Novel mechanism of aryl hydrocarbon receptor - mediated differential gene regulation

摘要 芳香烃受体(AhR)是异种生物毒性的媒介，被认为是一种配体激活的功能。 与异源反应元件结合的转录因子(XRE，GCGTG基序)与其相关 异二聚化伙伴，AhR核转运子(ARNT)蛋白。这项建议是建立在我们的 桂皮酸(CA)介导的AhR依赖的锡钙素2(Stc2)诱导减弱的观察 应激在体外和体内均可诱导细胞凋亡并保护肝损伤。获得了stc2诱导 对内源性AhR激动剂CA而不是经典的外源性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cyp1a1在肝细胞中表达，不调节异种代谢。因此，stc2和cyp1a1 基因表现出相互排斥的激动剂特异性AhR介导的转录反应，具有不同的 生理后果。作为我K01研究的一部分，我们观察到与转移相关的相互作用 蛋白2(MTA2)，一种已知的染色质重塑蛋白，其AhR仅适用于CA处理。AHR-MTA2 复合体只与stc2启动子结合，而不与cyp1a1启动子结合。此外，CA治疗能够表现出 组蛋白H4赖氨酸5在stc2启动子上的乙酰化伴随着AhR-MTA2复合体的招募。 我们的初步研究证实，除了初级DNA序列外，染色质的结构 显著促进了激动剂特异性差异基因的表达。以我们的初步研究为基础 这一建议检验了CA特异性AhR依赖的表观遗传修饰和染色质的假设 重组决定了stc2的转录调控，而stc2又负责以下的细胞保护 肝脏损伤。本申请的特定目标1也将描述CA和TCDD特定的表观遗传修饰 AS将识别招募到XRE边界AhR-MTA2的组蛋白修饰的读取器-写入器-擦除器 很复杂。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stc2调节中的作用 使用CRISPR/Cas9基因编辑。特定目标3将询问CA触发stc2的机制 识别下游细胞保护通路成分对乙醇诱导的细胞凋亡的保护作用 由stc2激活。因此，此应用程序的目标是解码CA特定的AhR中介 Stc2的转录调控并破译其细胞保护功能。鉴于STC2的S参与了 对肝损伤的细胞保护，了解其CA特异性调节可能是发展的关键 针对肝病的未来分子治疗。

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.