Data_Sheet_2_Functional Conservation of Divergent p63-Bound cis-Regulatory Elements.PDF

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.

转录因子p63是脊椎动物外胚层发育的核心调控因子，其调控范围涵盖表皮、四肢及颅面组织的发育过程。本研究围绕斑马鱼与人类的p63结合位点（binding sites，BSs）的进化保守性展开探究。首先，通过比对人类角质形成细胞与斑马鱼胚胎的染色质免疫共沉淀测序（ChIP-seq）数据，分析p63结合位点的序列保守性，结果显示二者的序列保守性极低。随后，我们比较了两个物种中p63调控的基因调控网络，发现二者存在高度重叠，这表明尽管序列存在差异且二者进化距离较远，但其功能保守性仍处于较高水平。最后，我们通过斑马鱼胚胎的转基因报告基因实验，对一组位于表皮发育相关基因附近的斑马鱼与人类等效p63结合位点进行了功能验证。人类与斑马鱼的结合位点均可驱动报告基因在p63表达结构域相关的多种常见组织中表达。综上，我们得出结论：尽管p63结合的调控元件存在高度序列差异，但p63所调控的基因调控网络在脊椎动物中仍保持高度保守性。