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

“组蛋白脱乙酰基酶”（HDAC）是一类催化乙酰化赖氨酸中乙酰基去除的酶，几乎参与所有细胞过程，包括细胞周期、DNA合成、DNA修复和基因表达。哺乳动物中有18种HDAC，最初可以将其分类为具有Zn 2+依赖性（I类、II类和IV类）或NAD+依赖性（III类-沉默调节蛋白）催化结构域;然后进一步根据额外N末端结构域的存在和组织特异性表达模式（II类和IV类）或短C末端尾部和普遍表达（I类）。经典地，I类HDAC（HDAC 1、2和3）被认为作为典型的共阻遏物复合物（例如Sin 3A、NuRD和CoREST）的催化核心参与基因阻遏过程。Sin 3a复合物被认为通过转录因子和Sin 3相关蛋白（SAP）的组合被募集到染色质中，然后介导组蛋白脱乙酰化和随后的染色质致密化。临床上，通用HDAC抑制剂（HDACi）用于治疗抑郁症（丙戊酸）和皮下T细胞淋巴瘤（SAHA）。尽管有这种临床重要性，但几乎对其作用方式一无所知。此外，这些通用HDACi的使用与许多使人衰弱的副作用相关，包括疲劳、腹泻、低血小板计数（血小板减少症）和高氨血症，其可导致脑损伤。因此，考虑到HDAC抑制在许多疾病状态中的积极治疗价值，以及通用HDACi的可怕副作用，合乎逻辑的前进方向是破坏个体HDAC复合物。例如，Sin 3A/HDAC 1复合物已被证明在细胞周期调节中起关键作用，因此抑制另外的HDAC 1/2复合物（NuRD、CoREST和MiDAC）对于阻止癌细胞的生长可能是不必要的。特异性抑制单个HDAC复合物的一个关键挑战是了解它们是如何组装的，以及哪些辅因子对其功能至关重要。迄今为止，大多数主要的蛋白质组学研究的Sin 3A复杂的哺乳动物中使用癌细胞系。在这项提案中，我们的目标是确定所有必要的辅因子和底物（包括非组蛋白）的Sin 3A复合物在一系列的主要细胞类型。为了实现这一目标，我们将采用最先进的蛋白质组学和转录组学方法，在考利和布雷肯实验室开发。我们将追求的具体目标如下：i）评估干细胞特异性SAP Fam 60 a和Tet 1对细胞中Sin 3A功能的需要，ii）测试Sin 3A的组成在不同类型的原代细胞中是否相同，和iii）询问乙酰细胞素组的什么部分（在大多数细胞类型中，Lys-ac的约4，000个位点）由Sin 3A复合物特异性调节。通过使用Sin 3A作为I类HDAC复合物的范例，我们期望在细胞背景下扩展我们对HDAC复合物的理解。通过了解HDAC复合物如何发挥作用的分子基础，我们可以利用这些知识设计新药来治疗各种疾病，包括癫痫，双相情感障碍，阿尔茨海默病和癌症。

'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.