Understanding the essential requirement for HDAC1 and HDAC2 in tissue development and homeostasis: implications for disease and therapy.

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'. My lab studies how HDACs do this and which, amongst the 25,000 genes in each cell, are selected for inactivation. HDAC enzymes also 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 are being further developed 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 need an improved understanding of how individual HDAC enzymes work in normal cells. In this study we intend study two closely related HDAC enzymes, HDAC1 and HDAC2. One of the best methods for understanding how an enzyme works is to generate mutant cells in which the specific enzyme has been inactivated, or 'knocked-out'. These 'knock-out' cells can then be examined for changes in their characteristics, lack of growth for instance, which can then be attributed to the function of that particular enzyme. Previously, we have generated 'knock-out' cells for HDAC1 and HDAC2 alone, but their function is overlapping, and so the effects on cell growth were small. To get around this, we have generated cells in which HDAC1 and 2 can be removed at the same time, so called 'double knock-out' cells. Early experiments indicate that loss of both enzymes causes cells to die, indicating that their activity is essential. Using DNA technology it is possible to add back normal or mutated forms of HDAC1 to prevent the double knock-out cells from dying and then ask, which parts of the enzyme are important for its function? In related experiments, we also intend to visualize the actual molecular structure of HDAC1 bound to a molecule called MTA1, using a technique called X-ray crystallography. The interaction of HDAC1 with other molecules in the cell is fundamental to their function. By understanding the molecular basis of these interactions we can better understand how HDAC enzymes work in normal and cancer cells, and potentially use that knowledge to design new drugs to prevent them from working. The ability to stop HDAC1 and 2 from working, as seen in our double knock-out cells, causes cells to stop growing and die, making them excellent drug targets in the search for improved anti-cancer agents.

Technical Summary

Histone deacetylases (HDAC) are important drug targets in the treatment of cancer and mental illness. However, the existing HDAC inhibitors used in the clinic (SAHA, valproic acid) inhibit all 11 Zinc-dependent HDAC enzymes (HDAC1-11) and their prescription is associated with a number of debilitating side-effects, including: fatigue, diarrhea, low platelet counts, and hyperammonemia, which can lead to brain damage. Knock-out (KO) mice and RNAi studies implicate HDAC1 and 2 (HDAC1/2) as the most clinically relevant targets, particularly in terms of growth inhibition and apoptosis. We propose that targeting HDAC1/2, rather than HDAC1-11, might retain the therapeutic benefits of current HDAC inhibitors while avoiding the side-effects associated with inhibiting the entire sub-family of enzymes. To explore this, we intend to identify the key pathways regulated by HDAC1/2 and determine the therapeutic value of specific inhibitors. Objective 1: Since HDAC inhibitors are used clinically to treat cutaneous T-cell lymphoma, we will assess the role of HDAC1/2 during T-cell development, using existing HDAC1/2 knock-out mice and a novel catalytically inactive HDAC1 mutant line. Objective 2: HDAC1/2 activity in vivo is regulated by incorporation into three specific co-repressor complexes: Sin3A, NuRD and CoREST. To determine molecular details of complex assembly we will solve the structures of MTA1-HDAC1/2 and Sin3A-HDAC1/2 using X-ray crystallography. Using this data, the functional necessity of complex incorporation will then be assessed by testing the ability HDAC1/2 mutants to rescue the viability of HDAC1/2 double KO cells. Objective 3: Define the physiological substrates and genomic targets of HDAC1/2 activity, using quantitative mass-spectrometry and ChIP-seq assays, in double HDAC1/2 KO cells. This multi-disciplinary program of work will clarify the essential roles of HDAC1/2, their mechanism of action and establish the therapeutic value of specific HDAC inhibitors.

Planned Impact

1. Commercial / Industrial -
There is a growing awareness within the pharmaceutical industry of histone modifying enzymes as potential drug targets. HDAC1 and 2 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. Given these essential biological roles, HDAC1 and 2 enzymes are strong candidates for pharmacological manipulation. The novel structural data of HDAC:co-repressor complexes, coupled with data from the novel HDAC1-Y303H mice and interaction studies, 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 to perturb HDAC1/2 function. 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 -
Inhibitors of HDAC1 and 2 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, will be necessary to understand the action of existing HDACi used clinically, and in the design of small molecules which inhibit HDAC function.

3. Animal Welfare - 3R's.
Reduction, refinement and replacement (3R's) of animals in experimental research is a commitment made by each of the research councils and major research institutes within the United Kingdom. This project will use cells with an inducible knock-out of HDAC1 and 2 (Objective 3), which were generated using 'embryo free' methodology. This contrasts with the traditional method of generating knock-out cell lines from mouse embryos. Recently, the large scale production of knock-out mice embryonic stem (ES) cell lines was begun by the KOMP (Knock-out Mouse Project) and EUCOMM (European Community Mutant Mouse project) consortia, in their attempt to make a mutant cell line for every protein-coding gene in the mouse genome. The KO cells generated by these schemes will be made freely available to the academic community. This project will promote the same ES cell knock-out technology, their suitability for study of gene expression and raise awareness of this alternative to animal based systems.

Publications

10 25 50
 
Description BBSRC - Project Grant 2016-18
Amount £507,944 (GBP)
Funding ID BB/N002954/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2016 
End 12/2018
 
Description BBSRC - project grant 2012-15
Amount £552,849 (GBP)
Funding ID BB/J009598/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2015
 
Title Hdac1/Hdac2 Double conditional knock-out ES cells 
Description Embryonic stem cells in which Hdac1 and Hdac2 can be deleted simultaneously following addition of Tamoxifen. 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact These cells have been published by my own group, Jamaladdin et al. (2014) PNAS and have been provided to our collaborator Georges Lacaud at the Patterson Institute in Manchester. 
 
Title Multiplex recombineering: a one-step method for vector construction in gene targeting applications 
Description We present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This single-step multiplex recombineering method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. Multiplex recombineering greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact This method will be published in the Journal of Visual Experiments in 2014 
 
Title Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells 
Description We describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact This improved method of producing protein complexes in mammlian cells was published in the Journal of Visual Experiemtns in 2014. 
URL http://www.jove.com/video/51897/recombinant-protein-expression-for-structural-biology-hek-293f
 
Description Characterization of HDAC1/2 in embryonic haematopoiesis 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated conditional knockout cells in which we can simultaneously inactivate the highly related histone deacetylase enzymes, Hdac1 and Hdac2.
Collaborator Contribution Dr Georges Lacaud has been using the Hdac1/2 double knockout cells to determine their roles in haematopoiesis.
Impact The work is still underway, but initial data looks good and we would expect to publish in then next 12 months.
Start Year 2012
 
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 Chromsome segregation in HDAC1/2 DKO cells 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided HDAC1/2 double knocjout cells to the Fry lab for them to analyze chromosome segregation.
Collaborator Contribution The Fry lab was able to take HDAC1/2 DKO cells and quantitate aberrant chromosome segregation using an immunofluoresence microscopy approach.
Impact This collaboration was instrumental in the publication of Jamaladdin et al. (2014) in PNAS
Start Year 2013
 
Description Understanding the regulation of the 'acetylome' in embryonic stem cells 
Organisation University of Sheffield
Department National Centre for III-V Technologies
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided cells, labelled with SILAC amino acids, in which individual HDAC enzymes have been deleted to address the regulation of Lysine-acetylation across all protein in stem cells.
Collaborator Contribution My collaborator at Sheffield has performed the mass-spectrometry analysis of complex samples and then informatics analysis of the data.
Impact We have generated proteome-wide dataset for individual sites of lysine-acetylation and the degree to which levels change following HDAC deletion. We intend to write up these findings and submit a manuscript in 2017.
Start Year 2016
 
Description Understanding the role of HDAC3 in stem cell differentiation 
Organisation Sanford-Burnham Medical Research Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution We have generated a conditional HDAC3 knock-out cell line which has been used in ChIP-seq to examine the relations ship between retinoid signalling and the pluripotent factor Oct4.
Collaborator Contribution Our collaborators have used our cells to confirm the role of the HDAC3/SMRT co-repressor complex in early embryonic stem cell differentiation.
Impact A manuscript "Oct4 acts as an integrator of pluripotency and signal-induced differentiation" has recently been written and submitted.
Start Year 2015
 
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/
 
Description School talks - Animals in Research (UoL) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As part of the outreach activities at the Animal Facility at the University of Leicester I have given talks to school children and their teachers about the practical, ethical and legal issues surrounding the use of animals in research.
Year(s) Of Engagement Activity 2015