Assessing conservation (enrichment) of binding sites across species. a. The best instance of the binding site being studied (one of the four in Figure 3) is identified upstream of each gene in the genome. b. The binding site is then permuted 100 times to generate 100 random sites with the same base composition as the real binding site. c. The best instance of each permuted site in the regions upstream of each gene is located in the same genome. d. The distribution of scores for the real site is compared to that of the permuted sites and score at the top one percentile of permuted sites is used as a cutoff for identifying high confidence hits for the real site. e. A p-value is computed representing the statistical significance of the overlap between genes whose upstream regions have hits for the real site and the list of genes in the process expected to be regulated by the site. f. The enrichment analysis (steps a-e) is repeated in all genomes and enrichment p-values are compared across species.