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.

Subtype-specific epigenomic landscape and 3D genome structure in bladder cancer

“…Our results show that the epigenetic regulation is a key contributor to molecular subtype assignment in both commonly used models of BLCA and in patient-tumors. [32] and TFAP2C [16] were previously reported to be a luminal and basal-squamous-specific genes, respectively, thereby validating our findings. Interestingly, binding motifs for AP-1 complex proteins FOXA1 and GATA3 are known to play a role in the development of urothelium [32] suggesting that their binding sites may be primed early during development.…”

“…[32] and TFAP2C [16] were previously reported to be a luminal and basal-squamous-specific genes, respectively, thereby validating our findings. Interestingly, binding motifs for AP-1 complex proteins FOXA1 and GATA3 are known to play a role in the development of urothelium [32] suggesting that their binding sites may be primed early during development. We also discovered that the stem-cell associated pioneering TFs such as KLF factors (KLF10/14) ATF factors (ATF1/2/4/7) and NANOG were enriched in basal bladder enhancers.…”

Repression of Transcription Factor AP-2 Alpha by Peroxisome Proliferator Activated Receptor Gamma Reveals a Novel Transcriptional Circuit in basal-squamous Bladder Cancer

“…As maintenance of a given molecular subtype in BC most likely results from the activity of a small subset of transcriptional master regulators (21) Figure S2). Our findings are in agreement with previous reports indicating a role for BORIS/CTCF (22) and KLF4 (23) in urothelial differentiation and BC, respectively and additionally suggest a role for TFAP2A in the emergence of a basal-squamous phenotype following PPARƔ inactivation in BC.…”

“…Although the presence of morphologic heterogeneity in BC and its relation to clinical outcomes has been recognized for decades, a series of recent studies definitively demonstrate the existence of intratumoral molecular heterogeneity in this common malignancy (3,5), as well as the potentially plastic nature of this heterogeneity. In keeping with specific tenants of the master regulator hypothesis (21), our in vivo and in vitro experimental studies (7,11,18,20,28), as well as additional foundational studies from other groups (6,9,10,22,(29)(30)(31)(32)(33)(34)(35)(36)(37) have identified a series of TFs apparently responsible for maintaining urothelial cell fate and establishing a luminal gene expression pattern in malignant disease.…”

The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

“…These findings have also been corroborated in Drosophila grainyhead (Grh), where approximately 50% of Grh binding sites were associated with regions of open chromatin in embryos (84). Enrichment of GRHL2 DNA binding motifs was observed in regions near upregulated genes in differentiated urothelium cells, suggesting the opening of chromatin by GRHL2 can lead to gene activation (85). We found that pS118-ER was similarly associated with upregulated genes in E2-treated MCF-7 cells.…”