Characterization of the HIF-binding motif. (A-B) HIF-1- and HIF-2-binding regions were examined for commonly occurring novel motifs using the Gibbs Motif Sampler module of CisGenome. Enrichment of motifs identified in this way was then examined by determining the relative occurrence of each motif in HIF-binding regions compared with a control set of regions (r1), of phylogenetically conserved motifs within phylogenetically conserved portions of HIF-binding regions compared with control regions (r2), and of phylogenetically conserved motifs within all portions of the HIF-1 binding and control regions (r3). The sequence logos40 for motifs with all 3 enrichment ratios greater than 2 are shown. The height of each letter is proportionate to its frequency, with the most frequent at the top. The height of each stack is then adjusted to signify the information content of the sequences at that position (measured in bits). (C) HIF-1 and (D) HIF-2-binding sites containing a single RCGTG motif were analyzed for sequence conservation beyond this core motif. Sequences were extended for 100 bp upstream and downstream of the RCGTG motif and aligned. At each position, the observed base frequency was compared with the expected using a χ2 test. The relative occurrence of each nucleotide at each position is indicated as a percentage of the total by the letter height. Positions showing nonrandom base frequency (P < .01 after Bonferroni correction) are highlighted in gray.
