Defining the molecular mechanisms of HDAC3 action in vivo

定义 HDAC3 体内作用的分子机制

• 批准号: 9910885
• 负责人: Amy Hauck
• 金额: $ 6.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910885
• 关键词: Acetylation; Address; Affinity; Age; Brown Fat; Cardiovascular Diseases; Cell physiology; Cessation of life; Complement; Complex; Data; Deacetylase; Diabetes Mellitus; Diet; Disease; Disease Progression; Embryo; Environment; Enzymes; Epitopes; Essential Genes; Etiology; Event; Fatty Liver; Gene Expression; Genes; Genetic Transcription; Goals; HDAC3 gene; HDAC4 gene; Histone Deacetylase; Histones; Homeostasis; Interruption; Knock-in Mouse; Knock-out; Knowledge; Laboratories; Light; Liver; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Control; Metabolic Diseases; Metabolic Pathway; Metabolic dysfunction; Metabolism; Methods; Modeling; Modification; Molecular; Mus; Mutation; Nuclear; Nuclear Extract; Nuclear Receptors; Obesity; Peptides; Phenotype; Physiological; Play; Process; Proteins; Proteomics; Regulation; Repression; Research; Role; Site; Stimulus; System; Techniques; Testing; Time; Tissues; Training; Transcript; Transcription Coactivator; Transcriptional Regulation; Work; career; cellular targeting; chromatin remodeling; environmental stressor; epigenome; functional genomics; genetic regulatory protein; genome-wide; genomic data; human disease; in vivo; innovation; insight; interest; metabolic phenotype; mouse model; mutant; novel; preference; protein protein interaction; response

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.

项目摘要 组织内的代谢功能障碍是许多最普遍的人类疾病的病因学中的成核事件。 今天的疾病拉扎尔实验室的主要目标是确定控制转录的转录机制， 代谢稳态和描绘这些系统是如何在疾病进展过程中被破坏。组蛋白 脱乙酰酶3（HDAC 3）是I类脱乙酰酶，其通过与核受体辅阻遏物相互作用 NCoR或SMRT驱动抑制性染色质重塑以转录调节关键代谢途径。 通过小鼠基因敲除研究证明了HDAC 3的基本功能;全身敲除是 而组织特异性缺失导致过多的适应不良表型， 棕色脂肪组织（BAT）中的不耐受和肝脏中的大量脂肪变性。然而，仍有大量 我们对这些表型背后的组织特异性机制的理解存在差距。最近的工作 表明HDAC 3不仅通过NCoR/SMRT抑制转录，而且也是激活转录所必需的。 在某些情况下，特定和必需基因的转录。虽然已经描述了镇压作用， HDAC 3如何激活特定基因座的转录尚不清楚。此外，拉扎尔实验室和其他人的工作 已经表明HDAC 3具有不依赖于其脱乙酰酶活性的不可缺少的功能。的 这项建议的目的是询问HDAC 3调节转录的具体机制， 以组织特异性方式的多种代谢途径。具体目标1是识别组织特异性 棕色脂肪组织和肝脏中HDAC 3蛋白-蛋白相互作用。拉扎尔实验室开创了一种新的 一种称为NEAT ChIP-MS（核提取亲和标记ChIP-mass spec）的方法，用于识别和定量 体内蛋白质-蛋白质相互作用。我们将利用这种方法来表征BAT中的HDAC 3相互作用组 并将其与肝脏相互作用组进行比较，以确定共同和组织特异性相互作用。在肝脏中， HDAC 3的敲除导致脂肪生成基因的去抑制，最终导致脂肪肝。HDAC3 BAT中的KO导致不能激活关键产热基因的表达。对这些组织的依赖 抑制性（肝脏）和激活性（BAT）HDAC 3活性的研究引起了极大的兴趣， 在这两种情况下的interactome与这些不同的功能产生重要的见解。具体目标二是 定义HDAC 3在体内的酶底物。HDAC 3缺失导致的脂肪肝在很大程度上得到了拯救 通过表达无催化活性的HDAC 3。相反，由于缺乏HDAC 3活性， NCoR和SMRT中的突变显示出致命的冷耐受，模拟BAT中的HDAC 3敲除。我们将使用 在WT、KO和突变体模型中亲和富集乙酰肽，然后进行蛋白质组学分析以确定， 第一次，HDAC 3在肝脏和BAT中的催化底物。这些创新的研究解决了长期 关于HDAC 3功能的常见问题。总之，最先进的“组学”方法 将产生新的见解的作用机制，这一关键的代谢调节。