697 Epidermal tissue differentiation requires the uncharacterized protein RED1

Abstract

The human genome encodes >4,000 uncharacterized open reading frames (ORFs) with undiscovered biological functions. To identify potentially novel mediators of epidermal differentiation from this candidate pool, RNA-Seq of primary human keratinocytes during calcium-induced differentiation was used to screen for uncharacterized ORFs whose transcript levels are increased during this process. The top up-regulated ORF encodes a 50 kD protein that we have termed Regulator of Epidermal Differentiation (RED) 1. An orthogonal screen also identified RED1 as highly down-regulated in primary human squamous cell carcinomas compared to patient-matched normal skin. As malignant neoplasms frequently demonstrate loss of differentiation, we hypothesized that RED1 is required for epidermal differentiation. To test this possibility, we undertook gain- and loss-of-function experiments in organotypic human epidermal tissue. Enforced expression of RED1 accelerated differentiation in regenerated human skin while CRISPR/Cas9-mediated depletion attenuated this process. In each case, whole transcriptome profiling to assess the global impact of altering RED1 expression identified gene groups that control skin differentiation, development, and function. Proximity-dependent biotin ligation assays in organotypic human epidermal tissue identified validated RED1 interactors including envoplakin (EVPL) and periplakin (PPL), genes known to be essential to skin differentiation, as well as genes not previously implicated in skin homeostasis, such as calcyclin binding protein (CACYBP). Gene set enrichment analysis suggests RED1 and its interacting proteins co-control distinct subsets of genes that induce epidermal differentiation and drive neoplastic transformation. Efforts are underway to study RED1s impacts on epidermal neoplasia. RED1 therefore represents a new regulator of epidermal differentiation with potential roles in skin carcinogenesis.