CBP Occupancy in Drosophila Embryos Figure 1. CBP and Dorsal co-occupy the genome more extensively than 39 other transcription factors in early Drosophila embryos. A) Percent overlap of CBP peaks in Drosophila 2–4 hour embryos with the 300 regions occupied most strongly by transcription factors at a similar stage of development. The peaks were selected with increasing cut-off, so that all CBP peaks are used for the comparison in the leftmost value on the x-axis, and increasing cut-offs leading to fewer, but stronger peaks along the x-axis. Note that long binding regions for one factor can overlap several CBP peaks. Only seven out of 40 transcription factors are shown. The CBP peaks overlap Dorsal bound regions more than any other factor. B) Percent overlap of the 300 strongest regions for seven transcription factors with all 3013 CBP peaks in wild-type and 1939 peaks in mutant embryos where Dorsal cannot enter the nucleus (gd7). Dorsal-binding regions overlap the CBP peaks better than any other factor in wild-type. C) Genomic regions where CBP occupancy is lost in gd7 mutant embryos (n = 191) were compared to all peaks for 40 transcription factors in wild-type embryos. Presence of a factor is indicated by red and absence by black. Only factors with .5% overlap are shown. Dorsal binds to 76% of the regions where CBP occupancy is lost in gd7 embryos. D) Dorsal can be co-immunoprecipitated with CBP from wild-type embryos. The CBP antibody and control immunoglobulin G (IgG) were used in immunoprecipitation from 2–4 hour old embryo extracts, separated on a gel together with 10% input, and probed with a Dorsal antibody. A fraction of Dorsal is associated with CBP. doi:10.1371/journal.pgen.1002769.g001 factors overlap the gd7 CBP peaks better than wild-type peaks. This indicates that CBP occupies regions bound by multiple factors to a larger extent in gd7 than in wild-type embryos (see below). In gd7 embryos that lack Dorsal in the nucleus, CBP continues to associate with 84% of Dorsal-binding regions, suggesting that other factors maintain CBP binding at these places in the absence of Dorsal. However, CBP occupancy at the top 300 Dorsal- binding regions is significantly lower in gd7 embryos compared to wild-type (paired T-test p = 5.2261029), showing that CBP occupies many Dorsal targets in gd7 embryos less strongly. We therefore extracted the regions where the CBP-peaks were completely lost in gd7 embryos, and compared them to all of the regions bound by the 40 factors in wild-type (not only the top 300 regions). High-confidence CBP peaks in wild-type with a CBP enrichment of less than 0.5 (in log2 scale) in gd7 were considered to be completely lost. It was found that 76% and 67% of the peaks that are lost in gd7 embryos overlap Dorsal or GAF respectively, but that no other factor overlaps these CBP-peaks to more than 22% (Figure 1C and Table S5). This indicates that Dorsal is important for targeting CBP to chromatin in Drosophila embryos, and since Dorsal and GAF co-occupy many of these regions (Figure 1C), that GAF may cooperate with Dorsal in specifying CBP binding. CBP and Dorsal can be co-immunoprecipitated from wild-type embryos (Figure 1D), indicating that Dorsal may directly bring CBP to its genomic binding sites. Taken together, our results suggest that Dorsal targets CBP to many sites throughout the genome. Dpp-target genes are highly occupied by CBP in embryos lacking nuclear Dorsal To identify Dorsal-independent transcription factor sites that are co-occupied by CBP, we next compared the CBP peaks in gd7 embryos to regions bound by the 40 transcription factors in wild- type embryos. Figure 2 shows that the best overlap of the strong CBP peaks in gd7 embryos is with the Smad4 protein Medea (Med), a transducer of Dpp-signaling (Figure 2A and Table S2). In gd7 embryos, all cells are converted to dorsal ectoderm, the tissue where Dpp-signaling occurs. Consequently, expression of dpp is expanded, whereas the expression of the Dpp-inhibitor sog is absent (Figure 2B). This results in expanded expression of Dpp- target genes in gd7 mutant embryos (u-shaped (ush) in Figure 2B, as well as Race and pannier in Figure S2). In wild-type embryos, these Dpp-target genes are expressed in a restricted number of cells in dorsal parts of the embryo, whereas in gd7 mutants they become expressed throughout the entire circumference of the embryo. Thus, Dpp-signaling occurs in the entire embryo in gd7 mutants, thereby providing a genetic background in which binding of CBP to Dpp-target genes can be visualized. PLoS Genetics | www.plosgenetics.org 3 June 2012 | Volume 8 | Issue 6 | e1002769