Identification of ELF3 as an early transcriptional regulator of human urothelium

TY - JOUR

T1 - Identification of ELF3 as an early transcriptional regulator of human urothelium

AU - Böck, Matthias

AU - Hinley, Jennifer

AU - Schmitt, Constanze

AU - Wahlicht, Tom

AU - Kramer, Stefan

AU - Southgate, Jennifer

N1 - Copyright © 2014 Elsevier Inc. All rights reserved.

PY - 2014/2/15

Y1 - 2014/2/15

N2 - 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.

AB - 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.

KW - Cell Differentiation

KW - DNA Primers

KW - DNA-Binding Proteins

KW - Electric Impedance

KW - Gene Expression Regulation, Developmental

KW - Gene Knockdown Techniques

KW - Hepatocyte Nuclear Factor 3-alpha

KW - Humans

KW - Immunohistochemistry

KW - Microarray Analysis

KW - Proto-Oncogene Proteins

KW - RNA, Small Interfering

KW - Real-Time Polymerase Chain Reaction

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Time Factors

KW - Transcription Factors

KW - Urothelium

U2 - 10.1016/j.ydbio.2013.12.028

DO - 10.1016/j.ydbio.2013.12.028

M3 - Article

C2 - 24374157

SN - 0012-1606

VL - 386

SP - 321

EP - 330

JO - Developmental Biology

JF - Developmental Biology

IS - 2

ER -