Potential bidirectional promoters. (A) Distribution of distances between paired proximal TSSs in opposite strands. The remarkable number of loci (total instances more than 600) with paired divergent TSSs at a distance of 60–160 bp suggests an abundance of potential bidirectional promoters. This range (60–160 bp) is consistent with the observation that motif sequences (candidate cis-elements) are typically enriched ∼50 bp upstream of the TSSs. In reality, many motifs, including novel ones, were observed frequently in those potential regions. (B) Example of candidate bidirectional promoter region. Two expressed representative outron-TSSs in the opposite strands were separated by 115 bp. A novel motif (TTCTCTCGGA), named “BiSL1-B” in Figure 5 (respectively, its reverse complement), was identified ∼50 bp (∼70 bp) upstream of the plus (minus) strand representative TSS. This is in accordance with the enrichment of the motif ∼30–50 bp (70–100 bp) upstream of the plus (minus) strand TSS obtained from our genome-wide analysis (see BiSL1-B in Fig. 5). Of note, the motif was highly conserved among the six allied species. The two outrons from the representative TSSs to their nearest downstream SL1 sites were different in length, demonstrating the difficulty of estimating outrons and TSSs solely from SL1 sites and the genomic sequence. (C) Another example of a bidirectional promoter. Two common motifs (SP1 and CpG motif 4) were highly conserved between two expressed outron-TSSs. Multiple highly expressed and isolated TSSs were seen in both strands, and the wide distribution of the occurrences of these motifs may account for the multiple outron-TSSs (SP1 in Fig. 4B and CpG motif 4 in Supplemental Fig. S3). Many more examples of regions harboring conserved motifs between expressed outron-TSSs were seen (Supplemental Fig. S9), which could function as potential bidirectional promoters (696 candidates in Supplemental Table S4). Expression levels between the pairs of genes have a very small similarity (Pearson's correlation coefficient <0.066).
