Challenging dogma: an alternative non-hierarchical, epigenetically regulated model of the urothelium.

Epithelial tissues occur at the interface of internal and external surfaces of the body, where they have evolved highly specialised forms and functions. For example, the skin has evolved for physical protection and to protect against dehydration, whereas the gut has specialised to digest food and absorb nutrients. Both these epithelial tissues show constant turnover, with specialised cells being replaced by others generated by proliferation and migration from a basal progenitor or stem cell pool. The hierarchical model of cells programmed to pursue a one-way journey from a stem cell to the specialised, differentiated cell is used to explain how tissues maintain a balance (homeostasis) between cell loss and gain, and even why cancer stem cells should be targeted in cancer therapy. This project challenges this generic model of epithelial regulation and differentiation, based on observations from a different epithelial tissue, the urothelium.

Urothelium is the specialised epithelium which lines the bladder and ureters and functions as a tight urinary barrier. Unlike gut or skin, there is no constant cycle of renewal: cells of the urothelium are long-lived and mitotically-quiescent, but retain the capacity to contribute to efficient urinary barrier repair and regeneration by rapid re-entry into the mitotic cycle. No specific stem cell has ever been identified in human urothelium and dividing cells can be observed in any of the three layers. Phenotypically and positionally, urothelial cells adopt one of four distinct cell types (each of which we can isolate). Current molecular knowledge is based on homogenised urothelial preparations and therefore lacks sufficient subtype-specific detail. Our hypothesis is that rather than being part of a linear differentiation programme, urothelial cells display the appropriate phenotype in response to exogenous cues or their "niche" within the tissue, including adaptation to change (eg damage). Our hypothesis predicts an intimate relationship between epigenetic and signal transduction machineries to effect changes in cell phenotype.

We will test our hypothesis using urothelium from the ureter and the bladder for comparison, as these are of different embryological derivations. From each source, we will isolate, to high purity, each of the four distinct urothelial cell phenotypes and perform an in depth characterisation of the transcriptome and epigenome of each subtype to determine differences and similarities. We will also analyse the separated cell types after adaptation to a non-permissive cell culture system where we predict that all cells will adopt a baseline or default reference squamous phenotype irrespective of derivation. We will use the cell culture system to investigate the signals of each "niche" and test how altering different regulatory pathways can modify cell phenotype, where we predict that the epigenetic machinery will play a hitherto unrecognised key role that can be manipulated and exploited. A deliverable from this project will be a data-rich, spatially-resolved urothelial map of the regulatory networks and machinery that defines the different urothelial subtypes.

The outcome of this study will be important new understanding of urothelial tissue homeostasis that will challenge longstanding models of tissue biology and bring new perspectives to chronic diseases of ageing that affect the bladder, including future therapeutic opportunities in tissue engineering and regenerative medicine.

Differentiation of urothelial cells can be investigated in vitro as a time-resolved process where nuclear receptor activation initiates a downstream network of transcription factors (TFs) that engage the urothelial transcriptome. Recent work indicates a non-hierarchical relationship where, alongside chromatin reorganisation, some TFs act independently of others in the network. These relationships were construed using conflated whole populations, whereas differentiated urothelium is organised into basal, intermediate and superficial cell zones, arguing a need for spatial resolution of the regulatory networks.

Study of separated basal versus suprabasal cells suggest that urothelial cell phenotype is plastic and determined by position or "niche". Rather than a staged hierarchical differentiation programme, we postulate individual urothelial cell phenotypes are modulated epigenetically in response to signalling from contextual cues within the tissue.

We will exploit technical advances to develop maps incorporating transcriptome (RNA-Seq), epigenome (DNA methylation) and chromatin accessibility and occupancy (ATAC-Seq) datasets for each of the phenotypically-distinct urothelial cell subtypes, including the distinct pedicled c-Kit+ cell. Integration of these datasets will enable us to identify the molecular events underpinning urothelial regenerative capacity and differentiation potential. Ontological analysis of the transcriptome data will be used to infer subtype-specific marker sets, including regulatory genes, methylation-modifying enzymes, receptors and signal transduction network expression. We will validate and exploit these new data to explore the epigenetic regulation, plasticity and function of the different phenotypes that contribute histioarchitecturally to urinary barrier function and repair. These data will bring new understanding of urothelial tissue homeostasis that will challenge longstanding models of tissue biology.

As expanded in the Academic Beneficiaries section, our research will be of interest to Academics from a broad range of fields including epigenetics, epithelial cell & tissue biology and stem cells. In addition, by developing epigenetic approaches to manipulate cell fate and phenotype as new avenues for translation and application, our research will be important to the Life Sciences industry.

The UK economy will benefit through the patent-protection of any IP generated during the project, which we will be vigilant in identifying, protecting and exploiting through our close partnership with Business Development Managers in the University of York/UCL Research and Industry Offices and through the Investigators' own involvement in commercial enterprise and industry collaborations (eg through BBSRC CASE studentships).

The project will be of immediate and substantial benefit to the cell biology and bioinformatics postdoctoral researchers supported on the project through the experience of research collaboration and the combining of skills and opportunity to learn from each other's ideas and approaches. Regardless of whether they pursue careers in industry or academia, these highly-skilled individuals will emerge in a strong position to contribute to the UK Biosciences.

The wider public will benefit via active engagement of the investigators and researchers in outreach, including school and charity events in which both investigators actively engage.

We can also consider the longer term impact of our research for health and well-being. The distinction between urothelium as a stem cell-regulated tissue undergoing a hierarchical programme of differentiation versus a tissue composed of cells whose phenotype is epigenetically-regulated and responsive to external cues holds major implications for understanding urothelial health and disease and for the future development of new therapies:
> In areas allied to Urology, researchers, clinicians, pathologists and pharmaceutical companies will have interest in the tissue-specific urothelial detail uncovered and the possibility of translating this into benefit for patients. This will include potential regenerative therapies for tissue reconstruction, differentiation and/or homeostasis-modifying therapies for benign chronic disorders. The existence or not of a hierarchical stem cell is also important for understanding whether the stem cell is the primary target for therapy. This obviously includes cancer, as well as chronic benign inflammatory conditions, such as interstitial cystitis, where the disease states can be described as an underlying failure of urothelial homeostasis.
> Moderate to severe urinary incontinence is found in 22.6% of women and 8.8% of men aged 40 years and 33.4% women and 23.5% men aged over 80 years. We anticipate our work will impact on the quality of life and health of the ageing population by contributing to the availability of effective regenerative and other therapies.
> Discovering how urothelial cell phenotypes are epigenetically-regulated will allow us to understand the role of environmental epigenetic modifiers in urothelial disease (eg by disrupting plasticity) and how to moderate or reverse this for therapy. This is likely to carry implications for the Health and Safety Executive and for Environmental Agencies who need to understand and limit the impact of epigenotoxins on human health.