Dynamic and physical clustering of gene expression during epidermal barrier formation in differentiating keratinocytes

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.

Figure 1. The EpiDerm™ model of keratinocyte differentiation.

The figure shows a schematic of the experimental design including the culture arrangements (A). The fully differentiated model (B) (MatTek Corporation) contains organized basal, spinous, granular, and cornified layers analogous to those found in normal human skin. The course of the experiment (C) includes biological triplicates taken for microarray analyses at five points, starting before the addition of calcium to the culture medium.

Figure 2. K-means clustering of genes differentially expressed during keratinocyte differentiation.

The mean abundance of each transcript at each of the 5 time points is shown for each of the 11 clusters of gene expression. Abundance levels are log base 2 with mean 0.

Figure 3. Cluster analysis of putative transcription factor binding sites for each expression cluster.

The figure shows potential binding site motifs for cis-acting regulatory factors in sequences 2 Kb upstream and 0.5 Kb downstream of transcripts in each expression cluster. Blue boxes indicate the significant presence of a putative binding site, with the TRANSFAC identifier to the right of the figure. Binding sites (rows) and expression clusters (columns) are ordered based on hierarchical clustering using a binary distance metric and complete linkage. The dendrogram at the top displays the distance between expression clusters as calculated from comparison of binding site profiles.

Figure 4. Physical clustering of genes differentially expressed during keratinocyte differentiation.

The human genome is depicted in two vertical panels with p and q arms labelled. All probe sets detected as differentially abundant across the time course are displayed at their mapped physical distance within each chromosome and their corresponding Smith-Waterman (SW) score (x axis). The significance threshold corresponding to a FDR of 1 false positive is shown in red. The maximum SW score of 4.1 was detected within the epidermal differentiation complex (EDC) on chromosome 1p21.

Figure 5. Expression of MHC genes during keratinocyte differentiation.

The figure shows abundance levels of those transcripts from the extended MHC which vary significantly during keratinocyte differentiation. Gene transcripts of increasing abundance are shown in the left panel, and transcripts of decreasing abundance on the right. The expression cluster containing each transcript is listed before each gene symbol.