Integrated genome analysis suggests that most conserved non-coding sequences are regulatory factor binding sites

More than 98% of a typical vertebrate genome does not code for proteins. Although non-coding regions are sprinkled with short (<200 bp) islands of evolutionarily conserved sequences, the function of most of these unannotated conserved islands remains unknown. One possibility is that unannotated conserved islands could encode non-coding RNAs (ncRNAs); alternatively, unannotated conserved islands could serve as promoter-distal regulatory factor binding sites (RFBSs) like enhancers. Here we assess these possibilities by comparing unannotated conserved islands in the human and mouse genomes to transcribed regions and to RFBSs, relying on a detailed case study of one human and one mouse cell type. We define transcribed regions by applying a novel transcript-calling algorithm to RNA-Seq data obtained from total cellular RNA, and we define RFBSs using ChIP-Seq and DNAse-hypersensitivity assays. We find that unannotated conserved islands are four times more likely to coincide with RFBSs than with unannotated ncRNAs. Thousands of conserved RFBSs can be categorized as insulators based on the presence of CTCF or as enhancers based on the presence of p300/CBP and H3K4me1. While many unannotated conserved RFBSs are transcriptionally active to some extent, the transcripts produced tend to be unspliced, non-polyadenylated and expressed at levels 10 to 100-fold lower than annotated coding or ncRNAs. Extending these findings across multiple cell types and tissues, we propose that most conserved non-coding genomic DNA in vertebrate genomes corresponds to promoter-distal regulatory elements.