Functional Conservation of Divergent p63-Bound cis-Regulatory Elements.

Lourdes Gallardo-Fuentes,José M Santos-Pereira,Juan J Tena

doi:10.3389/fgene.2020.00339

Abstract

The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence.

Highlights

Mutations affecting the activity of cis-regulatory sequences are thought to be the most prevalent cause of phenotypic divergence in animal evolution (Carroll, 2008; Stern and Orgogozo, 2008)
We have explored the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human
De novo motif discovery analysis identified a p53-like binding sequence that was virtually the same in both species despite their high evolutionary divergence (Figure 1A). These motifs represent incomplete versions of the p63 motif described for human keratinocytes (Kouwenhoven et al, 2010), which is enriched in the p63 peaks called in both species (Figure 1A)

Summary

INTRODUCTION

Mutations affecting the activity of cis-regulatory sequences are thought to be the most prevalent cause of phenotypic divergence in animal evolution (Carroll, 2008; Stern and Orgogozo, 2008). We have first analyzed the sequence conservation of p63 BSs of human keratinocytes and zebrafish embryos, showing a very poor conservation; about one third of the BS-associated genes are orthologous between these two species, suggesting that the p63 gene regulatory network is conserved. We validated this functional conservation using transgenic reporter assays in zebrafish and found that diverged enhancers with species-specific p63 binding from both species drove similar expression patterns. Our data suggest that the gene regulatory network regulated by p63 is conserved across vertebrates and despite the fact that CRE sequences diverged along species evolution

MATERIALS AND METHODS

RESULTS

DISCUSSION