mutant embryos, tld and zen are repressed. Surprisingly, although CBP binding is stronger in Tollrm9/rm10 than in Toll10B embryos (T-test of CBP at tld in Tollrm9/rm10 vs Toll10B, p = 0.037, but not significantly so at zen), there is equivalent amounts of histone acetylation in Toll10B embryos (Figure 6C, 6D and 6H, 6I). This may result from the high level of H3K27me3 in Tollrm9/rm10 relative Toll10B embryos (Figure 6E and 6J, T-test of H3K27me3 at tld in Tollrm9/rm10 vs Toll10B, p = 0.037, at zen in Tollrm9/rm10 vs Toll10B, p = 0.0016). Taken together, our analysis of Dorsal-target genes in different tissues shows that CBP can bind to these genes when they are silenced, but that this does not result in high levels of histone acetylation. In conclusion, our results suggest, 1) that CBP binding does not always correlate with gene expression or Dorsal binding, 2) that H3K18 acetylation levels closely follow CBP-binding, 3) that changes in histone acetylation can occur without a corresponding change in CBP binding, 4) that CBP binding is not prevented by the presence of H3K27me3 (Polycomb)-repressed chromatin, 5) but that H3K27me3-chromatin may restrict histone acetylation by CBP and other HATs. Discussion CBP and the related p300 protein are widely used transcrip- tional co-activators in metazoans that interact with transcription factors of all major families [reviewed in 1], and they are for this reason believed to be present at many transcriptional regulatory regions. Therefore, CBP/p300 is expected to contribute to gene activation by most transcription factors, and not selectively CBP Occupancy in Drosophila Embryos regulate a subset of transcriptional programs. By contrast, we found that the gene regulatory networks controlling dorsal-ventral pattering of the Drosophila embryo are associated with CBP to a larger extent than e.g. anterior-posterior patterning. CBP is primarily involved in dorsal-ventral patterning of early Drosophila embryos By comparison of CBP-bound regions in 2–4 hour old Drosophila embryos to previously mapped transcription factors [21,22], we found an extensive overlap of CBP peaks with the key activator of dorsal-ventral patterning, the Rel-family transcription factor Dorsal. We then determined the genome-wide distribution of CBP in embryos where Dorsal cannot enter the nucleus (gd7 mutants), and found that CBP peaks that overlap regions where Dorsal, but few other factors bind in wild-type are selectively reduced in gd7 mutant embryos. Instead, strong CBP-bound regions in gd7 mutants overlap best with regions bound by the Smad protein Medea, a mediator of Dpp-signaling. We and others have previously shown that signaling by the TGF-ß molecule Dpp is exceptionally sensitive to a small decline in the level of CBP in Drosophila embryos [10,11]. Our present results are consistent with a function for CBP in the genomic response to Dpp-signaling. Less overlap of the CBP peaks is found with mapped activators of anterior-posterior patterning such as Stat92E, Fushi-tarazu (Ftz), Paired, Caudal, and Bicoid (Table S2). Previous work has indicated that CBP may function as a Bicoid co-activator. When Bicoid and CBP are expressed in S2 cells, they can interact, and Bicoid-mediated activation of reporter genes in these cells is influenced by CBP levels [26,27]. We find that 43% of the 300 strongest Bicoid-binding regions overlap a CBP peak in wild-type embryos, indicating that CBP may participate in Bicoid-mediated activation in vivo. However, many of the Bicoid peaks are found in HOT regions that bind several transcription factors. Therefore, it may not be Bicoid that targets CBP to these sites. Furthermore, although the shape of the Bicoid gradient is slightly changed in embryos from the CBP hypomorph nej1, activation of Bicoid-target genes is not compromised by the decrease in CBP levels in nej1 embryos [28]. Consistent with a non-essential function for CBP in Bicoid-mediated activation, there is no co-occupancy of CBP and Bicoid at the known target genes hb, otd, kni, and eve (Figure S3). Thus, although CBP may contribute to Bicoid-mediated activation of some target genes, it seems to make a more widespread contribution to Dorsal-mediated activation. In conclusion, both genetic and genomic evidence points to a particularly important function for CBP in controlling the two key events in dorsal- ventral patterning of Drosophila embryos, the Dorsal gene regulatory network and Dpp-signaling. Perhaps CBP serves to coordinate the Dorsal and Dpp pathways in dorsal-ventral patterning (Figure 7A). CBP binds strongly to enhancers of upregulated genes in mutant embryos In embryos where Dorsal cannot enter the nucleus, we found places where CBP occupancy is increased, unchanged, decreased or lost. Regions that are unchanged bind several transcription factors, evident in their high HOTness, indicating that in the absence of Dorsal, other factors maintain CBP binding at these sites (Figure 3). Surprisingly, regions where CBP binding is increased are even HOTer, and therefore associated with even more factors in wild-type embryos. Although many CBP peaks in the genome are found where also GAF binds, the regions where CBP occupancy increases in gd7 embryos are lacking strong GAF binding, despite their high HOTness (Figure 3C). Perhaps binding PLoS Genetics | www.plosgenetics.org 9 June 2012 | Volume 8 | Issue 6 | e1002769