Bilateral BBSRC-SFI: Understanding the impact of divergent Sin3A/HDAC1 complex assemblies in gene regulation

'Histone deacetylase' (HDAC) enzymes, the class of enzymes which catalyse the removal of the acetyl group from acetylated lysines, have been implicated in almost all cellular processes, including cell cycle, DNA synthesis, DNA repair and gene expression. There are 18 HDACs in mammals, which can be categorized initially as having either a Zn2+-dependent (Class I, II and IV) or NAD+ dependent (Class III - Sirtuins) catalytic domain; and then further by the presence of additional N-terminal domains and a tissue specific expression pattern (Class II and IV) or a short C-terminal tail and ubiquitous expression (Class I). Classically, class I HDACs (HDAC1, 2 and 3) are thought to be involved in the process of gene repression as the catalytic core of canonical co-repressor complexes, such as Sin3A, NuRD and CoREST. The Sin3a complex is thought to be recruited to chromatin by a combination of transcription factors and Sin3-associated proteins (SAPs) where it then mediates histone deacetylation and consequent chromatin compaction. Clinically, generic HDAC inhibitors (HDACi) are used to treat both depression (Valproic acid) and subcutaneous T-cell lymphoma (SAHA). Despite this clinical importance, almost nothing is known about their mode of action. Furthermore, the use of these generic HDACi, is associated with a number of debilitating side-effects including, fatigue, diarrhoea, low platelet counts (thrombocytopaenia), and hyperammonemia, which can lead to brain damage. Therefore, given the positive therapeutic value of HDAC inhibition in numerous disease states, and the appalling side-effects of generic HDACi, the logical way forward is to disrupt individual HDAC complexes. The Sin3A/HDAC1 complex for instance, has been shown to play a critical role in cell cycle regulation, therefore inhibition of additional HDAC1/2 complexes (NuRD, CoREST and MiDAC) may be unnecessary to arrest the growth of cancer cells.

A key challenge to specifically inhibiting individual HDAC complexes will be to understand how they are assembled, and which co-factors are essential for their function. To date, most major proteomics studies of the Sin3A complex in mammals have used cancer cell lines. In this proposal, we aim to identify all essential co-factors and substrates (including non-histones) of the Sin3A complex in an array of primary cell types. To achieve this, we will employ state of the art proteomic and transcriptomic approaches, developed in the Cowley and Bracken labs. The specific aims we will pursue are the following: i) assess the requirement for the stem cell specific SAPs, Fam60a and Tet1, to the function of Sin3A in cells, ii) test whether the composition of Sin3A is the same in different types of primary cells, and iii) ask what fraction of the aceytlome (around 4,000 site of Lys-ac in most cell types) is regulated specifically by the Sin3A complex. By using Sin3A as an exemplar of a class I HDAC complex, we expect to extend our understanding of HDAC complexes in a cellular context. By understanding the molecular basis for how HDAC complexes function we can use that knowledge to design new drugs to treat a variety of diseases including, epilepsy, bipolar disorder, Alzheimer's disease, and cancer.

Histone deacetylase 1 and 2 (HDAC1/2) regulate global levels of lysine acetylation as common catalytic components of four distinct co-repressor complexes; Sin3A, NuRD, CoREST and MiDAC. These complexes have been implicated in almost all cellular process from cell cycle, DNA synthesis, DNA repair and gene expression. In the clinic, pan-HDAC inhibitors (HDACi, e.g. valproic acid) are used to treat cancer and depression, although their use is associated with a number of debilitating side-effects. More specific HDACi, such as Entinostat (which targets HDAC1/2), have been developed. However, these still have the issue that HDAC1/2 are present in all four complexes, which the genetic evidence tells us have unique functions. Therefore, given the positive therapeutic value of HDAC inhibition in numerous disease states, and the detrimental side-effects of generic HDACi, the logical way forward is to perturb individual HDAC complexes. Our goal is to define a network of target genes and substrates (including non-histones), for each of the four HDAC1/2 complexes, beginning (in this application) with Sin3A. Sin3A is an essential complex in all organisms and cell types. In addition to HDAC1/2, it contains a large number of accessory proteins which are thought to fine-tune its activity in cells. Using a combination of cutting edge techniques (enDIP-iBAQ, ChIP-RX, Bio-ID and SILAC/mass-spec) we will examine i) the requirement for individual accessory proteins to Sin3A function (Aim1), ii) the composition of Sin3A complexes in different primary cell types (Aim2) and iii) the 'portion' of the acetylome (7,668 Lys-Ac sites in ES cells) are specifically regulated by the Sin3A complex (Aim3). By utilizing Sin3A as the paradigm, we will have a greater understanding of HDAC1/2 function in a cellular context, information that will be critical to their future use as therapeutic targets in numerous disease types, including, bipolar disorder, Alzheimer's disease, and cancer.

BBSRC Strategic Priorities: This work focusses on a fundamental gene regulatory mechanism that underpins normal cell homeostasis and health (Priority 3 - Biosciences for Health). Understanding how HDAC complexes function is also key to future efforts to develop improved HDACi for the treatment of different cancers and depression. Our combination of cutting-edge genomics and proteomics approaches is core to Enabling Theme 2: Exploiting new ways of working. The project is made possible by the BBSRC-SFI partnership, which forms a key part of Enabling Theme 3.

1. Commercial / Industrial -
There is a growing recognition within the pharmaceutical industry of the importance histone modifying enzymes as effective drug targets with several in the clinic or in trials. Histone deacetylase 1 and 2 (HDAC1/2) function has been implicated in almost all cellular processes including cell cycle progression, DNA repair, differentiation and cancer. HDAC enzymes are largely inactive on their own and have little inherent substrate specificity until assembled into their cognate multiprotein complexes, such as Sin3a, NuRD and Co-REST. By examining each of the four canonical HDAC1/2 complexes individually (beginning with Sin3A in this application) we will have a greater understanding of their roles in a physiological context (target genes, accessory proteins, substrates, etc.), information which that will be critical to their future use as therapeutic targets. Our long-term goal is to be able to specifically target individual HDAC complex functions. The effectiveness of BET family bromodomain inhibitors (e.g. JQ1), is proof of concept that perturbing individual chromatin recognition motifs is a practicable method for drugging individual multiprotein complexes. The University of Leicester has a vigorous and experienced 'Research and Enterprise Division' (RED) 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. Similarly, Dr. Bracken has worked with TCD Technology Transfer Office to register patent applications for research conducted in his lab.

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. Sin3A), will be necessary to understand the action of existing HDACi used clinically, and in the design of small molecules which inhibit HDAC function.

3. Skills development -
This grant will directly lead to the training of one staff member in the UK and two in Ireland. Staff will gain expertise in genomics, proteomics and bioinformatics research areas that will expand the skills base of the UK. As is often the case, the combination of diverse research skills will likely lead to further discoveries and technological advances as this expertise is propagated though the workforce. The exchange of knowledge between groups in the UK and Ireland will be of benefit to the science base in both countries. Staff will also gain training in skills transferable to the wider economy, including time management, communication, presentation, IT and programming and university teaching.