To explore the possible uniqueness of noncoding ultraconserved elements, we identified a large number of human-rodent conserved elements that are under similar evolutionary constraint as regions containing ultraconservation. We compared the entire set of these elements, the majority of which lack perfectly conserved regions of ≥200 bp (that is, ultraconserved regions), to the small subset that overlapped ultraconserved elements in regard to their degree of constraint in other mammalian species and their enrichment near genes with certain functions. Moreover, we compared the ability of a genome-wide set of non-exonic ultraconserved elements and more than 200 extremely constrained human-rodent elements to drive tissue-specific expression in vivo, a property that has previously been observed to be a predominant function associated with noncoding ultraconservation^8–12.

In an initial comparative genomic assessment of ultraconservation, we found substitutions in 79% of these elements in other mammalian species (Fig. 1a), indicating that their absolute conservation between human and rodents is at least partially a matter of ascertainment bias, rather than absolute intolerance of nucleotide substitutions. This finding is consistent with the limited overlap among human-rodent, human-mouse-dog and human-chicken ultraconserved elements⁴ and supports the possibility that they represent only a subset of a larger group of elements with similar properties. We therefore used a statistical approach (Gumby¹³) with scoring parameters optimized through multiple genome-wide scans (Supplementary Methods and Supplementary Table 1 online) to identify a more general set of noncoding elements marked by extreme human-mouse-rat constraint. Conservation scores for individual elements were derived from log-transformed Gumby P values and reflect their length and constraint relative to the local neutral substitution rate. When we compared these elements to the distribution of non-exonic ultraconserved elements, we found that the constraint scores of regions with ultraconservation were distributed over a wide range, and that a much larger number of elements seem similarly constrained (Fig. 1b). We identified a population of 2,614 human-rodent constrained elements that overlap or include 234 (91%) of all 256 non-exonic ultraconserved elements (Supplementary Table 2 online). To quantify the extreme conservation of these elements independently from the scoring scheme used for their identification, we determined their branch length and rejected substitution counts⁶ in human, rodents and five additional mammalian species (Supplementary Fig. 1 online). We found that extremely constrained elements that contain or do not contain regions of ultraconservation have similar characteristics by these two widely used comparative genomic measures, indicating their ‘ultra-like’ conservation. Although an order of magnitude more numerous than non-exonic ultraconserved elements and located in the vicinity of a fivefold larger number of genes, the highly constrained noncoding regions identified here are enriched near genes belonging to a small subset of functional categories. As for ultraconserved elements, these functions include transcriptional regulation and development² and, in particular, development of the nervous system (Fig. 1c; see Supplementary Table 3 online for a list of all significantly enriched functions). In summary, comparative analyses, as well as the genome-wide distributions, suggest that ultraconservation merely defines a subset of genome regions that are under similar constraint and that are enriched near genes with similar functional properties.

To test whether such apparent equivalence at the sequence level is also associated with similar functional properties, we focused on transcriptional enhancer activity during embryonic development. We used a transgenic mouse assay to determine the embryonic enhancer activities of 155 human genome regions that contain non-exonic ultraconserved elements and combined these data with a previously reported smaller dataset¹⁰ to establish a genome-wide compendium of their enhancer activities. A total of 231 transgenic assays was considered, in which the tested human genome fragments included 245 of all 256 non-exonic ultraconserved elements (Supplementary Table 4 online). We found that half (115/231) of the ultraconserved regions drove reproducible reporter gene expression in various tissues of the developing mouse embryo, often in a tight spatially restricted manner and with subregions of the central nervous system among the most frequently targeted structures (Fig. 2a).

To determine whether such an enrichment in embryonic enhancers is specifically associated with the presence of ultraconserved regions, we also tested the enhancer activities of 206 extremely constrained human-rodent noncoding sequences that lack regions of ultraconservation. Of note, these regions were selected blind to evolutionary conservation depth in nonmammalian species, and purely based on their human-rodent constraint scores. Using identical scoring criteria as before, we found that 102 of these 206 elements (50%) acted as tissue-specific enhancers at embryonic day 11.5 (Supplementary Table 5 online). We did not observe significant differences between the ultraconserved and non-ultraconserved elements regarding the overall distribution of the targeted anatomical structures (Fig. 2a). We observed multiple cases of ultraconserved and non-ultraconserved elements driving virtually identical patterns when scrutinized at higher resolution (Fig. 2b), as well as dozens of patterns driven by non-ultraconserved elements for which no counterpart was found among ultraconserved elements (Supplementary Fig. 2 online). Our findings indicate that extreme human-rodent constraint identifies genome regions that are, in their entirety, highly enriched in embryonic enhancers, whereas the ultraconserved subset within this population was neither found to be enriched in enhancers targeting specific tissues nor found to be generally more enriched in developmental enhancers.

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