CBP Occupancy in Drosophila Embryos Figure 4. Differences in CBP occupancy between wild-type and gd7 mutant embryos correlate with mean changes in gene expression. A) Differences in CBP occupancy between wild-type and gd7 mutant embryos (no Dorsal in the nucleus) was compared to published gene expression changes in pipe mutant embryos (no Dorsal in the nucleus) versus Toll10B mutant embryos (high level of Dorsal in all nuclei) [25]. Squares indicate mean expression in pipe versus Toll10B mutant embryos, and whiskers correspond to 95% confidence intervals. There is a mean up- regulation of transcription at genes where CBP occupancy is increased at least 2-fold in gd7 mutant embryos (gd7 Up), and a mean decrease in expression where CBP occupancy is decreased or lost. The small decline in mean expression of unchanged regions is due to an overrepresentation of regions with CBP occupancy decreased less than 2-fold in the gd7 Unchanged group. B) In gd7 mutant embryos, a mean decrease in CBP occupancy at Dorsal mesoderm target genes (down-regulated in pipe and gd7), and a mean increase in CBP occupancy at Dorsal-target genes in the dorsal ectoderm (up-regulated in pipe and gd7) is observed. Mesoderm and ectoderm targets are defined in [25]. Squares indicate mean CBP binding in gd7 versus wild-type embryos, and whiskers correspond to 95% confidence intervals. doi:10.1371/journal.pgen.1002769.g004 wild-type or that had a CBP region within 500 bp were considered. This shows that at sites where CBP occupancy is increased in gd7 mutants compared to wild-type, the corresponding genes are on average up-regulated in pipe mutant embryos. Genes associated with regions where CBP occupancy does not change in gd7 embryos do not alter their expression significantly between pipe and Toll10B mutants. By contrast, at regions where CBP occupancy is reduced or lost in gd7 mutants compared to wild-type, mean gene expression is decreased (Figure 4A). We then examined the difference in CBP occupancy between gd7 mutants and wild-type at regions that overlap Dorsal binding at mesoderm-targets (down- regulated in pipe and gd7) and dorsal ectoderm-targets (up- regulated in pipe and gd7) as defined in [25]. Figure 4B shows that mean CBP occupancy is decreased at Dorsal-targets in the mesoderm and increased at targets in the dorsal ectoderm in gd7 mutant embryos. Taken together, our results show that differences in CBP occupancy correlate well with changes in gene expression genome-wide. Binding of CBP to silent genes does not lead to histone acetylation We wanted to compare changes in CBP occupancy to histone acetylation levels, and therefore looked closer at some of the best known Dorsal targets in the mesoderm and dorsal ectoderm. We compared histone modifications with CBP binding at these genes in gd7, Tollrm9/rm10, and Toll10B mutant embryos by ChIP-qPCR. In these mutant backgrounds the entire embryo is converted to dorsal ectoderm (gd7), neuroectoderm (Tollrm9/rm10), or mesoderm (Toll10B). We normalized the binding of Dorsal, CBP, and histone modifica- tions to two intergenic regions, selected based on the absence of protein binding and histone modifications, and plotted the fold enrichment relative these intergenic sites. The histone modifications were additionally normalized to histone H3 levels (Table S6). In the mesoderm, Dorsal targets such as twi and sna, are activated by high levels of Dorsal. These genes are therefore not expressed in gd7 and Tollrm9/rm10 mutant embryos that contain no Dorsal or an intermediate concentration of nuclear Dorsal in the entire embryo. In Toll10B mutant embryos Dorsal is present in high amounts and twi and sna are therefore expressed throughout the embryo (Figure 5A and 5F). Less CBP and less histone acetylation on H3K9, H3K18, H3K27, and on histone H4 is found at the twi promoter in gd7 embryos as compared to wild-type (Figure 5B–5D, T-test of all four histone acetylations in gd7 vs wild-type, p = 0.0042). This is consistent with the genome-wide correlation of reduced CBP occupancy with lower gene expression and lack of Dorsal protein (Figure 3 and Figure 4). Interestingly, in Tollrm9/rm10 embryos, there is a further reduction in histone acetylation without a corresponding decrease in CBP binding, as compared to gd7 embryos (Figure 5D, T-test of histone acetylations in Tollrm9/rm10 vs gd7, p = 0.020). This shows that the amount of CBP bound to a genomic region is not the only determinant of histone acetylation levels. In Toll10B embryos, twi expression is turned on in the entire embryo, and CBP as well as histone acetylations are present in high amounts (Figure 5B–5D, T-test of CBP in gd7 vs Toll10B, p = 0.0028, histone acetylations in gd7 vs Toll10B, p = 0.00072). Unexpectedly, CBP binding to the sna promoter is not decreased in gd7 mutant embryos compared to wild-type (Figure 5G and 5H), although Dorsal is absent and the gene not expressed (Figure 5F). By contrast, with the exception of H3K18ac, histone acetylation levels change in the different genetic backgrounds (Figure 5I, T-test of H3K9ac, H3K27ac, plus H4ac in wild-type vs gd7, p = 0.000011, gd7 vs Tollrm9/rm10, p = 0.037, gd7 vs Toll10B, p = 0.00033, Tollrm9/rm10 vs Toll10B, p = 0.041). We therefore measured histone lysine methylation at Dorsal-target genes, a modification mutually exclusive to lysine acetylation. Interestingly, we observed high amounts of H3K27me3 in gd7 and Tollrm9/rm10 embryos (Figure 5E and 5J). PLoS Genetics | www.plosgenetics.org 7 June 2012 | Volume 8 | Issue 6 | e1002769