The mineralocorticoid receptor in glucocorticoid-mediated gene regulation: MR/GR interactions and chromatin accessibility as mechanisms

Cortisol is a natural hormone that circulates through the blood and acts on the brain to regulate the ways brain cells signal to each other and processes such as learning and memory. It is also a major factor in the regulation of brain centres that regulate mood, anxiety and wellbeing. Disruptions in the normal regulation of cortisol have been linked to psychiatric disease including major depression and post-traumatic stress disorder.Stress plays a major role in the activation of cortisol release and has itself been linked directly with the onset of psychiatric illness. There is now a large body of evidence that changes in the pattern of cortisol secretion is a major factor in increasing vulnerability to these increasingly common disorders.

Brain cells respond to cortisol though a protein called the glucocorticoid receptor (GR) which has been well studied, and by the mineralocorticoid receptor (MR) protein about which much less is known. It's believed that the balanced activity of MR and GR is important for normal brain function and responses to stressful situations, and that imbalances lead to psychiatric symptoms. Yet we know very little about how by working together the MR and GR produces 'normal' brain function by correctly reading and interpreting the genetic blue print in DNA. We know even less about how this process goes wrong when the receptor levels are imbalanced, when the cortisol pattern changes, or when synthetic hormones similar to cortisol are used to treat patients.

Because MR and GR are often present in the same brain cells we predict the normal brain environment is produced via the cooperative action of both when cortisol turns them on. We would like to study how far reaching this MR/GR cooperation is in a cell, and how it arises at a level of MR/GR interacting with the cells library of stored instructions (DNA). The bulk of our work here will aim to understand what is 'normal', but experiments will also hint at how the normal situation can change with abnormal patterns and types of synthetic hormones; or abnormal levels of MR/GR. This work therefore hints at how such changes can contribute to psychiatric disease.

MR and GR regulate the copying of DNA instructions that control the cells function, like a photocopier copies pages from a manual. We'll first discover which parts of the DNA have their copying controlled by both MR and GR, and how widespread cooperative activity is by looking at where on DNA they are found together. We'll next look at selected examples of how cooperative function might occur and the effect this has on the copying process. A physical interaction of MR/GR is one possibility that could produce a unique outcome. We'll determine if this is the case and also define the number of MRs and GRs within the interaction, assessing whether this number can change under different circumstances. MR/GR might also interact with a site independently meaning the balance of actions through each produces the final outcome. One of the proteins may carefully control the ability of the other to interact with the DNA for example, or the activities maybe complementary or opposite to each other. These possibilities will also be tested. These are not easy questions to address inside a living cell so we have travelled to the USA to learn new ways to study these questions and will return these methods to Britain as we define ways in which MR/GR cooperativity normally works.

Finally, the pattern of cortisol secretion likely produces specific times at which MR and GR can function cooperatively so we will determine where in this pattern MR is active at the same time as GR. This will provide additional insight into how disease-associated cortisol patterns and concentrations, or the presence of synthetic hormones or imbalanced MR/GR levels, misdirects the MR/GR cooperativity mechanisms leading to altered interpretation of cellular instructions that may begin disease proces

A role for MR in the glucocorticoid response of the brain has been recognised for many years. How MR cooperates with GR to regulate transcriptional responses to glucocorticoid is however poorly understood. We hypothesize that the glucocorticoid response in co-expressing cells is mediated through a dual receptor system comprising both MR and GR. We further hypothesize that MR/GR display overlapping DNA binding activities at a significant percentage of sites, and at such sites, a variety of mechanistic possibilities exist to co-regulate gene targets. Our program will significantly extend understanding of MR/GR gene regulation beyond the heterodimer hypothesis. Our program will examine these possibilities by:
1) MR/GR ChIP-seq genome-wide in co-expressing cells. Bioinformatics analysis determines properties of unique and overlapping sites making mechanistic predictions.
2) Describing the temporal dynamics of MR-GR interactions by defining the response of MR to the ultradian rhythm of glucocorticoid secretion. Dynamics of GR DNA binding during this pattern are already known while MR data is necessary to define temporal conjunctions of MR/GR at DNA where cooperative regulation of gene targets can be achieved.
3) Clarifying the functional role and structural properties of the MR-GR interaction in the living cell nucleoplasm and at DNA. Preliminary data with advanced microscopy techniques suggests the true heterodimer model is incomplete or inaccurate (example of a mechanism involving direct MR/GR interaction).
4) Exploring whether MR and GR can contribute to each others' DNA loading by modifying chromatin accessibility in a mechanism known as assisted loading (example of an alternative mechanism not requiring direct MR/GR contact).
5)Define how cell type specificity and clinically relevant manipulations of receptor ratio or hormone milieu influence the function of the MR/GR system in accordance with the balance hypothesis of psychiatric disease.

Knowledge/Research: Our collaborative and multi-disciplinary program will deliver the highest quality science published in leading research journals, having wide implications for research across multiple fields. In addition to the brain, the concepts of MR/GR balance and cooperative function have major implications in the fields of immunology, metabolism research, and cardiovascular function. Understanding MR/GR cooperativity will be a scientific advancement that has been difficult to achieve by conventional approaches. Thus our work incorporates new techniques and their dissemination to the wider community as an important part of the proposed program.

Economic: UK pharmaceutical companies may develop new drugs or delivery methods duplicating physiological patterns of hormone, capable of normalizing aspects the MR/GR system in disease, or normalising downstream effectors contributing to symptoms. Several companies are already taking an interest in our ultradian rhythm work, and we already have one patent and ongoing collaboration with a number of pharmaceutical companies. We anticipate manufacture and sale of new therapeutics and research products; creating jobs, revenues, and an economic boost. Although this process may take several years jobs in pharmaceutical companies and subsequent clinical testing of produces will contributeto the UK economy by investing in creative assets and new knowledge. Mini-pump technologies to duplicate pulsatile hormone deliveries are already available and under continuous development by UK companies. In addition to products needed for our immediate work, it should be noted our program incorporates a period in the USA to learn novel next-generation microscope and sequencing techniques. Dissemination of these approaches in the UK will occur through collaboration and conference attendance as Dr. Pooley moves back to the UK. Genomics centres and companies making instruments for these methods will benefit through increased business, boosting economic performance.

Society: As clinical practitioners become increasingly aware of the importance of the pattern of treatment, and how synthetic glucocorticoids influence the dual MR/GR system, doctors should increasingly be able to tailor treatment regimes to minimize side effects. Patients will receive more effective treatments and improved quality/quantity of life,with reduced time off work and reduced costs to the NHS.Furthermore in the light of the current projections of increased burdens of psychiatric disorders on the NHS any new approach to reduce mortality and morbidity will be of great help. Development of next-generation technologies will also support job creation.. Dr. Pooley also has a strong interest in public engagement in science and will work to communicate advances to the public via special seminars, and possibly via other media such as television/school visits.

Investment in People:Coupled with our efforts to match the USA on their use and development of next-generation approaches, Dr. Pooley will train in the USA to work with these techniques and develope a unique skill set. On his return to the UK these skills will be available for others in Oxford and Bristol, then more widely by conference attendance and collaboration. The capacity and quality of UK research increases concordantly. Dr. Pooley will work with biologists, institutions, bioinformaticians and computer programmers to ensure availability to the UK research community, beginning a pipeline of people continuing to innovate and develop such approaches long-term, possibly resulting in commercially viable software. The career of Dr. Pooley will gain significant enhancement if this project is supported, allowing growth toward an independent investigator acquiring management and teaching skills that will support competitiveness for further funding, help to train students in the UK, and develop his public engagement .