ChIP-Chip Designs to Interrogate the Genome of Xenopus Embryos for Transcription Factor Binding and Epigenetic Regulation Robert C. Akkers1., Simon J. van Heeringen1., J. Robert Manak2,3, Roland D. Green2, Hendrik G. Stunnenberg1, Gert Jan C. Veenstra1* 1 Department of Molecular Biology, Faculty of Science, Radboud University Nijmegen, Nijmegen, The Netherlands, 2 Roche Nimblegen, Inc., Madison Wisconsin, United States of America, 3 Department of Biology, University of Iowa, Iowa City, Iowa, United States of America Abstract Background: Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos. Principal Findings: Here we report on two genome tile path ChIP-chip designs for interrogating the Xenopus tropicalis genome. In particular, a whole-genome microarray design was used to identify active promoters by close proximity to histone H3 lysine 4 trimethylation. A second microarray design features these experimentally derived promoter regions in addition to currently annotated 59 ends of genes. These regions truly represent promoters as shown by binding of TBP, a key transcription initiation factor. Conclusions: A whole-genome and a promoter tile path microarray design was developed. Both designs can be used to study epigenetic phenomena and transcription factor binding in developing Xenopus embryos. Citation: Akkers RC, van Heeringen SJ, Manak JR, Green RD, Stunnenberg HG, et al. (2010) ChIP-Chip Designs to Interrogate the Genome of Xenopus Embryos for Transcription Factor Binding and Epigenetic Regulation. PLoS ONE 5(1): e8820. doi:10.1371/journal.pone.0008820 Editor: Ferenc Mueller, University of Birmingham, United Kingdom Received November 30, 2009; Accepted December 30, 2009; Published January 21, 2010 Copyright: 2010 Akkers 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 United States National Institutes of Health (grant 5 R01 HD054356, www.nih.gov) and the Netherlands Organization of Scientific Research (NWO-ALW VIDI grant 864.03.002, www.nwo.nl) to GJCV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Roche NimbleGen, Inc. contributed reagents and expertise for the experiments presented in this paper and may benefit financially from publication of the results. JRM and RDG were employed by Roche NimbleGen, Inc. * E-mail: g.veenstra@ncmls.ru.nl . These authors contributed equally to this work. Introduction Chromatin immunoprecipitation combined with either micro- array hybridization (ChIP-chip) or sequencing (ChIP-seq) allows to determine genomic association of DNA binding proteins and to analyze epigenetic regulation [1,2,3]. Active transcription coincides with epigenetic features like the presence of the histone H3 lysine 4 trimethylation (H3K4me3) and acetylation of lysine 9 of histone H3 (H3K9ac) at the 59 end of transcriptionally active genes [4,5,6]. In the context of development little is known about epigenetic marks in embryos using ChIP profiling. H3K4me3 ChIP-chip using zebra- fish embryos identified actively transcribed embryonic genes [7]. The latter mark was also detected in Drosophila embryos at promoter regions [8]. ChIP sequencing of H3K4me3 and H3K27me3, an inactive histone mark, was used to enhance 59 gene annotation in Xenopus and to analyze spatial regulation of gene expression during gastrulation [9]. The two marks only appear after the onset of transcription at the mid-blastula transition with a hierarchy in deposition of H3K4me3 and H3K27me3, respectively. The challenge in the near future is to elucidate epigenetic transitions and transcription networks that underlie early verte- brate development, the analysis of which will be facilitated by genome binding site analyses using either ChIP-chip or ChIP-seq. Genome tiling arrays are most cost-efficient if a dedicated design covering the relevant regulatory regions is used. Such a design can be made on the basis of annotation, or alternatively using experimental data of histone modifications like H3K4me3 that identify these regulatory regions. Here we present results obtained using a five array whole-genome tiling X. tropicalis design and a promoter microarray design based on chromatin decorated with H3K4me3. The data show that both microarray designs are suitable for studies of histone modifications and transcription factor binding events in early Xenopus embryogenesis. Results Genome-Wide ChIP-Chip Using X. tropicalis Gastrula Embryos To explore epigenetic features of gastrula-stage Xenopus tropicalis embryos, chromatin was harvested (Nieuwkoop-Faber stage 11– 12) and immunoprecipitated using H3K4me3 antibodies. The purified DNA of H3K4me3-ChIP was amplified using a T7 PLoS ONE | www.plosone.org 1 January 2010 | Volume 5 | Issue 1 | e8820