Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos Per-Henrik Holmqvist1, Ann Boija1, Philge Philip2,3, Filip Crona1, Per Stenberg2,3*, Mattias Mannervik1* 1 The Wenner-Gren Institute, Developmental Biology, Stockholm University, Stockholm, Sweden, 2 Department of Molecular Biology, Umea˚ University, Umea˚, Sweden, 3 Computational Life Science Cluster (CLiC), Umea˚ University, Umea˚, Sweden Abstract CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP–seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso- ventral patterning and that CBP binds silent genes without causing histone hyperacetylation. Citation: Holmqvist P-H, Boija A, Philip P, Crona F, Stenberg P, et al. (2012) Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos. PLoS Genet 8(6): e1002769. doi:10.1371/journal.pgen.1002769 Editor: Norbert Perrimon, Harvard Medical School, Howard Hughes Medical Institute, United States of America Received February 9, 2012; Accepted May 2, 2012; Published June 21, 2012 Copyright: 2012 Holmqvist et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council to MM and by grants from the Carl Tryggers, Erik-Philip S o¨rensens, and A˚ ke Wiberg Foundations to PS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mannervik@devbio.su.se (MM); per.stenberg@molbiol.umu.se (PS) Introduction CREB-binding protein (CBP) and its paralog p300 are widely used transcriptional co-regulators with histone acetyltransferase (HAT) activity (reviewed in [1]). Over 400 interaction partners have been described for these proteins, including transcription factors of all major families, and they are therefore believed to be present at many transcriptional regulatory regions. Indeed, chromatin immunoprecipitation (ChIP) of p300/CBP has been used to successfully predict novel enhancers (e.g. [2,3]). Although p300/CBP can interact with most transcription factors in vitro, it is not known whether p300/CBP preferentially associates with some factors in vivo. Here, we use the early Drosophila melanogaster embryo to compare the genomic distribution of p300/CBP with 40 transcription factors involved in embryonic patterning and cell differentiation. Drosophila has one CBP/p300 ortholog, also known as nejire [4]. Chromatin binding of Drosophila CBP has recently been used to identify novel enhancers that are active in embryos [5]. By comparing CBP occupancy at different stages of Drosophila development, around 14 000 CBP peaks were identified that may represent regulatory DNA sequences. CBP binding was found to correlate with active chromatin, including histone acetylation and H3K4 methylation [5]. Drosophila CBP has been implicated in Hedgheog, Wnt, and TGF-ß signaling, as well as in dorsal-ventral patterning of early embryos [reviewed in 6]. The loss of function allele nejire3 (nej3) is cell-lethal, whereas the hypomorphic nej1 allele reduces CBP expression approximately two-fold, and causes embryonic patterning phenotypes [7–11]. These can be attributed to reduced signaling by the TGF-ß molecule Decapentaplegic (Dpp), in turn caused by impaired expression of the Tolloid (Tld) protease in nej1 embryos [10]. In the absence of Tld, the Short-gastrulation (Sog) inhibitor prevents the Dpp ligand from signaling through its receptors. Interestingly, the acetyltranferase activity of CBP appears dispensable for tld gene activation [9]. Embryonic dorsal-ventral patterning is controlled by an intra- nuclear concentration gradient of Dorsal, a Rel-family tran- scription factor related to NF-kB. Over 50 Dorsal target genes are known, constituting one of the best understood gene regulatory networks in animal development (reviewed by [12]). Dorsal enters ventral nuclei at high levels in response to signaling by the transmembrane receptor Toll. The Toll ligand Spa¨tzle is present in the periviteline space surrounding the embryo, at high concentrations on the ventral side and progressively lower concentration in lateral and dorsal regions [13]. A proteolytic cascade is responsible for generating active Spa¨tzle ligand, and mutations that disrupt this cascade, such as in the Pipe sulfotransferase and in the protease Gastrulation defective (gd), result in absence of Toll signaling and failure of Dorsal to enter the nucleus (reviewed in [14]). In such mutants, the entire embryo is converted to presumptive dorsal ectoderm PLoS Genetics | www.plosgenetics.org 1 June 2012 | Volume 8 | Issue 6 | e1002769