the change in CBP occupancy in gd7 versus wild-type is plotted against HOTness. Remarkably, there is an almost perfect correlation between the difference in CBP occupancy in gd7 versus wild-type embryos and mean HOTness. Thus, in gd7 embryos, CBP is present at regions where many factors are bound, but decreases or is lost at regions bound by only few factors. However, the highest mean CBP occupancy in wild-type is found at regions where CBP binding decreases in gd7 embryos (Figure 3A), suggesting that HOTness alone does not determine how much CBP that binds a particular genomic region. Rather, the HOTness determines if CBP occupancy will change in the absence of Dorsal. We then compared the different classes of CBP-bound regions with the top 300 regions bound by the 40 transcription factors, as well as with histone modifications, gene features, and gene expression. We divided the CBP peaks into three bins of increasing cut-off, so that fewer but stronger CBP peaks are shown along the x-axis. The first bin represents all CBP peaks within the respective class, and the third bin the ,5% strongest peaks. For the second bin we used an enrichment cut-off midway between the cut-offs for bin one and three. The bins were used to calculate % overlap with other genomic features. Regions where CBP binding increases in gd7 mutants overlap most factors extensively, consistent with the gd7 Up regions having the highest HOTness (Figure 3A and 3C). However, the gd7 Up regions do not overlap the top 300 GAF-binding sites (Figure 3C). As expected from the analysis in Figure 2, CBP-peaks that increase in gd7 show a high overlap with Medea. Surprisingly, the gd7 Up peaks that are strongest in wild-type overlap the Sox-protein Dichaete even better than binding regions for Medea (Figure 3C). Dichaete is involved in both anterior-posterior patterning by regulating pair-rule gene expression, and in dorsal-ventral pattering where it is expressed in medial and lateral regions of the neuroectoderm [reviewed by 24]. Our results indicate that Dichaete-regulated genes become associated with CBP in gd7 embryos. CBP-peaks that increase in strength in gd7 embryos (gd7 Up) strongly overlap H3K4me1, a histone modification associated with enhancer sequences, as well as the ‘‘active’’ histone marks H3K18ac and H3K27ac, but are depleted of H3K27me3- repressed chromatin (Figure 3D). Almost all of the gd7 Up CBP- peaks map to introns and intergenic sites, and very few to promoter sequences (Figure 3E). Interestingly, mean expression in 2–4 hour wild-type embryos for genes associated with gd7 Up regions are higher than for genes associated with regions where CBP binding decreases or is lost in gd7 (Figure 3F). Furthermore, there is a decrease in mean expression of genes associated with gd7 Up regions during the course of development, whereas expression of genes where CBP binding is reduced or lost increases during development (Figure 3F). Taken together, these analyses suggest that the genomic regions where CBP-binding increases in gd7 embryos are HOT intronic and intergenic enhancer sequences of highly expressed genes regulated by Medea and Dicheate. Regions where CBP binding does not change between wild-type and gd7 overlap best with Dorsal, GAF, and Medea, but some overlap is also found with other factors, in agreement with the high HOTness of these regions. Unchanged regions overlap well with most histone modifications, including H3K4me1 (enhancers) and H3K4me3 (active promoters), histone acetylation, but also with H3K27me3 (Polycomb-repressed chromatin) (Figure 3D). Un- changed peaks are found in introns and intergenic regions, but they are also common in promoters (Figure 3E). Like gd7 Up regions, mean expression for genes associated with unchanged regions is high in 2–4 hour wild-type embryos, and decreases at later stages of development. CBP Occupancy in Drosophila Embryos Regions where CBP binding is decreased in gd7 embryos overlap mainly Dorsal and GAF (Figure 3C). Especially the gd7 Down CBP peaks that are strongest in wild-type overlap Dorsal extensively, but show little overlap with other factors. Thus, in embryos where Dorsal fails to enter the nucleus (gd7), CBP binding is selectively reduced at regions where Dorsal, but few other factors bind. Interestingly, strong CBP-peaks that are decreased in gd7 embryos (gd7 Down) overlap more with the inactive chromatin mark H3K27me3 than with the active histone marks H3K18ac, H3K27ac, and H3K4me3 (Figure 3D). The regions where CBP binding decreases in gd7 embryos are more often than other CBP regions found in promoters, but they also occur frequently in intronic and intergenic sequences (Figure 3E). Regions where CBP is reduced in gd7 embryos are associated with genes that are medium-expressed in wild-type 2–4 hour embryos, but whose expression increase during development. Together, this indicates that regions where CBP-binding decreases in gd7 embryos are associated with Dorsal-regulated tissue-specific genes that are silenced by repressive chromatin in tissues where they are not expressed (compare with the Dorsal-target gene twist below). Unexpectedly, we found that the CBP peaks that are lost in gd7 overlap virtually none of the top 300 sites for the 40 transcription factors (Figure 3C and Table S2), although 76% of these peaks overlap a Dorsal-binding region (Figure 1C). Thus, CBP binding is lost in gd7 embryos from regions where Dorsal binds weakly in wild-type. Surprisingly, CBP-bound regions that are lost in gd7 poorly overlap all types of histone modifications (Figure 3D). The gd7 Lost regions are found in introns and intergenic sequences, but also in promoters (Figure 3E). Genes where CBP occupancy is lost in gd7 show the lowest mean expression in wild-type 2–4 hour embryos, but increases at later stages of embryogenesis (Figure 3F). It appears that regions where CBP occupancy is lost in gd7 embryos are found in low- and non-expressed genes that bind few other factors except Dorsal and GAF, and which are depleted in histone modifications. Together, these analyses suggest that the four categories of CBP peaks occur at very different genomic sites. Regions where CBP occupancy is increased in gd7 embryos are found in intronic and intergenic HOT regions of highly expressed Medea- and Dicheate-regulated genes, and are depleted of GAF. Unchanged regions are found in both promoters and enhancers of highly expressed genes, and are associated with many different factors. Regions with decreased CBP occupancy in gd7 embryos are found in both promoters and enhancers of medium expressed genes. They are found predominantly where Dorsal, but few other factors bind and are regulated by H3K27 methylation. Finally, regions where CBP occupancy is lost are found in genes with low expression at regions devoid of chromatin modifications and most transcription factors except Dorsal and GAF. Differences in CBP binding between wild-type and mutant embryos correlate with changes in gene expression Dorsal-regulated genes have previously been identified by comparing the difference in gene expression in pipe versus Toll10B mutant embryos [25]. In pipe mutants, the proteolytic cascade leading to Toll ligand activation is not initiated, and just as in gd7 mutants, Dorsal protein does not enter the nuclei in these embryos. Toll10B mutants on the other hand contain high levels of Dorsal in all nuclei. The difference in gene expression between the two represents Dorsal-dependent expression, and was plotted against changes in CBP occupancy between wild-type and gd7 mutant embryos (Figure 4A). All genes on the arrays used by Stathopoulos et al. [25] that overlapped a CBP bound region in PLoS Genetics | www.plosgenetics.org 5 June 2012 | Volume 8 | Issue 6 | e1002769