Histone acetylation is a key epigenetic mechanism controlling chromatin structure and DNA accessibility. The levels of acetylation are controlled by the opposing activities of histone acetyl transferases (HAT) and histone deacetylase (HDAC) enzymes. Approximately 50% of the total HDAC activity in cells is contributed by HDAC1 and HDAC2 (HDAC1/2), highly homologous proteins found in one of four multi-protein complexes (Sin3, NuRD, CoREST and MiDAC). Incorporation into these complexes is crucial for HDAC1/2 function as they activate enzymatic activity and determine substrate specificity. Inhibition of HDAC1/2, using knock-outs and knock-down, has indicated important roles in cell cycle progression, DNA damage repair, DNA synthesis and the regulation of gene expression. HDAC inhibitors have shown positive effects in a number of diseases states (cancer, epilepsy, neurodegenerative disease, HIV infection, etc.), but their use in patients is associated with debilitating side-effects. We propose to target HDACs more specifically by targeting individual HDAC complexes, starting with Sin3.
Sin3A and Sin3B (Sin3A/B) are essential genes, which are ubiquitously expressed, and have roles in spermatogenesis, kidney, bone, muscle and T-cells. These wide-ranging properties make Sin3A/B attractive therapeutic targets. Our hypothesis is that inhibition of key domains will result in a loss of HDAC1/2 functionality associated with the Sin3 complex. This project therefore aims to better understand how the Sin3 complex is put together in order to develop tools to pull it apart.
Aim 1: Sin3A/B contain four protein:protein docking motifs called ‘PAH’ domains, a four helical bundle which bind to short (12aa) helical Sin3 interaction domains (SID) of transcription factors (Mxd1, PLZF, etc.) and epigenetic regulators (Tet1) with high affinity and specificity. We propose to generate protein based ‘PAH-blockers’ to target the Sin3 complex therapeutically in a range of cancer cell lines.
Aim2: The catalytic core of Sin3A/B is a ternary complex consisting of the HDAC interaction domain (HID) of Sin3, SUDS3 and HDAC1. We have optimized expression of the ternary complex in 293F cells, and used this for biophysical analysis. We plan to extend this work by solving an atomic resolution structure of the complex using the Titan Krios cryo-electron microscope recently awarded to the Leicester Institute of Structural and Chemical Biology (LISCB).
Closing date for applications: 11th February 2018
Interview date: February 2018, TBC