ChIP-Chip in Xenopus Figure 3. Analyses of H3K4me3 triplicate. (A) Distributions of H3K4me3 at annotated Xtev genes. (B) ChIP-qPCR validation of randomly selected enriched H3K4me3 regions on three new generated biological replicates. 16 out of 17 of H3K4me3 enriched regions are validated as true positives with an average enrichment .2.5-fold over background (genomic locus on scaffold_1:6458583-6458633). Asterisk indicates a locus with ,2.5-fold enrichment (experimental FDR ,0.06). Error bars represent the SEM of three independent ChIP experiments. (C) Overlap of H3K4me3-enriched regions and Xtev genes (p,10225). (D) Overlap of H3K4me3 ChIP-chip and ChIP-seq peaks. Of the peaks found with ChIP-chip, 88% are also detected with ChIP-seq [9] (p,10225). (E) ‘Orphan’ H3K4me3 peaks linking different scaffolds. Schematic overview of orphan linkage of two scaffolds using H3K4me3 signals and the EST database. The start site is determined by the H3K4me3 peak and the transcription unit combined with the EST information of the other scaffold. (F) RT-PCR validation of ESTs linking two scaffolds using primers that align to two different scaffolds: scaffold 649 and 145 (EST AL847047), scaffold 675 and 1278 (EST DC172702), scaffold 716 and 1312 (EST CX475139) and scaffold 1393 and 307 (uxs1). cDNA used for validation was generated from RNA isolated from stage 11–12 embryos. doi:10.1371/journal.pone.0008820.g003 known function was bound by TBP in the presence of H3K4me3 (Figure 4A; right panel). We determined the correlation of H3K4me3 peak regions and TBP to reveal the relation between epigenetic marks and transcription initiation factors (Figure 4B). Although correlation is less than observed for the H3K4me3 replicates, many H3K4me3-positive loci are bound by TBP and the correlation is highly significant (r = 0.40, p,2.2 * 10-16). Discussion Our results present ChIP microarray designs for X. tropicalis. So far gene regulatory networks and epigenetic regulation during vertebrate embryogenesis have not extensively been studied using genome-wide binding analysis. By contrast, many epigenetic profiles have been established for human cells in culture [12], providing a solid conceptual framework to analyze epigenetic regulation in other systems. The presence of H3K4me3 at the 59 end of transcribed genes is consistent in many experimental systems [5,7,8,9,13,14,15,16,17,18]. The number of H3K4me3-enriched regions in Xenopus gastrula embryos based on ChIP-chip or ChIP-seq [9] is comparable (88% overlap). For Xenopus, a promoter microarray design based on current Joint Genome Institute gene annotation would lose valuable information regarding the TSS, since a large number of H3K4me3- Figure 4. TBP-enrichment at loci of promoter microarray design. (A) Independent genes visualized in the UCSC Genome Browser for H3K4me3 (green) and TBP (pink). Left panel represents tubulin, alpha 1c (tuba1c) and the right panel human chromosome 12 open reading frame 41 (c12orf41). (B) Correlation of H3K4me3 ChIP-chip and TBP promoter ChIP-chip. Spearman rank r = 0.40 (p,2.2 * 10216). doi:10.1371/journal.pone.0008820.g004 PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8820