CBP Occupancy in Drosophila Embryos Figure 7. CBP may coordinate dorsal-ventral axis specification in Drosophila embryos. A) The Drosophila embryo is divided into segments along its anterior-posterior axis, whereas different germlayers arise along the dorsal-ventral axis. CBP occupancy genome-wide is over-represented at targets for the two key morphogens in dorsal-ventral patterning, Toll/Dorsal and Dpp/Medea. By contrast, anterior-posterior activators such as Bicoid and Caudal overlap the genomic distribution of CBP to a much smaller extent. B) Summary of histone modifications, Dorsal, and CBP occupancy at Dorsal-target genes in different tissues. An average of the four histone acetylations tested is colored green and H3K27 tri-methylation blue, CBP is colored red and Dorsal (dl) light blue. The size of the spheres represents protein/histone modification abundance and is drawn to scale. Dorsal represses tld and zen together with Groucho (Gro), whereas Zelda and possibly other factors (?) activate these genes. Although CBP binding is associated with gene expression and Dorsal binding genome-wide, CBP can also associate with silent genes. One reason that silent CBP-bound genes are not activated could be that H3K27me3-repressed chromatin restricts histone acetylation. doi:10.1371/journal.pgen.1002769.g007 of GAF to these sites is not compatible with proper regulation of the corresponding genes. Instead, many of these regions bind Medea and Dichaete, especially the places where CBP binding is strong already in wild-type. We show that in gd7 embryos, Dpp/ Medea-regulated genes are expressed in more cells, resulting in increased CBP signal (Figure 2). Our data indicate that also Dichaete-regulated genes are more highly expressed in gd7 mutants, and that CBP-binding therefore increases at these regions. Unexpectedly, median gene expression level of genes associated with gd7 Up regions is high in wild-type embryos. Most genes associated with these regions increase in expression even further in the absence of Dorsal (Figure 4), in most cases probably due to an expansion in the number of cells expressing the gene. We therefore expected that these CBP-binding sites would be situated in promoter regions, and the increase in CBP binding a consequence of increased gene activity. However, we found that these sites are mainly found in intronic and intergenic regions associated with H3K4me1, a mark of transcriptional enhancers. This indicates that CBP becomes recruited to these enhancers to mediate gene activation, rather than passively associating with active gene regions. CBP occupancy in mutant embryos is related to HOTness and dependent on Dorsal CBP occupancy in gd7 embryos is reduced at regions where only few factors bind. The bigger the reduction in CBP occupancy compared to wild-type, the fewer the factors that are associated with such a region in wild-type, i.e. the lower the HOTness of the region (Figure 3B). CBP peaks that are reduced in gd7 embryos are much more common at regions where Dorsal binds in wild-type compared to other factors, consistent with a requirement for Dorsal in targeting CBP to chromatin. Although not all of the gd7 Down CBP peaks overlap the top 300 Dorsal-binding regions, 92% overlap Dorsal when all Dorsal-binding regions are considered (Table S5). Peaks where CBP is reduced in gd7 embryos are found in several known Dorsal target genes, such as twi, brk, htl, and Mef2 (Figure 5 and Figure S4). Furthermore, 10 of the 20 strongest Dorsal peaks overlap a region where CBP binding is reduced in gd7 embryos. Together, these data show that in early embryos, chromatin binding of CBP to many sites in the genome is dependent on Dorsal. We found a number of genomic regions where CBP occupancy in gd7 embryos is reduced to a level approaching background, the gd7 Lost regions. These regions are mostly devoid of histone modifications and occupied by very few or none of the 40 transcription factors (Figure 3). The factors found at these regions bind at very low levels, indicating that they may not contribute to regulation of the corresponding genes at this stage of development. Further, most genes associated with the gd7 Lost regions are expressed at very low levels or completely silent. These CBP- binding regions may therefore represent regulatory sequences that are poised for subsequent activation. Consistent with this interpretation, mean expression of the corresponding genes increases at later stages of development (Figure 3F). Why is CBP occupancy lost from these regions in gd7 embryos? Perhaps these genes are not, and will not be expressed in the dorsal ectoderm, and are therefore not associated with CBP in gd7 mutants that convert the entire embryo into dorsal ectoderm. Alternatively, CBP binding to these regions is dependent on Dorsal. Although binding is weak, Dorsal occupies many of these regions in wild- type (Figure 1C). It is possible that even small amounts of Dorsal is sufficient and necessary for CBP recruitment to these sites, and that CBP binding is consequently lost in the absence of Dorsal. Although CBP occupancy is reduced predominantly at Dorsal- binding regions in gd7 mutant embryos, expression of Dorsal target genes is also altered. The decrease in CBP occupancy in mutant embryos may therefore be a consequence of transcriptional inactivity, rather than a lack of recruitment by Dorsal. Indeed, as shown in Figure 4, CBP occupancy is on average reduced at down-regulated genes and increased at up-regulated genes. Therefore, although Dorsal and CBP occupancy often coincide, Dorsal may not directly recruit CBP to regulatory DNA sequences. However, there are also places where CBP occupancy is reduced without a corresponding change in gene expression. One such example is at the promoter of the caudal (cad) gene, which is co-occupied by Dorsal and CBP but where CBP binding is reduced more than two-fold in gd7 embryos (Table S7), although PLoS Genetics | www.plosgenetics.org 11 June 2012 | Volume 8 | Issue 6 | e1002769