Dynamic and Physical Clustering of Gene Expression during Epidermal Barrier Formation in Differentiating Keratinocytes

Jennifer M Taylor,William O C M Cookson,Patrick J Hayden,Miriam F Moffatt,Jotun Hein,Richard Copley,Gina Stolper,Richard Mott,Lizhong Hao,Teresa L Street,Martin S Taylor

doi:10.1371/journal.pone.0007651

Abstract

The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease.

Highlights

Keratinocytes develop continuously from a basal layer replenished by stem cells in hair follicles
We identified a group of 1,282 probe sets showing statistically significant differences in levels of expression between the five times with the Significance Analysis of Microarrays (SAM) method [2], using a stringent threshold false discovery rate (FDR),0.01%
The study of a single human cell type in controlled conditions has shown a high level of organisation of co-ordinate gene expression with physical clustering and sharing of distinctive TF binding sites among co-expressed transcripts

Summary

Introduction

Keratinocytes develop continuously from a basal layer replenished by stem cells in hair follicles. Primary keratinocytes can be induced to form a differentiated model of the epidermis by the addition of calcium and culture at an air-liquid interface on membrane inserts. We harvested cells from the model at five times, starting before calcium was added to the medium (0 days), immediately before the cultures were lifted to the air-liquid interface (3 days) and three subsequent time points at five, seven and ten days after calcium addition (Figure 1). Each time was tested in triplicate from different cultures We analysed this time-series gene expression data to identify the transcripts that varied most significantly during differentiation, and we used a clustering algorithm to find genes with correlated expression patters. In order to understand the biological significance of our results, we examined the gene ontology (GO) content of each cluster and used known genes to suggest the function of other cluster members

Methods

Results

Conclusion