title: Heterarchy of transcription factors driving basal and luminal cell phenotypes in human urothelium.
abstract: Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq was used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.
authors.affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: Carl
authors.initials: C
authors.lastname: Fishwick
authors.affiliation: Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
authors.forename: Janet
authors.identifier: 0000-0002-7560-2212
authors.initials: J
authors.lastname: Higgins
authors.affiliation: Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
authors.forename: Lawrence
authors.identifier: 0000-0001-7725-9203
authors.initials: L
authors.lastname: Percival-Alwyn
authors.affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: Arianna
authors.initials: A
authors.lastname: Hustler
authors.affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: Joanna
authors.initials: J
authors.lastname: Pearson
authors.affiliation: Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Bastkowski
authors.affiliation: Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Moxon
authors.affiliation: Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Swarbreck
authors.affiliation: School of Computing Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Chris D
authors.initials: CD
authors.lastname: Greenman
authors.affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5DD, UK.
authors.forename: Jennifer
authors.identifier: 0000-0002-0135-480X
authors.initials: J
authors.lastname: Southgate
mesh_terms: D000076246:CCCTC-Binding Factor / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D005557:Formaldehyde / Q000737:chemistry; D050990:GATA3 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D051541:Hepatocyte Nuclear Factor 3-alpha / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D017423:Sequence Analysis, RNA; D015398:Signal Transduction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019459:Urothelium / Q000166:cytology / Q000378:metabolism
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D002843:Chromatin; D004268:DNA-Binding Proteins; C496499:FOXA1 protein, human; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; C470830:GRHL2 protein, human; D051541:Hepatocyte Nuclear Factor 3-alpha; D047495:PPAR gamma; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D005557:Formaldehyde