Uncovering p63 targets involved in hereditary malformations

Abstract

p63 transcription factor plays an important role during epidermis and neural crest derivatives formation. Mutations affecting its coding sequence or its target genes lead to the development of malformations mainly characterized by cleft palate, ectodermal dysplasia and limb malformation. To date, the search of genetic events underlying these malformations has been mainly focused on exonic regions. However, impairment on the transcriptional regulation of genes related to these phenotypes might cause similar diseases. The major problem of the studies focused on transcription factors binding sites is the difficulty to associate a certain regulatory region with its target gene. In general, this association is solely established having into account the proximity between these elements, which might lead to wrong assignments and waste of effort and resources in deeply studying the incorrect genes. To overcome this problem, we designed an approach based on binding sites and chromatin 3D interactions, which will allow us to properly associate transcriptional regulatory elements with their target genes. First, we performed a ChIP-seq in zebrafish to find p63 binding sites, and then 4C-seq experiments centered in some of these putative regulatory regions to uncover their regulatory landscape. Finally, we moved to genome-wide scale by means of a novel technique, the HiChIP, which allowed us to obtain a global map of contacts from every active promoter and assign the most probable target gene to each binding site. The integration of these data will provide us a list of genes contacting p63-binding regions, which could potentially belong to the p63 genetic network and be involved in p63-related diseases.