Identification of ELF3 as an early transcriptional regulator of human urothelium

Abstract

Despite major advances in high-throughput and computational modelling techniques, understanding of the mechanisms regulating tissue specification and differentiation in higher eukaryotes, particularly man, remains limited. Microarray technology has been explored exhaustively in recent years and several standard approaches have been established to analyse the resultant datasets on a genome-wide scale. Gene expression time series offer a valuable opportunity to define temporal hierarchies and gain insight into the regulatory relationships of biological processes. However, unless datasets are exactly synchronous, time points cannot be compared directly. Here we present a data-driven analysis of regulatory elements from a microarray time series that tracked the differentiation of non-immortalised normal human urothelial (NHU) cells grown in culture. The datasets were obtained by harvesting differentiating and control cultures from finite bladder- and ureter-derived NHU cell lines at different time points using two previously validated, independent differentiation-inducing protocols. Due to the asynchronous nature of the data, a novel ranking analysis approach was adopted whereby we compared changes in the amplitude of experiment and control time series to identify common regulatory elements. Our approach offers a simple, fast and effective ranking method for genes that can be applied to other time series. The analysis identified ELF3 as a candidate transcriptional regulator involved in human urothelial cytodifferentiation. Differentiation-associated expression of ELF3 was confirmed in cell culture experiments and by immunohistochemical demonstration in situ. The importance of ELF3 in urothelial differentiation was verified by knockdown in NHU cells, which led to reduced expression of FOXA1 and GRHL3 transcription factors in response to PPARγ activation. The consequences of this were seen in the repressed expression of late/terminal differentiation-associated uroplakin 3a gene expression and in the compromised development and regeneration of urothelial barrier function.

FOXA1, GATA3 and PPARɣ Cooperate to Drive Luminal Subtype in Bladder Cancer: A Molecular Analysis of Established Human Cell Lines

“…3A). Of these 662 differentially expressed transcription factors, 32 were differentially expressed between luminal and basal cell lines in the CCLE data set, including several markers of urothelial differentiation such as GATA314, ELF31420, GRHL315, FOXA19, and PPARɣ11 (q < 0.1, t-statistic from linear model, Fig. 3B).…”

“…The “basal” subtype often exhibits squamous differentiation, is biologically aggressive, and is found almost exclusively in invasive disease48. Evidence suggests that putative markers and transcriptional regulators of urothelial differentiation are upregulated in the luminal molecular subtype and downregulated in the basal subtype of bladder cancer234591011121314151617181920. However, there is limited experimental data (with some notable exceptions221) identifying a direct contribution of these transcription factors to molecular subtype.…”

Genetic Alterations in the Molecular Subtypes of Bladder Cancer: Illustration in the Cancer Genome Atlas Dataset

“…These observations support the emerging conclusion that FGFR3 mutations mark the luminal MIBCs that correspond to the papillary NMIBCs that have progressed to become muscle invasive. Alterations affecting several transcription factors that appear to play important roles in urothelial terminal differentiation [84,85] (PPARG , GATA3 , RXRA , and ELF3) were also enriched in luminal cancers. Biological effects of these alterations will need to be explored in future functional studies.…”

Concomitant downregulation of the imprinted genes DLK1 and MEG3 at 14q32.2 by epigenetic mechanisms in urothelial carcinoma

“…Upon culturing, normal urothelial cells acquire a considerable degree of plasticity, including the ability to differentiate into epidermis-like as well as urothelial-like structures [42]. Upon immortalization by telomerase expression, further changes ensue, in particular, deregulation of key epigenetic regulators [43].…”