Transcription factor regulation in urothelial-type differentiation and reprogramming

For bladder tissue-engineering, there is a need for a surrogate epithelial cell source for patients whose own bladder epithelial cells (urothelial cells) are compromised by disease or cancer. Current work in the field has focused on attempting to differentiate various types of stem cells into urothelial cells using conditioned medium, co-culture or specific factors. These approaches have lacked clear focus, making it obvious that a better understanding of the transcription factors underlying urothelial lineage, development, and cell differentiation is necessary. The aim of the work presented in this thesis was to acquire specific knowledge regarding transcription factors involved in urothelial cell differentiation, which could be applied to an in vitro transdifferentiation approach of cell reprogramming. Since buccal mucosa is readily available, and already used as a grafted tissue for urology surgical applications, buccal epithelial cells were identified as a potential starting cell type. An in vitro comparison of human buccal epithelial cells to urothelial cells revealed that buccal epithelial cells had weak transcript and protein expression of four transcription factors that play key roles in urothelial cell differentiation: ELF3, FOXA1, GATA3 and PPARγ. Since ELF3 and FOXA1 have been previously shown to act downstream of PPARγ in urothelial cells, PPARγ and GATA3 were chosen as key upstream transcription factors to overexpress in buccal epithelial cells. Investigation into PPARγ expression in human urothelial cells revealed that of the two main PPARγ protein isoforms, PPARγ1 was most critical for urothelial cell differentiation. Knockdown of GATA3 expression in urothelial cells using specific siRNA revealed an important role for GATA3 in maintaining the differentiated state of urothelium. Individual overexpression of GATA3 and PPARγ1 in human buccal epithelial cells was unable to cause complete transdifferentiation to urothelial cells, but each resulted in the upregulation of downstream genes that play critical roles in urothelial cell differentiation and barrier formation. This work revealed key differences in transcription factor expression between buccal epithelial cells and urothelial cells, allowing for a better understanding of transcription factors involved in urothelial cell differentiation. The work provides a starting point for future urothelial-type cell reprogramming efforts which will likely require combined overexpression of several of these identified transcription factors to achieve complete transdifferentiation

Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation

Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signalling cues were found to display inherent similarities and differences of phenotype. Investigation of transcription factors implicated in driving urothelial-type differentiation revealed buccal epithelial cells to have minimal or absent expression of PPARG, GATA3 and FOXA1 genes. Retroviral overexpression of GATA3 or PPARG1 coding sequences in buccal epithelial cells resulted in nuclear immunolocalisation of the respective proteins, with both transductions also inducing expression of the urothelial differentiation-associated claudin 3 tight junction protein. PPARγ1 overexpression alone entrained expression of nuclear FOXA1 and GATA3 proteins, providing objective evidence of its upstream positioning in a transcription factor network and identifying it as a candidate factor for urothelial-type transdifferentiation or reprogramming

Heterarchy of Transcription Factors Driving Basal and Luminal Cell Phenotypes in Human Urothelium

Cell differentiation is effected 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 were used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor PPARG 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 FOXA1, P63, GRHL2, CTCF and GATA3. In addition, co-occurrence of GATA3 motifs was observed within sub-sets of differentiation-specific peaks containing P63 or FOXA1 after induction of differentiation. 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