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

点击翻译按钮获取中文摘要

'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.