Exploring the role and implications of a Histone Deacetylase III (HDAC3) and NCOR/SMRT in cancer and embryonic stem cell differentiation

组蛋白去乙酰化酶（HDAC）通过去除组蛋白尾部赖氨酸残基的乙酰基片段，恢复其正电荷，收紧染色质以阻止基因转录，从而调节染色质的整体可及性。高度相关的i类hdac及其相关的共抑制因子复合物（Sin3, NuRD, CoREST， MiDAC和NCoR）在许多生物学过程中发挥作用，包括但不限于胚胎发育，细胞周期调节和细胞凋亡。本研究的主要目的是创建具有降解TAG （dTAG）的HDAC3，以阐明其在胚胎发育和癌症进展中的作用。该系统使用CRISPR/Cas9技术将突变体FKBP12F36V标签整合到目标蛋白（POI）的基因中，这样得到的融合蛋白（HDAC3-dTAG）可以被PROTAC分子靶向。蛋白靶向嵌合体（PROTACs）是一种异质双功能分子，它结合了高选择性E3连接酶和FKBP12F36V配体，利用细胞降解系统去除POI。HDAC3的快速降解（约2小时）将使我们能够在与基因调控和DNA合成相同的时间框架内检查其作用，这在以前的敲除模型中是不可能的。我们期望扩展我们对i类HDAC在细胞增殖和体内平衡中的功能的理解，这将为它们作为多种疾病类型的药物靶点提供信息。

Histone deacetylase (HDAC) enzymes regulate global chromatin accessibility by removing the acetyl moiety from Lys residues in histone tails, restoring their positive charge and tightening chromatin to prevent gene transcription. The highly related class-I HDACs and their associated co-repressor complexes (Sin3, NuRD, CoREST, MiDAC and NCoR) have roles in many biological processes including, but not limited to, embryonic development, cell cycle regulation and apoptosis. The primary aim of this research is to create a HDAC3 with a degradation TAG (dTAG) to elucidate its role in embryonic development and cancer progression. The system uses CRISPR/Cas9 technology to incorporate a mutant FKBP12F36V tag into the gene of the protein of interest (POI), such that the resulting fusion protein (HDAC3-dTAG) can be targeted with a PROTAC molecule. Protein targeting chimeras (PROTACs) are hetero-bifunctional molecules that incorporate highly-selective E3 ligase and FKBP12F36V ligands to exploit a cells degradation system to remove the POI. The rapid degradation of HDAC3 (~2 hours) will allow us to examine its role within the same time-frame as gene regulation and DNA synthesis, something not previously possible with knockout models. We expect to extend our understanding of class-I HDAC function in cell proliferation and homeostasis that will inform their use as drug targets in numerous disease types.