Defining the molecular mechanisms of HDAC3 action in vivo

项目摘要 在许多最常见的人类疾病的病因学中，组织内的代谢功能障碍是一种成核事件 今天的疾病。Lazar实验室的一个主要目标是定义控制 代谢动态平衡，并描绘这些系统是如何在疾病进展期间被破坏的。组蛋白 脱乙酰基酶3(HDAC3)是一种I类脱乙酰基酶，通过与核受体辅阻遏物相互作用 NCoR或SMRT，驱动抑制染色质重塑，从转录上调节关键的代谢途径。 通过小鼠基因敲除研究证明了HDAC3的基本功能；全身基因敲除是 致命的，而组织特异性的缺失会导致过多的不适应表型，包括致命的感冒 棕色脂肪组织不耐受(BAT)和肝脏大量脂肪变性。然而，仍然有很大的 我们对这些表型背后的组织特异性机制的理解存在差距。近期工作 表明HDAC3不仅通过NCoR/SMRT抑制转录，而且也是激活 特定关键基因在特定环境中的转录。虽然压制作用已经被描述过了，但它 目前尚不清楚HDAC3是如何激活特定位点的转录的。此外，来自Lazar实验室和其他人的工作 已经证明HDAC3具有不依赖于其脱乙酰酶活性的不可或缺的功能。这个 这项建议的目的是询问HDAC3调节A基因转录的具体机制 不同的代谢途径阵列以组织特有的方式。具体目标1是识别组织特异性 棕色脂肪组织和肝脏中HDAC3蛋白-蛋白的相互作用。Lazar实验室开创了一种新的 核提取亲和标记芯片-质谱仪鉴定定量的方法 体内的蛋白质-蛋白质相互作用。我们将利用这种方法来表征BAT中的HDAC3互动组 并将其与肝脏相互作用组进行比较，以确定共同的和组织特异性的相互作用。在肝脏中， HDAC3基因敲除导致脂肪生成基因的抑制，最终导致脂肪肝。相反，HDAC3 蝙蝠体内的KO导致不能激活关键的生热基因的表达。对这些组织的依赖 关于抑制(肝脏)和激活(BAT)HDAC3的活性对HDAC3的兴趣和比较 在这两个背景下的相互作用组对这些不同的功能产生了重要的见解。具体目标2是 确定体内HDAC3的酶底物。HDAC3缺失导致的脂肪肝在很大程度上得到了挽救 通过表达催化失活的HDAC3。相反，缺乏HDAC3活性的小鼠由于 NCoR和SMRT的突变显示出致命的耐寒，类似于蝙蝠的HDAC3基因敲除。我们将使用 在WT、KO和突变模型中亲和富集乙酰多肽，然后进行蛋白质组学分析以确定， 首次报道了HDAC3在肝脏和BAT中的催化底物。这些创新的研究解决了Long 有关HDAC3功能的长期问题。总而言之，最先进的“组学”方法的结合 将对这一关键代谢调节剂的作用机制产生新的见解。

Project Summary Metabolic dysfunction within a tissue is a nucleating event in the etiology of many of the most prevalent human diseases today. A primary goal of the Lazar laboratory is to define the transcriptional mechanisms that control metabolic homeostasis and delineate how these systems are disrupted during disease progression. Histone deacetylase 3 (HDAC3) is a class I deacetylase that, through interaction with the nuclear receptor corepressors NCoR or SMRT, drives repressive chromatin remodeling to transcriptionally regulate critical metabolic pathways. The essential function of HDAC3 is demonstrated through murine knockout studies; whole body knockout is lethal while tissue specific deletions result in a plethora of maladaptive phenotypes including lethal cold intolerance in brown adipose tissue (BAT) and massive hepatic steatosis in liver. However, there are still large gaps in our understanding of the tissue-specific mechanisms that underlie these phenotypes. Recent work indicates that HDAC3 not only represses transcription via NCoR/SMRT, but is also necessary to activate the transcription of specific and essential genes in certain contexts. While the repressive role has been described, it is unknown how HDAC3 activates transcription at specific loci. In addition, work from the Lazar lab and others has shown that HDAC3 has indispensable functions that are not dependent upon its deacetylase activity. The goal of this proposal is to interrogate the specific mechanisms by which HDAC3 regulates transcription of a diverse array of metabolic pathways in a tissue-specific manner. Specific Aim 1 is to identify tissue specific HDAC3 protein-protein interactions in brown adipose tissue and liver. The Lazar lab has pioneered a new method called NEAT ChIP-MS (Nuclear Extraction Affinity Tag ChIP-mass spec) to identify and quantitate protein-protein interactions in vivo. We will utilize this method to characterize the HDAC3 interactome in BAT and compare it to the liver interactome in order to define common and tissue-specific interactions. In the liver, knockout of HDAC3 leads to de-repression of lipogenic genes, ultimately causing fatty liver. Conversely, HDAC3 KO in BAT leads to the inability to activate expression of critical thermogenic genes. The reliance of these tissues on repressive (liver) and activating (BAT) HDAC3 activity is of great interest and comparison of the HDAC3 interactome in both contexts with yield important insights on these divergent functions. Specific Aim 2 is to define the enzymatic substrates of HDAC3 in vivo. Fatty liver as a result of HDAC3 deletion is largely rescued through the expression of catalytically inactive HDAC3. Conversely, mice lacking HDAC3 activity due to mutations in NCoR and SMRT display lethal cold intolerance, mimicking HDAC3 knockout in BAT. We will use affinity enrichment of acetyl peptides followed by proteomic analysis in WT, KO, and mutant models to determine, for the first time, the catalytic substrates of HDAC3 in liver and BAT. These innovative studies address long standing questions regarding HDAC3 function. Together, the combination of state-of-the-art ‘omics’ approaches will generate new insights into the mechanism of action of this critical metabolic regulator.