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. 2009 Feb 12;457(7231):854-8.
doi: 10.1038/nature07730.

ChIP-seq accurately predicts tissue-specific activity of enhancers

Axel Visel  1 , Matthew J Blow, Zirong Li, Tao Zhang, Jennifer A Akiyama, Amy Holt, Ingrid Plajzer-Frick, Malak Shoukry, Crystal Wright, Feng Chen, Veena Afzal, Bing Ren, Edward M Rubin, Len A Pennacchio
Affiliations

ChIP-seq accurately predicts tissue-specific activity of enhancers

Axel Visel et al. Nature. .

Abstract

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here we present the results of chromatin immunoprecipitation with the enhancer-associated protein p300 followed by massively parallel sequencing, and map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases demonstrated reproducible enhancer activity in the tissues that were predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities, and suggest that such data sets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

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Figures

Figure 1
Figure 1. Tissue dissection boundaries, overview of the ChIP-seq approach and summary of p300 results
Tissue dissection boundaries are indicated in a representative unstained E11.5 mouse embryo. For each sample, tissue was pooled from more than 150 embryos and ChIP-seq was performed with a p300-antibody. Reads obtained for each of the three tissues that unambiguously aligned to the reference mouse genome were used to define peaks (FDR < 0.01). A more comprehensive overview of sequencing and mapping results is provided in Supplementary Table 1. fb, forebrain; li, limb; mb, midbrain.
Figure 2
Figure 2. p300 binding accurately predicts enhancers and their tissue-specific activity patterns
Bar height indicates the frequency of in vivo enhancers (reproducible at E11.5) that are active in any tissue (grey bars and coloured bars) and the fraction of enhancers in which the pattern includes or is restricted to reproducible forebrain (blue bars), midbrain (red bars) or limb activity (green bars). In each case, candidate elements predicted by p300 peaks in forebrain, midbrain or limb were compared to the frequency of the respective pattern in a background set of 528 previously tested sequences identified through extreme evolutionary conservation (combined data sets from refs and 11). The component activities of elements predicted to be active in several tissues were counted separately. *P < 0.00005, Fisher's exact test, one-tailed.
Figure 3
Figure 3. Examples of successful prediction of in vivo enhancers by p300 binding in embryonic tissues
a, Coverage by extended p300 reads in forebrain (blue), midbrain (red) and limb (green). Asterisks indicate significant (FDR < 0.01) p300 enrichment in chromatin isolated from the respective tissue. Multispecies vertebrate conservation plots (black) were obtained from the UCSC genome browser. Grey boxes correspond to candidate enhancer regions. Numbers at the right indicate overlapping extended reads. b, Representative LacZ-stained embryos with in vivo enhancer activity at E11.5. Reproducible staining in forebrain, midbrain and limb is indicated by arrows. Numbers show the reproducibility of LacZ reporter staining. Additional embryos obtained with each construct and genomic coordinates are available using the enhancer ID in the bottom portion of a at the Vista Enhancer Browser.
Figure 4
Figure 4. In all tissues examined, p300 is enriched at highly conserved non-coding regions
We used a genome-wide set of 50,000 extremely constrained non-coding sequences identified in human-mouse-rat genome alignments to assess the correlation between p300 enrichment and non-coding sequence conservation. Even though only subsets of the constrained non-coding elements are expected to be active enhancers in any given embryonic tissue, we observe strong enrichment in p300 binding in all three tissues compared to input DNA. *P < 1 × 10−100, Fisher's exact test. Relative p300 coverage near random sites and internal exons is shown for comparison. Brown bars indicate the median sizes of conserved elements or exons (124 bp in both cases). For further details, see Supplementary Table 7.
Figure 5
Figure 5. p300 peaks are enriched near genes that are expressed in the same tissue
We compared the genome-wide distribution of p300-enriched regions in forebrain tissue at E11.5 with microarray expression data for forebrain at the same stage. Eight-hundred-and-eighty-five genes were forebrain-specifically overexpressed, and 495 genes were underexpressed relative to whole embryo RNA at the selected thresholds. Promoters (defined as 1 kb upstream and downstream of transcription start sites) were excluded from the analysis. Blue bars denote comparison to 2,435 forebrain-derived peaks, grey bars denote comparison to 2,435 random sites. a, Ten-kilobase bins up to 91 kb away from forebrain-overexpressed genes were significantly enriched in forebrain p300 peaks. b, No peak enrichment was observed for forebrain-underexpressed genes. Error bars indicate the 90% confidence interval on the basis of 1,000 iterations of randomized distribution. *P < 0.05, **P < 0.01, both one-tailed.
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