Functional evaluation of noncoding regulatory control provides novel insights into their constraint, distribution, and overlapping functions

Functional non-coding DNA has important roles in the temporal, spatial, and quantitative expression of genes in development, disease, homeostasis, and inter-species variation. One of the most powerful tools to detect functional non-coding DNA is evolutionary sequence conservation-based metrics. However, the extent to which this approach may systematically overlook functional non-coding sequences remains unclear. To address this, we have tiled sequence across the zebrafish phox2b gene, evaluating the sequence enhancer activity in zebrafish.Post hoc analyses of this interval utilizing multiple measures of evolutionary constraint demonstrate that all systematically overlook regulatory sequences. These established algorithms detected only 29-61% of our identified regulatory elements, consistent with the suggestion that many regulatory sequences may not be readily detected by metrics of sequence constraint. Collectively, these data demonstrate that the non-coding functional component of vertebrate genomes may far exceed estimates predicated on evolutionary constraint.To determine whether these conclusions could be extended beyond the zebrafish phox2b locus, we explored these observations in vivo at the human PHOX2B locus. Analysis of human sequences conserved with previously identified zebrafish phox2b regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control and provides evidence that non-conserved regulatory elements are more likely to be positioned proximal to gene promoters. Novel analyses of extant data from the ENCODE project on noncoding DNA regions revealed that these trends were present genome-wide.Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Here, we examine the efficacy of injecting pooled constructs to increase the effective rate that large numbers of regulatory sequences can be assayed and reduce subjectivity of transgene study by restricting analysis to stably integrated transgenic zebrafish at the human ASCL1 locus. Using this technique we find numerous transgenepositive zebrafish, transmitting one or more independent constructs that display ASCL1-like regulatory control. These data demonstrate the power of this system and its potential for higher throughput application.