the gene continues to be expressed [25]. Furthermore, as shown in Figure 1, Dorsal and CBP associate in vivo. We believe, therefore, that Dorsal may directly recruit CBP to many sites in the genome. Dorsal-independent CBP binding As summarized in Figure 7B, there are also genomic sites where CBP occupancy is not dependent on either Dorsal or gene expression. Several known Dorsal target genes, including sna, neur, ind and ths, continue to associate with CBP in gd7 embryos (Figure 5 and Figure S4). Although in general, HOTness is major determinant of CBP occupancy (Figure 3B), there is no big difference in HOTness of the Dorsal target gene regions where CBP-binding is reduced (e.g. twi, htl, brk) compared to Dorsal target gene regions where CBP binding is not changed (e.g. sna, ind, ths). What maintains CBP binding on these genes in the absence of Dorsal is not clear. Presumably, other factors recruit CBP to these sites in the absence of Dorsal, but we have not found a common factor for the regions where CBP binding is unchanged. We note, however, that GAGA-factor (GAF) associates with many of the CBP-binding regions in wild-type embryos, but much less with CBP-binding regions in gd7 embryos. It is possible that GAF contributes to the recruitment of CBP to chromatin. Dorsal is converted to a repressor when it binds in proximity to AT-rich sequences, and thereby prevents expression of dorsal ectoderm target genes in the neuroectoderm and mesoderm [17– 19]. Consequently, these target genes, e.g. dpp, zen, and tld, are activated in all cells of gd7 mutant embryos (Figure 2 and Figure 6). As expected, CBP occupancy increases at these target genes in gd7 embryos, since more cells express the genes. The Zelda protein is a maternally contributed activator of these genes [29–31]. We have previously shown that in nej1 embryos containing reduced amounts of CBP, tld expression is diminished, whereas dpp and zen expression remains unaffected [10]. It is possible, therefore, that more activators than Zelda contribute to activation of tld, zen, and dpp in the dorsal ectoderm. Until these factors are identified, it may not be possible to explain why tld expression is particularly sensitive to a reduction in CBP amount in early embryos. Silent genes bound by CBP are hypoacetylated When Dorsal functions as a repressor, it recruits the Groucho co-repressor [19]. The yeast Tup1 protein, which is related to Groucho, was recently shown to block recruitment of co-activators to target genes [32]. By contrast, we find that CBP continues to associate with the tld and zen genes in the neuroectoderm although they are being repressed by Dorsal/Groucho (Figure 6). Groucho binds the histone deacetylase Rpd3 (HDAC1), which may be important for repression [33]. Indeed, we find that when tld and zen are repressed by Dorsal in the neuroectoderm and mesoderm, the genes are hypoacetylated despite the presence of CBP (Figure 6). Contrary to the general trend, some genes recruit CBP even though they are silent. Why are these genes not activated? In the cases we have examined, histone acetylation is low despite the presence of CBP when the genes are not expressed (summarized in Figure 7B). Since lysine methylation and acetylation are mutually exclusive, we measured histone methylation at CBP-bound regions and found that Polycomb-repressed H3K27me3 chromatin is present at Dorsal-target genes in some tissues where these genes are not expressed. Although H3K27me3-decorated chromatin restricts DNA accessibility [34], we find that H3K27me3- chromatin does not preclude CBP binding, but restrains histone acetylation at these CBP-bound genomic sites. Interestingly, all histone acetylations that we measured are blocked by H3K27me3- chromatin, not only the mutually exclusive H3K27ac. This CBP Occupancy in Drosophila Embryos indicates that despite the ability of CBP to bind to genes enclosed in H3K27me3-chromatin, the histones are not accessible for acetylation by CBP and other HATs. Our data are consistent with a model for Polycomb silencing that allows access of proteins and pol II to DNA, but that restrains pol II elongation [reviewed in 35]. Perhaps high levels of histone acetylation are necessary for release of pol II from the promoter, for example by recruiting the bromodomain protein Brd4 that brings in the P-TEFb kinase to phosphorylate pol II [36]. Regulation of CBP’s HAT activity In cells depleted of CBP and p300, global levels of H3K18ac and H3K27ac are greatly diminished whereas other histone acetylations remain unaffected, suggesting that these are in vivo targets of CBP acetylation [37,38]. CBP can also acetylate H3K56, which occurs in response to DNA damage [39]. We find that H3K18ac and H3K27ac levels do not always correlate with changes in CBP occupancy at Dorsal target genes, although H3K18ac levels are most similar to CBP abundance (Figure 5 and Figure 6). In part, this can be explained by the presence of H3K27me3-chromatin, that precludes histone acetylation. How- ever, in the neuroectoderm (Tollrm9/rm10 embryos), the twi promoter contains less histone acetylation than in the dorsal ectoderm (gd7 embryos) although H3K27me3 levels are reduced and CBP binding not decreased compared to dorsal ectoderm (Figure 5). Together, our results show that CBP’s HAT activity is regulated by substrate availability, but that it may also be regulated by genomic context or signaling. Implications for enhancer prediction Genome occupancy of CBP/p300 and H3K4me1 can be used to predict cis-regulatory DNA sequences [reviewed in 40]. However, what fraction of regulatory sequences that can be identified in this way is not known. We find that CBP binding to many known enhancer sequences that are active in early embryos is below our cut-off for high-confidence peaks, although we determined average CBP occupancy to be 1.73 times the genomic background at 97 previously described early embryonic enhancers [41]. Our results also show that CBP binding differs greatly between wild-type and mutant embryos, and that some gene regulatory networks rely on CBP to a much larger extent than others. Together, these results suggest that although CBP/p300 binding can be used to successfully identify transcriptional regulatory sequences, many enhancer sequences will be missed because they are not bound by CBP/p300 or bound at levels below criteria for high-confidence peaks. Even though mapping CBP/p300 binding in different cell-types will increase the number of putative regulatory sequences, we anticipate that a substantial number of enhancers will require alternative strategies for their identification, e.g. genome occupancy of other HATs [42]. In conclusion, we show that association of CBP with the genome is dependent on the number and types of transcription factors that bind the DNA sequence, that CBP preferentially associates with some gene regulatory networks, that CBP binding correlates with gene activity, but that CBP also binds silent genes without causing histone hyperacetylation. Materials and Methods Antibodies A GST-dCBP amino acids (aa) 2540–3190 fusion protein was used to immunize rabbits and the resulting serum affinity-purified as described in [9]. This antibody has been used by modEN- CODE to map CBP binding during Drosophila development [5]. Its PLoS Genetics | www.plosgenetics.org 12 June 2012 | Volume 8 | Issue 6 | e1002769