Understanding the unique properties of the Sin3A histone deacetylase complex in transcription and cell viability

Histone deacetylases (HDACs) are a family of enzymes which help regulate histones, the packaging material for our genetic material, DNA. There are 18 individual HDACs in human cells all with a subtly different functions. Our group studies HDAC1 and HDAC2 (HDAC1/2), two highly related enzymes that are found together as components of large, multi-protein constructs, known as complexes. As part of four distinct complexes, HDAC1/2 helps regulate which of the 20,000 genes in our DNA are switched on, and just as importantly, which ones are switched off. The combination of genes on, and genes off, is what makes each particular cell type unique, and why we don't have teeth in our eyeballs..!

Drugs which inhibit HDACs, known as HDAC inhibitors (HDACi) are used to treat epilepsy, depression and leukemia. However, the use of HDACi in patients is associated with debilitating side-effects. Given the positive therapeutic value of HDAC inhibition in numerous diseases, and the detrimental side-effects of our existing drugs, there is a strong imperative to design novel HDACi with improved specificity and alternative modes of action. Our long-term goal is to develop novel inhibitors against each of the four HDAC1/2 complexes using a PROTAC approach, to understand their function in cells and develop novel therapeutics. PROTACs are a new type of drug that both inhibit an enzyme AND cause it to be marked for destruction by the cells internal rubbish disposal system. We have recently published and patented the first PROTACs target against HDAC1/2. To extend this work, we have engineered cells in which a known PROTAC drug is able to target an individual HDAC1/2 complex, known as, Sin3A.

Sin3A binds directly to HDAC1 and together they help control the accessibility of DNA, by modifying histones, which helps regulate our genes and maintains DNA integrity. The advantage of our new cells is that we are able to destroy the Sin3A/HDAC1 complex by simply adding a drug to cells. It is degraded in just 2 hours, enabling us to perform experiments that look directly at its role in gene-regulation (switching genes on and off), and in the generation of new DNA strands prior to cell division. In addition, using specific technology known as, mass-spectrometry, we will be able to identify the precise sites of action in histones. By identifying the Sin3A-dependent activity, which genes are affected by its loss, other proteins it interacts with, which sites in histones are modified, we will make significant strides to understanding its role in cells. This information informs the use of HDACi already in use in the clinic, and potentially extends the roles of HDAC1/2 complexes like Sin3A, so that they might be the drug targets of tomorrow.

Histone deacetylase 1 and 2 (HDAC1/2) regulate global histone-acetylation levels as common components of four distinct multi-protein complexes: Sin3A, NuRD, CoREST and MiDAC. These complexes have definitive roles in all nuclear processes, including DNA repair, DNA synthesis and gene expression. Pan-HDAC inhibitors (HDACi) are used in the clinic to treat cancer and depression, although their use is associated with several debilitating side-effects. Our goal is to develop small-molecule inhibitors against each of the four HDAC1/2 complexes using a PROTAC approach, to understand their function in cells and develop novel therapeutics. PROTACs are small hetero-bifunctional molecules which incorporate a known binding moiety to the protein of interest (POI, e.g. an inhibitor), coupled to a ligand for an E3 ubiquitin ligase. Direct recruitment of the E3 ligase to the POI via the PROTAC, targets it for ubiquitination and ultimately degradation. As a proof-of-concept, we have tagged both alleles of Sin3A in embryonic stem cells (ESCs) with an FKBP12-F36V domain, so that it can be degraded by a known PROTAC, dTAG-13. Addition of dTAG-13 to the culture media results in loss of Sin3A protein within 2 hours and a loss of ESC viability by 72 hours. Loss of viability is unique to Sin3A among the range of HDAC1/2 complexes and we would like understand why, at molecular level. Using Sin3A-FKBP12-F36V cells, we now have the ability to address absolutely fundamental questions of Sin3A biology for the first time: (i) what are the direct transcriptional targets of Sin3A and how do these contribute to active transcription; (ii) understand the requirement for Sin3A/HDAC1 in DNA replication and genome stability; and (iii) define specific sites of Lys-acetylation regulated by Sin3A/HDAC1. Furthermore, by using a comprehensive range of deletion mutants we can identify the critical Sin3A domains (PAH1, HID, etc.), and protein-protein interactions they mediate, required for these activities.