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

Lead Research Organisation: University of Leicester
Department Name: Biochemistry

Abstract

'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.

Technical Summary

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.

Planned Impact

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.
 
Description 1) We are in the process of solving the Cryo-EM structure for the tetameric mitotic deacetylase complex, MiDAC complex. 2) Generated knock-out mice for each of the MiDAC familty memebrs (Dnttip1, Elmsan1, Trerf1 and ZnF541). 3) Generated IPMK-KO cell line in which we can regulate inositol phosphate levels using a TET-inducible rescue system. Each of these outputs should be published within the next 12-24 months.
Exploitation Route Knowledge of histone deacetylase complexes is key to understanding the role in the cell and exploiting them as therapeutic targets.
Sectors Education,Pharmaceuticals and Medical Biotechnology

 
Description Presentation of research to schools and colleges as part of open days and school visits.
First Year Of Impact 2017
Sector Education
 
Description Characterizing the Sin3A/HDAC1 complex: an alternative strategy for the precise inhibition of deacetylase activity in cells. 
Organisation LifeArc
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Characterization of the Sin3/HDAC1 complex. In particular the PAH domains of Sin3A, a unique protein-protein interaction motif which could be targeted as a more precise way of inhibiting HDAC activity in cells.
Collaborator Contribution Knowledge of drug development and converting basic research into therapeutics.
Impact Collaboration was able to secure a BBSRC iCASE PhD studentship
Start Year 2017
 
Description Department of Molecular and Cell Biology Open Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The Department of Molecular Cell Biology held its inaugural 'open day' in October 2016, hosting 120 year 12 students from schools across Leicestershire and Northamptonshire for talks, demonstrations and lab tours.
Year(s) Of Engagement Activity 2016
 
Description Host Yr10 work placement student 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact My lab has hosted a year 10 work placement student annually since 2014. The student works as a research technician in the lab for 2 weeks as part of work experience programme.
Year(s) Of Engagement Activity 2014,2015,2016,2017
 
Description STEM ambassador 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact In 2015 I registered as a STEM (Science, Technology, Engineering and Mathematics Network) ambassador in the East Midlands network and through the activities of the Leicestershire Education Business Company. I participated in a 'walking careers library' at Lancaster Boys school in Leicester - the idea is that the boys have the opportunity to walk around the hall and meet with and discuss possible careers with adults from different sectors. The event took place with the whole of Year 9 (approx. 180 pupils) following on from their careers assembly
Year(s) Of Engagement Activity 2015,2016,2017
URL http://www.leics-ebc.org.uk/