Studying the role of the Sin3A/HDAC1 complex in DNA replication and Mitosis using novel therapeutic approaches

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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.