Metrics of sequence constraint overlook regulatory sequences in an exhaustive analysis at phox2b

Abstract

Despite its recognized utility, the extent to which evolutionary sequence conservation-based approaches may systematically overlook functional noncoding sequences remains unclear. We have tiled across sequence encompassing the zebrafish phox2b gene, ultimately evaluating 48 amplicons corresponding to all noncoding sequences therein for enhancer activity in zebrafish. Post hoc analyses of this interval utilizing five commonly used measures of evolutionary constraint (AVID, MLAGAN, SLAGAN, phastCons, WebMCS) demonstrate that each systematically overlooks 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. However, we were able to discriminate functional from nonfunctional sequences based upon GC composition and identified position weight matrices (PWM), demonstrating that, in at least one case, deleting sequences containing a subset of these PWMs from one identified regulatory element abrogated its regulatory function. Collectively, these data demonstrate that the noncoding functional component of vertebrate genomes may far exceed estimates predicated on evolutionary constraint.

Conserved regulatory architecture underlies parallel genetic changes and convergent phenotypic evolution

“…We were concerned that a search guided only by sequence conservation might generate an incomplete picture of the D. virilis svb cis-regulatory architecture (21). For example, new enhancers may have evolved in the D. virilis lineage.…”

Enhancer turnover and conserved regulatory function in vertebrate evolution

“…Can zebrafish transgenesis be used to study mammalian enhancers that are not conserved in teleosts? The answer to this question is yes, as it has been found in some experiments that mammalian enhancers without obvious teleost orthologues can nevertheless drive specific transgene expression in zebrafish embryos [39,43,44], indicating that at least part of the underlying cis-(TFBS composition or order) as well as trans-(transcription factors) elements are conserved in mammals and teleosts in many diverged enhancers. It is likely that many of these non-conserved enhancers do possess orthologues that cannot be readily identified owing to the turnover of neutral and redundant nucleotide sites within the enhancers in each lineage (TFBS turnover).…”

“…There are, however, other attractive vertebrate models that can be used to study enhancers by transgenesis, including the teleost zebrafish, which allows for quicker and cheaper transgenic assays [37]. Mammals and zebrafish share thousands of CNEs that can work as enhancers, and zebrafish transgenesis plays an important role in the analysis of such conserved genetic elements [14,15,[38][39][40][41].…”

Integration of ChIP-seq and machine learning reveals enhancers and a predictive regulatory sequence vocabulary in melanocytes

“…We used an established pipeline for analyzing putative enhancers in zebrafish (Fisher et al 2006a,b;McGaughey et al 2008;Prasad et al 2011), which we have previously used to analyze melanocyte regulatory elements at Sox10 (Antonellis et al 2008) and GPNMB . The 10 putative enhancers tested were chosen at random from the 50 analyzed in vitro as described above.…”