Open Chromatin Profiling of Differentiating Human Urothelium Reveals Heterarchy of Transcription Factors Driving Basal and Luminal Cell Phenotypes

Cell specialisation during lineage differentiation is effected by interactions of the chromatin landscape with networks of transcription factors, which 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 were used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor PPAR? as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq as having altered accessibility during differentiation were enriched with sequence-specific binding motifs for transcription factors, including P63, GRHL2, CTCF and GATA3. These were predicted to be transcription factors driving basal-like and differentiated urothelial phenotypes. Changes in chromatin-association of GRHL2, GRHL3, GATA3, and P63 were observed by immunoblotting of chromatin extracts. Transient siRNA knockdown of P63 and GATA3 influenced expression of transcription factors and urothelial differentiation markers, revealing that P63 favoured a basal-like phenotype by inhibiting urothelial differentiation, whereas GATA3 promoted urothelial differentiation. The transcriptional relationships around GATA3 demonstrated positive feedback on upstream PPARG, but independence from FOXA1, which functioned laterally and cooperatively with GATA3 to promote urothelial differentiation. This approach provides new insight for identifying drivers of luminal and basal-like urothelial cancer phenotypes and suggests that as a transcriptionally-regulated programme, urothelial differentiation operates as a heterarchy.