Figure 2.
Binding profiles and their effects on statistical tests. (a) ChIP-seq data sets of several regulatory proteins show that the majority of binding events lie well outside the proximal promoter, both for sequence-specific transcription factors (SRF and NRSF, ref. 8; Stat3, ref. 43) and a general enhancer-associated protein (p300, refs. 33,43). Cell type is given in parentheses: H, human; M, mouse. (b) When not restricted to proximal promoters, the gene-based hypergeometric test (red) generates false positive enriched terms, especially at the size range of 1,000–50,000 input regions typical of a ChIP-seq set. Negligible false positive enrichment was observed for the region-based binomial test (blue). For each set size, we generated 1,000 random input sets in which each base pair in the human genome was equally likely to be included in each set, avoiding assembly gaps. We calculated all GO term enrichments for both hypergeometric and binomial tests using GREAT’s 5+1 kb basal promoter and up to 1 Mb extension association rule (see Results). Plotted is the average number of terms artificially significant at a threshold of 0.05 after application of the conservative Bonferroni correction. (c) GO enrichment P values using the genomic region-based binomial (x axis) and gene-based hypergeometric (y axis) tests on the SRF data8 with GREAT’s 5+1 kb basal promoter and up to 1 Mb extension association rule (see Results). b1 through b10 denote the top ten most enriched terms when we used the binomial test. h1 through h10 denote the top ten most enriched terms when we used the hypergeometric test. Terms significant by both tests (B ∩ H) provide specific and accurate annotations supported by multiple genes and binding events (Table 3). Terms significant by only the hypergeometric test (H\B) are general and often associated with genes of large regulatory domains, whereas terms significant by only the binomial test (B\H) cluster four to six genomic regions near only one or two genes annotated with the term (Supplementary Table 46).