ChIP-Chip in Xenopus Figure 2. Promoter microarray H3K4me3 ChIP-chip. (A) Part of scaffold_1 is visualized using the UCSC Genome Browser. The four H3K4me3 enriched regions; Fas associated factor 1 (faf1), cyclin-dependent kinase inhibitor 2C (cdkn2c), ring finger protein 11 (rnf11) and an unknown protein are preserved in the three independent experiments. (B) Correlation of the genome-wide tiling and promoter microarray design ChIP-chip experiments; Pearson correlation r = 0.72, 0.70 and 0.94 respectively (p,2.2 * 10216). doi:10.1371/journal.pone.0008820.g002 experiments using the promoter microarray design (r = 0.72 and r = 0.70 respectively, p,2.2 * 10216). The correlation between the promoter microarray experiments was r = 0.94 (p,2.2 * 10216). This shows that ChIP-chip experiments based on genome-wide tiling or a promoter microarray design are highly reproducible and that the biological variance between different batches of embryos is relatively low. Analyses of H3K4me3 Triplicate We determined the profile of the H3K4me3 signal over the 59 ends of X. tropicalis experimentally validated genes (Xtev [9]). H3K4me3 is predominantly found at the transcription start site (TSS; Figure 3A) as expected. The distribution of the H3K4me3 mark over Joint Genome Institute FilteredModels genes shows a similar profile (Figure S1). We determined the number of H3K4me3-enriched regions using TileMap [11]. In total 10,179 H3K4me3 positive regions were detected. To validate this peak set, randomly selected targets were tested in ChIP-qPCR using three new biological ChIP samples (Figure 3B). Using two negative loci on scaffold 1 (scaffold_1:6458583-6458633) and scaffold 919 (scaf- fold_919:126357-126407) to determine the genomic background, 16 out of 17 regions were enriched more than 2.5-fold (Figure S2, FDR ,0.06). The H3K4me3 peak set shows a high degree of overlap with 59 ends of Xtev genes (within 1 kb of the TSS, p,10225; Figure 3C). Up to 89% of the H3K4me3-enriched regions (9,031 of 10,179) are within 1kb of genes. A list of all H3K4me3-enriched regions and associated Xtev, JGI FilteredMo- dels and RefSeq genes is supplied as a supplemental table (Table S1). The peaks of genome-wide ChIP-chip were compared to ChIP- seq of H3K4me3 of gastrula-stage embryos [9]. 88% of the peaks determined by ChIP-chip are also detected by ChIP-seq (p,10225; Figure 3D). These results show that H3K4me3-based ChIP-chip experiments are highly reproducible and that both experimental platforms identify an almost identical collection of H3K4me3- enriched genomic sequences. Moreover, integration of ChIP and EST data allows linking of ‘orphan’ H3K4me3 peaks to gene models on different genomic scaffolds in cases where promoter and coding regions were placed on different sequence contigs during genome assembly (schematic overview, Figure 3E). Both 59 and 39 exons can be located on different scaffolds. To test this, using the EST information, RT-PCR primers were designed that align to different scaffolds to amplify gastrula X. tropicalis cDNA. Four random chosen examples were validated by this approach as transcription units that go together but are assembled to different scaffolds (Figure 3F). Importantly, genuine 59 ends of genes can be identified by their enrichment for H3K4me3, even if the 59 end and the gene body are located on different scaffolds. In total, for 991 loci we detected H3K4me3-enrichment and EST annotation to two scaffolds (Table S2). We also tested the enrichment for H3K9ac for a limited number of genomic regions and found that 34 out of 43 (79%) H3K4me3 regions are also enriched for H3K9ac (data not shown). These results show that the genome-wide ChIP-chip design is a useful tool to interrogate gene regulatory regions in Xenopus. TBP-Enriched Regions To examine the application value of our promoter microarray design for transcription factors we studied TATA-binding protein (TBP), a key initiation factor that is known to bind to promoter regions. T7 amplified TBP ChIP and input DNA was hybridized to the promoter microarray. Many TBP-enriched regions colocalize with H3K4me3 enriched regions as is seen for example for tubulin alpha 1c (Figure 4A; left panel). TBP is also found at H3K4me3-positive regions that lack gene annotation. For example, the promoter region of an open reading frame with no PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8820