Development and application of dual fluorescent reporting systems for HIF-1alpha and other transcription factor pathways

Abstract

The ability to detect and quantify transcription factor (TF) activity has a broad range of utilities when probing for molecular interactions that intersect with their respective pathways or physiological function. This thesis describes the creation, optimisation, and application of the dFLASH (dual FLuorescent transcription factor Activity Sensor for Histone integrated reporting) system used to target a range of different TF pathways in a robust and sensitive manner with a focus on Hypoxia Inducible Factor 1 alpha (HIF-1a). HIF-1a is major regulator of the low oxygen response and is tightly regulated by a canonical post-translation pathway that, when inhibited, facilitates stabilisation of HIF-1a. Activation of its gene program drives processes such as erythropoiesis and vascularisation, and this has led to interest in targeting HIF-1a with small molecule regulators. Correspondingly, HIF-1a dysregulation has been implicated as a promoter of tumorigenesis and cell survival in hypoxic tumour microenvironments, although to date no small molecule inhibitor against HIF-1a has been successful in clinical trials. Conversely, pharmacological stabilisation of HIF-1a has been demonstrated to be beneficial in chronic anaemia. As a result, we established a dFLASH reporter for HIF-1a that was applicable in a high throughput context, utilising high content imaging and flow cytometry to demonstrate detection of HIF-1a activation at scale. We then leveraged dFLASH to perform a pilot screen utilising a library of ~1600 compounds in a live-cell bimodal arrangement, allowing detection of novel activator and inhibitor compounds, with efforts made to validate their mode of action. We further utilised an 800-compound library to refine our highthroughput screening approach, with an interest in combining the dFLASH HIF-1aspecific readout with phenotypic measurements of the live cells that contain the reporter. Specifically, we combined nuclear morphology scoring with dFLASH reporting to refine our lead selection process. Finally, we utilised dFLASH to probe for interactions between the microbial metabolome and the HIF-1a pathway in two distinct disease contexts, diabetic foot ulcers and Inflammatory Bowel Disease (IBD). HIF-1a has been implicated as a protective factor in both cases, and in both disease cases, changes in the bacterial species present in the local environment have been observed. Given there are a number of described physiological and metabolite-mediated interactions with the HIF-1a pathway, we therefore probed both individual bacterial species and complex co-cultures for activation and inhibition of the pathway. As a result, we described a novel downregulatory event with the HIF-1a dFLASH system by an IBD microbiome culture. As a result, we developed and undertook an exploratory pipeline that further utilised dFLASH, 16s sequencing, and metabolomics in an effort identify the causative agent.