Understanding the contribution of inositol phosphate signalling to class-1 HDAC complex function

'Histone deacetylase' (HDAC) enzymes are present in all cells of the body. Their function is to switch genes 'off', and make sure they stay 'off'. In many respects shutting a gene down is every bit as important as switching a gene on. HDAC enzymes represent an exciting medical opportunity because they are 'druggable'. Already, drugs which inhibit HDAC activity are being used in the clinic as anti-cancer agents, and in the laboratory for their beneficial effects on dementia and anti-inflammatory properties. There is therefore a compelling applied, as well as academic, motivation for studying their physiological roles in order to assess their potential as pharmacological targets. We use a technique called 'X-ray crystallography' which allows us to determine the shape and structure of HDAC enzymes at a molecular level. Once we have determined their shape it allows us to understand the way that HDACs bind to other proteins and small molecules such as inositol phosphate (IP). We recently showed in vitro (i.e. in a test tube) that the enzymatic activity of HDACs was dependent upon the binding of IP. Following on from this discovery, the purpose of this project is to understand the importance of IP to HDAC function in cells and in mice. To do this we have three main objectives: 1) We plan to generate cells with low, medium and high levels of IP and test whether these correlate with level of HDAC activity. 2) In cells, HDACs interact with other proteins to form a multi-protein 'complex'. The complex most sensitive to the presence of IP is called, MIDAC and it consists of three proteins bound together (HDAC1, DNTTIP1 and MIDEAS). To understand the regulation of HDAC complexes by IP we plan to solve the structure of MIDAC using X-ray crystallography. 3) The role of MIDAC in cells is completely unknown and so we aim characterize the physiological activity of MIDAC, using cells lacking one of the three members of the complex, DNTTIP1. By understanding the molecular basis of HDAC complex function we can use that knowledge to design new drugs to prevent them from working. The ability to stop HDACs from working has beneficial effects on a wide-range of diseases, including epiplepsy, bipolar dissorder and Alzheimer's disease, making them excellent drug targets.

Class-1 histone deacetylase enzymes (HDAC1, 2 and 3) are ubiquitous, long-lived, nuclear enzymes which regulate chromatin structure as the catalytic core of at least 5 distinct multi-protein co-repressor complexes. The enzymes are largely inactive and have little inherent substrate specificity until assembled into their cognate complexes. Therefore the complex into which they are incorporated is critical to their functional context. We were the first to solve the structure of HDAC1 and HDAC3 bound to their co-repressor proteins, which led to the finding of a molecule of inositol tetraphosphate (IP4) bound between the two protein components when the complex was purified from mammalian cells. Subsequently, we showed that class-1 HDAC activity could be regulated 10-20 fold by the addition or removal of IP4. Although we have shown that IP4 regulates HDACs in vitro, it remains to be determined to what extent IP4 signalling regulates the activity of class-1 HDAC complexes in vivo. To address this major outstanding question in HDAC biology we have three objectives. Objective 1: Using cells either lacking the major IP4 kinase (IPMK), or over-expressing the IP phosphatase, SopB we will determine the extent to which IP levels regulate class-1 HDAC complex activity in cells. Objective 2: Of the major class-1 HDAC complexes, it is the novel MiDAC complex which is most sensitive to the addition of IP4 (up to 20-fold). We therefore plan to solve the structure of the ternary MiDAC complex (HDAC1/DNTTIP1/MIDEAS), expressed and purified from mammalian 293F cells, to help us understand the mechanism by which IP4 stimulates HDAC activity. Objective 3: to define a physiological role for MiDAC using tissue culture cells and transgenic mice. Using a multi-disciplinary approach (cell lines, X-ray crystallography, transgenic mice) this study will significantly improve our understanding of HDAC1/2 complex function and their regulation via inositol phosphates.

1. Commercial / Industrial -
There is a growing awareness within the pharmaceutical industry of histone modifying enzymes as potential drug targets. Class-1 HDAC (1, 2 and 3) function has been implicated in almost all cellular processes including, cell cycle progression, DNA repair, differentiation and cancer. Furthermore, mouse knock-out studies have demonstrated that HDAC1/2 have essential roles in the development of the heart, neurons, skin and B-cells. The enzymes themselves are largely inactive and have little inherent substrate specificity until assembled into their cognate complexes. Therefore the complex into which they are incorporated is critical to their functional context. We were the first to solve the structure of HDAC1 bound to its co-repressor protein, MTA1; part of a larger programme to determine the structure of all FOUR HDAC1/2 complexes (Sin3A, NuRD, CoREST and MiDAC). The novel structural data of the MiDAC complex generated from this study will be of great value in the design of conventional HDAC inhibitors (focussed on the active site), and the long-term goal of using small molecules to inhibit the protein-protein interactions of these complexes in vivo. In addition, if we are able to confirm a link between IP levels and HDAC activity, then it would also provide an alternative and, importantly, more specific method of HDAC inhibition. We have already begun collaborations with synthetic chemists to design novel 'dual' HDAC inhibitors, which incorporate both active site inhibitors (i.e. hydroxamic acids) with an inositol phosphate group. The University of Leicester has a vigorous and experienced 'Enterprise & Business Development' team and an embedded unit ('The Biobator'), dedicated to exploitation of activities arising from work in biomedical research. Outputs from the project will be used by BIOBATOR to establish partnerships with industrial collaborators to exploit these findings.

2. Societal -
HDAC inhibitors are currently used in the clinic to treat depression and cancer. It is only a matter of time before their application becomes more extensive, enhancing the well-being of society as a whole. In the laboratory, inhibition of HDAC1 and 2 reactivates alpha-globin (the foetal globin isoform) in human erythroid progenitors, making them potential therapeutic targets for the treatment of sickle cell disease. Inhibition of HDAC activity has ameliorative effects in mice models of dementia and muscular dystrophy. The essence of our project is basic science, and the therapeutic payoff long-term. However, an understanding of HDAC enzymes in their cellular context, incorporated into diverse co-repressor complexes (e.g. MiDAC), will be necessary to understand the action of existing HDACi used clinically, and in the design of small molecules which inhibit HDAC function.