Pairs of Adjacent Conserved Noncoding Elements Separated by Conserved Genomic Distances Act as Cis-Regulatory Units

Comparative genomic studies have identified thousands of conserved noncoding elements (CNEs) in the mammalian genome, many of which have been reported to exert cis-regulatory activity. We analyzed ∼5,500 pairs of adjacent CNEs in the human genome and found that despite divergence at the nucleotide sequence level, the inter-CNE distances of the pairs are under strong evolutionary constraint, with inter-CNE sequences featuring significantly lower transposon densities than expected. Further, we show that different degrees of conservation of the inter-CNE distance are associated with distinct cis-regulatory functions at the CNEs. Specifically, the CNEs in pairs with conserved and mildly contracted inter-CNE sequences are the most likely to represent active or poised enhancers. In contrast, CNEs in pairs with extremely contracted or expanded inter-CNE sequences are associated with no cis-regulatory activity. Furthermore, we observed that functional CNEs in a pair have very similar epigenetic profiles, hinting at a functional relationship between them. Taken together, our results support the existence of epistatic interactions between adjacent CNEs that are distance-sensitive and disrupted by transposon insertions and deletions, and contribute to our understanding of the selective forces acting on cis-regulatory elements, which are crucial for elucidating the molecular mechanisms underlying adaptive evolution and human genetic diseases.