Next generation approaches to understand tissue specific regulation of the glucocorticoid response

Understanding how the regulators of steroid hormone receptors work together to process the cells' response to glucocorticoids is fundamental to several fields of biology, including gene regulation, signaling responses and biological heterogeneity. These systems are highly relevant as not only are glucocorticoids naturally produced by the body, they are also some of the most commonly prescribed therapeutics worldwide because of their low-cost and anti-inflammatory effects. Therefore, throughout our lives, cells are constantly coming into contact with steroids, either produced by the body or therapeutically.

The Glucocorticoid Receptor (GR) is a protein found in almost every cell in the body. The GR responds to the body's production of the steroid hormone cortisol and translates this signal into action by activating genes that control a diverse range of cellular responses, including development, metabolism and immune response.

The GR has been extensively studied at the molecular level. Such research has revealed how cortisol activates the GR and leads it to enter the nucleus. Once in the nucleus the GR binds directly to the promoters and enhancers of genes, activating them. We have also learnt that the GR does not work alone: the receptor is regulated by a complex network of interactions including co-activators, co-repressors and epigenetic modifiers, along with other nuclear receptors.

The range of responses caused by the presence of different regulators within the different cell types can be huge. For example, while GR activation promotes the growth of mammary tissue, the same activation of the GR causes lymphocytes within the blood to rapidly reduce in number.

In order to precisely control the cellular response to steroids it is vital that we understand how the differences in these responses are controlled by these regulators. Once understood we will be able to reprogram one cell type to respond more like another. Given the ubiquitous nature of GR activating compounds this has far-reaching implications: enabling us to block an unwanted cellular response, activating novel responses in cells to selectively direct them to survive or die, or precisely target a specific tissue to gain a specific outcome.

It is this knowledge gap that the present project will address.

Typically, studies that focus on complex systems like the GR are extremely time consuming as each part of the system must be identified and studied in turn. Instead, we will apply leverage to the development of single cell experimental techniques to enable the study of the GR as part of a much larger system.

The first step will be to detect the co-regulators that interact with the GR in response to glucocorticoids in multiple tissues. These will define the parts of the system that the GR interacts with and how they change between the tissue.

The second step will be to delete the genes that encode for these parts, the regulators of the GR, and then monitor how it alters the cells' response to activating the GR. State-of-the-art single cell sequencing technology makes this possible by enabling us to increase the number of regulator genes we can target and increases the amount of data we can collect. Additionally, because single-cell sequencing requires fewer cells than previous methods, the technology enables us to undertake the work in cells from healthy volunteers.

The data we generate that describes the GR responses in these gene-edited cells will be fitted, using current and future computational methods, to build a functional and experimentally testable representation of the network of co-regulators that modulate GR signaling in each tissue. This will effectually provide us with a wiring diagram of the different tissues.

Finally, we will use the models we have generated to establish how to precisely alter the cells' response to steroids in each tissue.

Whilst Cortisol is naturally produced by the body in response to stress and other stimuli, the same hormone and its derivatives are also found in commonly prescribed therapeutics. Understanding the regulation of cell responses to the hormone, and how the response varies so widely between cell types, is a highly relevant biological question with extensive implications.

The Glucocorticoid Receptor (GR) is a steroid hormone receptor that regulates the cellular response to cortisol. On activation, the receptor dimerises and binds DNA, acting as a transcription factor and activating a tissue-specific transcriptional response. Key to this process is that at each genomic locus the receptor binds, it recruits a host of other proteins and epigenetic remodellers. This network of interactions is critical for regulating the tissue-specific response.

This project will answer the following question: "How do the co-regulators interact with the GR to drive the tissue-specific response?". This will enable future research to precisely control specific functions of the GR transcriptional programme.

In this NIRG, I will use a systems approach to visualise these regulatory interactions as a functional network and compare the differences in the networks between the tissues in primary cells collected from healthy volunteers. Until recently these strategies were limited to model organisms, e.g. yeast, because of the requirement for the organism to be easily genetically modifiable and strict minimum requirements in terms of the number of cells needed.

Coupling CRISPR/Cas9 methods with single-cell sequencing enables us to bypass these limitations and fulfil a timely opportunity to apply these systems methods cells from healthy volunteers. These methods, therefore, enable us to monitor the differences in the GR response between tissues using cells from human donors, avoiding the need for model organisms and enabling us to acquire data that closely represent human physiology.

This research is fundamentally important to furthering our understanding and manipulation of the function of steroid hormone receptors. These receptors play a vital modulatory role in cellular differentiation, cancer and inflammation. The outcomes of this research and the methods developed will have a critical impact on efforts in regenerative medicine to maintain healthy ageing and fight chronic disease, in addition to translational use of steroid hormones therapeutically in prostate and breast cancer, and the use of steroids in primary care.

In the medium to long term, the results of this work will inform evidence-based policy and will have the potential to change the global culture and practice of steroid use as an intervention. This is crucial, as while hydrocortisone and its derivatives are widely prescribed and are considered the most effective anti-inflammatory treatments available (Scheschowitsch, 2017), there is a critical knowledge gap in our system's understanding of how these compounds function across tissue types.

The cultural and policy transformations inherent in this research hold great promise for improving the health and well-being of society, both directly and indirectly, through advancing practices in steroid use and generating knowledge and methods to inform future research and medical interventions.

The research programme will establish the PI in an independent position and train a highly-skilled researcher in state-of-the-art computation and experimental research skills, thus supporting a future generation of cross-discipline computational and experimental biologists. This is an important resource as modern research outcome measures are increasingly focused on 'omics and big data.

The project will also provide training in the use of primary human cell culture, enabling the use of donated normal human cells as a more relevant model system compared to established cell lines. Enabling research on primary cells provides a vital alternative to animal models, thereby supporting the three Rs (replacement, reduction and refinement).

Through training new researchers and in meeting the research outcomes stated in the Case For Support, this research supports the knowledge-based economy and these benefits will extend beyond the funding period.

Dr Holding holds a research and teaching contract (initially at a low-level of teaching to support the establishment of his research group); funding this research will consequently improve teaching and learning as his role includes disseminating the latest research as part of the taught undergraduate and graduate courses within the Department of Biology at the University of York.

The data from this research programme will be made available openly and will therefore be available for use as a key example of the application of systems biology. This builds on the previous teaching by the PI using his own previous package research data to ensure the relevance and impact of the material he teaches.

The research project innovates by combining diverse research strategies and multiple biological systems to form an integrated research programme. Through the strategic choice of collaborators, the work can make use of state-of-the-art methods, applying perturbation coupled single-cell sequencing, gene regulatory network analysis, molecular and computational biology to established (and therefore de-risked) methods in primary cell culture and molecular manipulation. The interdisciplinary nature of this research will promote knowledge transfer of the methods and outcomes across these fields of researchers and facilitate the application of the methods employed more widely.

Finally, through the collaboration of individuals from multiple disciplines and the use of these technologies, the research promotes global academic advancement in both the dissemination of the strategies employed and in the knowledge that will result from these experiments.