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

组蛋白去乙酰化酶1和2（HDAC 1/2）调节全局组蛋白乙酰化水平，作为四种不同的多蛋白复合物的共同组分：Sin 3A，NuRD，CoREST和MiDAC。这些复合物在所有核过程中具有决定性作用，包括DNA修复，DNA合成和基因表达。泛HDAC抑制剂（HDACi）在临床上用于治疗癌症和抑郁症，尽管它们的使用与几种使人衰弱的副作用有关。我们的目标是使用PROTAC方法开发针对四种HDAC 1/2复合物的小分子抑制剂，以了解它们在细胞中的功能并开发新的治疗方法。PROTAC是小的异源双功能分子，其掺入与E3泛素连接酶的配体偶联的与目的蛋白质（POI，例如抑制剂）的已知结合部分。通过PROTAC将E3连接酶直接募集到POI，使其靶向泛素化并最终降解。作为概念验证，我们已经用FKBP 12-F36 V结构域标记了胚胎干细胞（ESC）中Sin 3A的两个等位基因，使得它可以被已知的PROTAC dTAG-13降解。向培养基中加入dTAG-13导致Sin 3A蛋白在2小时内丧失，ESC活力在72小时丧失。在HDAC 1/2复合物的范围中，生存力的丧失是Sin 3A所独有的，我们希望在分子水平上了解原因。使用Sin 3A-FKBP 12-F36 V细胞，我们现在有能力首次解决Sin 3A生物学的绝对基本问题：（i）Sin 3A的直接转录靶点是什么，以及这些靶点如何促进活性转录;（ii）了解Sin 3A/HDAC 1在DNA复制和基因组稳定性中的需求;（iii）定义由Sin 3A/HDAC 1调节的Lys乙酰化的特定位点。此外，通过使用广泛的缺失突变体，我们可以鉴定关键的Sin 3A结构域（PAH 1，HID等），以及它们所介导的蛋白质-蛋白质相互作用，这是这些活动所必需的。

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.