overlap using an increasing cut-off until about five percent of the binding regions of dataset X remained. In plots where only three cut-offs are shown, the cut-offs are; 1) no cut-off (all regions included), 2) an average of cut-offs 1 and 3, 3) the highest cut-off where only the ,5% most enriched regions remained. Defining regions that have an altered CBP enrichment in gd7 embryos CBP peak values in gd7 embryos were calculated (as described above) within each CBP bound region from WT embryos. In regions for which the gd7 peak values were below 0.5 (data in log2 scale) CBP was considered lost. Next, a ratio of gd7 and WT peak values was calculated. Regions with less than a two-fold difference (21 to 1 in log2 ratio) were considered unchanged. Regions with more than two-fold higher gd7 peaks were defined as going up and regions with more than two-fold higher WT peaks where defined as going down. When CBP peak values in WT embryos were determined within CBP bound regions in gd7 embryos we found only two regions where no CBP could be detected in WT. We therefore did not define a class of regions unique for gd7 embryos. Statistics All statistics was performed using Statistica 10.0 (Statsoft). All reported T-tests are two-tailed without assuming equal variance. When multiple histone acetylations were compared between genotypes, all replicates from each acetylation were treated as one sample. Accession number The ChIP-seq data is deposited in GEO under accession number GSE34221. Supporting Information Figure S1 The affinity-purifed rabbit serum is CBP specific. A– B) Western blot and Coomassie-staining of CBP RNAi-treated Drosophila S2 cells. Primers with 59T7 RNA polymerase-binding sites (Table S8) were used in PCR amplification of CBP and GFP cDNA. The 1002 bp (CBP) and 700 bp (GFP) PCR products were used to generate double stranded RNA for RNA interference using the Megascript RNAi kit (Ambion). 26106 S2 cells were washed twice in serum-free medium and resuspended in 750 ml serum-free medium before treatment with CBP dsRNA or GFP dsRNA. 37 mM of dsRNA was added and the cells were incubated for one hour at 25uC followed by adding 1.5 ml 15% FCS medium. After three days the cells were collected and washed in serum-free medium followed by a second dsRNA treatment, and harvested three days later. Cells were washed twice in PBS and resuspended in lysis buffer (50 mM Tris, pH 7.8, 150 mM NaCl, 1% Nonidet P-40, EDTA-free protease inhibitors) and lysed for 30 minutes at room temperature. Centrifugation at 13000 rpm for 5 min followed, and the supernatant was saved. The protein concentration was measured by the BCA protein assay kit (Thermo scientific). SDS-PAGE sample buffer was added, the samples denatured at 95uC for 5 min and centrifuged at 13000 rpm for 5 min before loading on 7.5% SDS-PAGE gels. The gel was either Coomassie-stained (B) or transferred to PVDF membrane (Bio Rad laboratories) at 30 V over night (A). The membrane was blocked in PBS containing 5% non-fat dry milk and incubated with the affinity-purified rabbit anti-dCBP serum (1:200 in PBS containing 1% BSA) over night. The membrane was washed with PBS three times and incubated with HRP-coupled anti-rabbit antibody (1:10000, DAKO) for one hour followed by ECL detection (GE Healthcare), and exposure to a Luminiscent CBP Occupancy in Drosophila Embryos Image Analyzer (LAS-1000plus, Fujifilm). Expression of the loading controls that we used to re-probe the membrane with was affected by the CBP RNAi treatment. We therefore compared total protein concentration between samples on Coomassie-stained gels. Arrow in A) points to full-length CBP, the other bands represent degradation products since they are also reduced in strength by CBP RNAi treatment. C) Comparison of ChIP signals obtained with the rabbit anti-dCBP aa 2540–3190 serum with ChIP signals from a guinea-pig anti-dCBP aa 1–178 serum [9]. Two to four hour old wild-type embryos were used for ChIP, and CBP targets with different levels of occupancy in ChIP-seq were analyzed by qPCR. Occupancy is plotted as enrichment relative the average of two negative control loci (intergenic regions). Mean fold enrichment and standard deviations from 3 independent biological replicates are shown. (PDF) Figure S2 CBP occupies Dpp-target genes in gd7 mutant embryos. A) In situ hybridization of Dpp target genes Race and pnr in wild-type (w1118) and gd7 derived embryos. Two to four hour old embryos were hybridized with digoxigenin-labeled probes and are oriented with anterior to the left, and dorsal up. Note the expanded expression of the Dpp target genes in gd7 mutant embryos. B) ChIP-qPCR of CBP recruitment to Dpp target genes Race and pnr. Values are presented as fold over CBP binding at Mi- 2 whose expression and CBP binding is unaffected by the levels of Dorsal. As a negative control, the average of background CBP binding at two intergenic loci that do not bind CBP is included (IG). C–F) CBP and Medea occupancy overlap at the Dpp-target gene loci Race (Ance) (C), pnr (D), GATAc (grn) (E), and tup (F). CBP ChIP-seq peaks (as defined in Materials & Methods) in wild-type (wt) and gd7 mutant embryos, as well as Medea ChIP-chip peaks in wt are shown. Occupancy is plotted as log2-fold enrichment over input. (PDF) Figure S3 CBP and Bicoid do not co-occupy Bicoid-target genes. A–D) CBP and Bicoid occupancy does not overlap at the Bicoid-target gene loci eve (A), hb (B), kni (C), and otd (oc) (D). CBP ChIP-seq peaks (as defined in Materials & Methods) in wild-type (wt) and gd7 mutant embryos, as well as Bicoid ChIP-chip peaks in wt are shown. Occupancy is plotted as log2-fold enrichment over input. (PDF) Figure S4 CBP occupancy at Dorsal-target genes. In gd7 embryos that lack nuclear Dorsal, CBP occupancy is reduced at some Dorsal-target genes (A–C), but relatively unaffected at other Dorsal-target genes (D–F) compared to wild-type. ChIP-seq peaks for CBP in wild-type (wt) and gd7 mutant embryos (raw data without cut-off), as well as Dorsal ChIP-chip peaks in wild-type are shown for the Mef2 (A), htl (B), brk (C), neur (D), ind (E), and ths (E) loci. Occupancy is plotted as log2-fold enrichment over input. (PDF) Table S1 Overlap between previously published CBP ChIP-seq peaks in 0–4 hour embryos [5] and regions bound by 40 sequence specific transcription factors [21,22]. Overlap for all transcription factor binding sites or only the 300 most strongly occupied sites are shown in separate sheets. (XLSX) Table S2 Overlap between CBP ChIP-seq peaks from 2 to 4 hour old wt or gd7 embryos and the 300 most strongly occupied regions for 40 sequence specific transcription factors [21,22]. Overlap of ChIP-seq peaks with increasing cut-off is shown. Also, overlap of CBP peaks with the transcription factors after PLoS Genetics | www.plosgenetics.org 14 June 2012 | Volume 8 | Issue 6 | e1002769