Stem Cell Reports Repor t Conserved Two-Step Regulatory Mechanism of Human Epithelial Differentiation Jayant K. Rane, 1 Alastair P. Droop, 1,2,5 Davide Pellacani, 1 Euan S. Polson, 1,6 Matthew S. Simms, 3,4 Anne T. Collins, 1 Leo S.D. Caves, 2 and Norman J. Maitland 1, * 1 YCR Cancer Research Unit, Department of Biology, University of York, York, North Yorkshire YO10 5DD, UK 2 York Centre for Complex Systems Analysis and Department of Biology, University of York, York, North Yorkshire YO10 5DD, UK 3 Department of Urology, Castle Hill Hospital, Cottingham, Humberside HU16 5JQ, UK 4 Hull York Medical School, University of Hull, Hull, Humberside HU6 7RX, UK 5 Present address: Leeds Institute of Cancer and Pathology, St James’s University Hospital, Beckett Street, Leeds LS9 7TF, UK 6 Present address: Stem Cells and Brain Tumour Group, Section of Oncology and Clinical Research, Leeds Institute of Cancer and Pathology, Wellcome Trust Brenner Building, St James’s University Hospital, Leeds LS9 7TF, UK *Correspondence: n.j.maitland@york.ac.uk http://dx.doi.org/10.1016/j.stemcr.2014.01.001 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. SUMMARY Human epithelia are organized in a hierarchical structure, where stem cells generate terminally differentiated cells via intermediate progen- itors. This two-step differentiation process is conserved in all tissues, but it is not known whether a common gene set contributes to its regu- lation. Here, we show that retinoic acid (RA) regulates early human prostate epithelial differentiation by activating a tightly coexpressed set of 80 genes (e.g., TMPRSS2). Response kinetics suggested that some of these genes could be direct RA targets, whereas others are probably responding indirectly to RA stimulation. Comparative bioinformatic analyses of published tissue-specific microarrays and a large-scale tran- scriptomic data set revealed that these 80 genes are not only RA responsive but also significantly coexpressed in many human cell systems. The same gene set preferentially responds to androgens during terminal prostate epithelial differentiation, implying a cell-type-dependent interplay between RA and tissue-specific transcription factor-mediated signaling in regulating the two steps of epithelial differentiation. INTRODUCTION Differentiation of self-renewing adult human epithelial stem cells into rapidly proliferating progenitor cells is a conserved early event in human tissue development and homeostasis. Systematic and coordinated regulation by master transcription factors, noncoding RNA-mediated networks, or the establishment of progressive epigenetic marks have been proposed as likely regulatory mechanisms (Hanna et al., 2010). Identification of control mechanisms will enable the basic understanding of epithelial dynamics and could identify common perturbations leading to the disruption of epithelial homeostasis in cancers. We have investigated the nature of stem cell regulation during early human epithelial differentiation using patient-derived prostate epithelium as our primary experimental tool. In human prostate, stem-like cells (SCs) of a basal phenotype and their early differentiated progeny (transit amplifying [TA] and committed basal cells [CB]) can be reproducibly enriched from patient-derived benign and malignant tis- sues by selecting for differential CD133, CD44, and a 2 b 1 in- tegrin expression (Collins et al., 2005; Richardson et al., 2004). Transcriptional profiling of prostate SCs and CBs identified consistent gene expression changes associated with differentiation and carcinogenesis (Birnie et al., 2008). We have now investigated the expression pattern and the regulation of genes that are differentially expressed in the SC and CB populations, supplemented by a large- scale analysis of published transcriptomic experiments, to identify shared signaling pathways that regulate general epithelial differentiation. RESULTS AND DISCUSSION Identification of Genes Coregulated during Prostate Stem Cell Differentiation Coexpression analysis was performed on differentially expressed Affymetrix probes between SC and CB from the Birnie et al. (2008) data set (Figure 1A) to identify func- tionally related and coregulated genes without intro- ducing observer or selection bias (Lee et al., 2004). Probes with high pairwise correlation (Pearson’s correlation coef- ficient, R R 0.8 or R % 0.8) were clustered into four distinct groups (A–D) (Figures 1B and 1C). All the correla- tions (above the threshold) were positive and no signifi- cant connections between probes within the four groups were seen. Analysis of independent published microarrays (Shepherd et al., 2008) showed similar changes in the expression of group A–D genes during prostate stem cell differentiation (data not shown). Gene ontology (GO) analysis further revealed that the genes in each group are functionally related. Groups B, C, and D genes were prin- cipally enriched for differentiation-associated processes Stem Cell Reports j Vol. 2 j 1–9 j February 11, 2014 j ª2014 The Authors 1 Please cite this article in press as: Rane et al., Conserved Two-Step Regulatory Mechanism of Human Epithelial Differentiation, Stem Cell Reports (2014), http://dx.doi.org/10.1016/j.stemcr.2014.01.001