Novel mechanism of aryl hydrocarbon receptor - mediated differential gene regulation

芳烃受体介导差异基因调控的新机制

• 批准号: 10612044
• 负责人: Aditya D Joshi
• 金额: $ 26.1万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-12 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10612044
• 关键词: ARNT gene; Acetylation; Acids; Address; Affect; Agonist; American; Apoptosis; Architecture; Aryl Hydrocarbon Receptor; Attenuated; Binding; Biology; CRISPR/Cas technology; CYP11A1 gene; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoprotection; DNA; DNA Binding; DNA Sequence; Data; Development; Dioxins; Epigenetic Process; Ethanol; Exhibits; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hepatocyte; Heterodimerization; Histone H4; Histones; In Vitro; Induction of Apoptosis; Intervention; Libraries; Ligand Binding; Ligands; Liver diseases; Lysine; Mass Spectrum Analysis; Mediating; Mediator; Metastasis-associated protein; Modification; Molecular; Molecular Conformation; Monitor; Oxidative Stress Induction; Pathway interactions; Peptide Signal Sequences; Physiological; Post-Translational Protein Processing; Promoter Regions; Property; Proteins; Quantitative Reverse Transcriptase PCR; RNA Interference; Reaction; Reader; Regulation; Research; Research Personnel; Response Elements; Role; Signal Pathway; Specificity; Stress; Testing; Tetrachlorodibenzodioxin; Therapeutic; Tissue-Specific Gene Expression; Toxic effect; Transcriptional Regulation; Tryptophan; Up-Regulation; Xenobiotic Metabolism; Xenobiotics; aryl hydrocarbon receptor ligand; chromatin immunoprecipitation; chromatin remodeling; cofactor; crosslink; epigenetic regulation; experimental study; histone modification; in vivo; liver injury; molecular targeted therapies; novel; promoter; receptor binding; recruit; response; screening; stanniocalcin 2; stressor; transcription factor; translational research program

ABSTRACT Aryl hydrocarbon Receptor (AhR), a mediator of xenobiotic toxicity, is known to function as a ligand-activated transcription factor that binds to a xenobiotic response element (XRE, GCGTG motif) in association with its heterodimerization partner, the AhR nuclear translocator (Arnt) protein. This proposal is built on our observation that cinnabarinic acid (CA) mediated AhR-dependent induction of stanniocalcin 2 (stc2) attenuates stress induced apoptosis and protects against liver injury both in vitro and in vivo. stc2 induction was achieved in response to endogenous AhR agonist CA but not the classic exogenous AhR ligand 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Moreover, CA in contrast to TCDD, was unable to trigger prototypical AhR target gene, cyp1a1 in hepatocytes and did not regulate xenobiotic metabolism. Therefore, stc2 and cyp1a1 genes exhibit mutually exclusive agonist-specific AhR-mediated transcriptional responsiveness with distinct physiological consequences. As a part of my K01 research, we observed interaction of Metastasis-associated protein 2 (MTA2), a known chromatin remodeling protein, with AhR exclusively upon CA treatment. AhR-MTA2 complex was recruited only to stc2, but not cyp1a1 promoter. In addition, CA treatment was able to exhibit histone H4 lysine 5 acetylation specifically at stc2 promoter concomitant with AhR-MTA2 complex recruitment. Our preliminary studies identified that chromatin architecture in addition to the primary DNA sequence significantly contributes to the agonist-specific differential gene expression. Building on our preliminary studies this proposal tests the hypothesis that CA-specific AhR-dependent epigenetic modifications and chromatin restructuring dictates transcription regulation of stc2, which in turn is responsible for cytoprotection following liver injury. Specific Aim 1 of this application will profile CA and TCDD specific epigenetic modifications as well as will identify the readers-writers-erasers of the histone modifications recruited to the XRE bounds AhR-MTA2 complex. Specific Aim 2 will determine role of chromatin architecture in agonist specific stc2 regulation by using CRISPR/Cas9 gene editing. Specific Aim 3 will interrogate mechanism by which CA-triggered stc2 protects against ethanol-induced apoptosis by identifying downstream cytoprotective pathway components activated by stc2. Therefore, the objective of this application is to decode CA-specific AhR-mediated transcription regulation of stc2 and decipher its cytoprotective function. Given stc2’s involvement in cytoprotection against liver injury, understanding its CA-specific regulation may serve as a key to developing future molecular therapeutics targeting hepatic diseases.

摘要 芳烃受体（AhR）是异生物质毒性的介质，已知其作为配体激活的受体发挥功能。 与异生素反应元件（XRE，GCGTG基序）结合的转录因子， 异二聚化配偶体，AhR核转运蛋白（Arnt）。这项建议是建立在我们的 观察到朱砂酸（CA）介导的斯钙素2（stc 2）的AhR依赖性诱导减弱了 应激诱导细胞凋亡，并在体外和体内保护肝损伤。实现了stc 2诱导 响应于内源性AhR激动剂CA，但不响应经典的外源性AhR配体2，3，7，8- 四氯二苯并对二恶英（TCDD）。此外，与TCDD相比，CA不能触发原型AhR 靶基因cyp 1a 1在肝细胞中表达，不调节外源性代谢。因此，stc 2和cyp 1a 1 基因表现出相互排斥的激动剂特异性AhR介导的转录反应性， 生理后果。作为我的K 01研究的一部分，我们观察到转移相关的 蛋白2（MTA 2），一种已知的染色质重塑蛋白，仅在CA处理后与AhR一起。AhR-MTA 2 复合物仅募集到STC 2，而不是CYP 1A 1启动子。此外，CA处理能够表现出 组蛋白H4赖氨酸5乙酰化特异性地在stc 2启动子处伴随AhR-MTA 2复合物募集。 我们的初步研究表明，除了主要的DNA序列外， 显著地有助于激动剂特异性差异基因表达。根据我们的初步研究 这一提议验证了CA特异性AhR依赖的表观遗传修饰和染色质 重组决定了stc 2的转录调节，stc 2反过来又负责细胞保护， 肝损伤本申请的具体目标1也将描述CA和TCDD特异性表观遗传修饰 这将确定XRE边界AhR-MTA 2招募的组蛋白修饰的读者-作者-擦除者 复杂.特异性目标2将通过以下方式确定染色质结构在激动剂特异性stc 2调节中的作用： 使用CRISPR/Cas9基因编辑。具体目标3将询问CA触发stc 2的机制 通过鉴定下游细胞保护途径组分来防止乙醇诱导的细胞凋亡 由STC 2激活。因此，本申请的目的是解码CA特异性AhR介导的细胞凋亡。 stc 2的转录调控，并破译其细胞保护功能。鉴于stc 2参与了 对肝损伤的细胞保护作用，了解其CA特异性调节可能是开发 未来针对肝病的分子治疗。