Epigenetic regulation of hormone action in Tribolium and Aedes

项目总结 人们一直需要开发针对特定目标的昆虫控制方法。 保幼激素(JH)和蜕皮激素(20-羟基蜕皮酮，20E是最有效的形式)是主要的 调节昆虫许多生理和发育过程的激素。因为这些荷尔蒙 它们不存在于脊椎动物中，它们是开发昆虫控制方法的诱人靶标。 阻碍这一努力的是对这些激素作用的分子基础缺乏了解。那里有 最近在理解20E和JH作用的分子基础方面取得了很好的进展。关键人物， 包括参与这些作用的受体、共激活子、反应元件和靶基因 荷尔蒙已经被识别出来。已发现非甾体蜕皮激素激动剂和JH类似物，以及 用于控制害虫和病媒的商业化。该计划支持的研究表明， 组蛋白乙酰转移酶调控的表观遗传修饰，特别是组蛋白的乙酰化水平 组蛋白脱乙酰酶(HDAC)和组蛋白脱乙酰酶(HAT)调节JH和20E的作用。荷尔蒙的作用需要多层 因为相同的激素循环水平在不同的组织和组织中发挥不同的功能 在不同的生理条件下。基于已发表的文献和包含在 应用，我们假设表观遗传调节因子HDACs、HATS、组蛋白赖氨酸甲基转移酶 (HKMT)和组蛋白去甲基酶(HDM)调节JH和20E在胚胎后调控中的作用 发展和繁殖。这项提议的主要目的是通过研究 表观遗传修饰剂在JH和20E中的作用机制利用卡氏锥虫、TCA细胞、Ae.埃及伊蚊 以Aag2细胞为模型系统。该提案的两个具体目标是：1.确定和描述 20E和JH调控胚胎后发育和雌性所需的表观遗传修饰物 生殖和2.研究20E和JH调控所需的表观遗传修饰物的作用机制 胚胎后发育和雌性生殖。在第一个具体目标中，RNAi、CRISPR/Cas9 基因组编辑方法、RNA测序、生物信息学、RT-qPCR、酶分析和细胞培养 将使用各种方法来确定和确定以下各项所需的帽子、HDAC、HKMT和HDMS的特征 卡氏锥虫和Ae.胚胎后发育和雌性生殖。埃及伊蚊。在第二个具体例子中 目的：利用蛋白质印迹和芯片技术研究10-12种表观遗传修饰剂的作用机制。 记者，双杂交和下拉分析，以及质谱学方法。建议的结果 研究将通过为媒介控制方法的发展提供信息来影响医学。这 信息可能有助于理解癌症等人类疾病的表观遗传学。结果来自于 拟议的研究可能有助于改变我们对病媒内分泌学的理解。

PROJECT SUMMARY There has been a continuous demand for development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids (20-hydroxyecdysone, 20E is the most active form) are the major hormones that regulate many physiological and developmental processes in insects. Since these hormones are not present in vertebrates, they represent attractive targets for development of insect control methods. Hindering this effort is the lack of understanding on the molecular basis of action of these hormones. There has been excellent recent progress in understanding the molecular basis of 20E and JH action. Key players, including receptors, co-activators, response elements and target genes involved in the action of these hormones have been identified. Non-steroidal ecdysone agonists and JH analogs have been discovered, and commercialized for the control of pests and disease vectors. Research supported by this program showed that epigenetic modifications, especially acetylation levels of histones regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC) modulate JH and 20E action. Hormone action requires multiple layers of regulation because the same circulating levels of hormones exert diverse functions in different tissues and under different physiological conditions. Based on the published literature and preliminary data included in this application, we hypothesize that epigenetic regulators HDACs, HATs, histone lysine methyltransferases (HKMT) and histone demethylases (HDM) modulate JH and 20E action in the regulation of postembryonic development and reproduction. The main goal of this proposal to test this hypothesis by studying the mechanisms of action of epigenetic modifiers in JH and 20E action using T. castaneum, TcA cells, Ae. aegypti and Aag2 cells as model systems. The two Specific Aims of the proposal are 1. To identify and characterize epigenetic modifiers required for 20E and JH regulation of postembryonic development and female reproduction and 2. To study mechanisms of action of epigenetic modifiers required for 20E and JH regulation of postembryonic development and female reproduction. In the first Specific Aim, RNAi, CRISPR/Cas9 genome editing methods, RNA sequencing, bioinformatics, RT-qPCR, enzyme assays and cell culture approaches will be used to identify and characterize HATs, HDACs, HKMTs and HDMs required for postembryonic development and female reproduction in T. castaneum and Ae. aegypti. In the second Specific Aim, the mechanisms of action of 10-12 epigenetic modifiers will be studied using western blots and ChIP, reporter, Two-hybrid and pull-down assays, and mass spectrometry approaches. Results from the proposed research will impact medicine by providing information for the development of vector control methods. This information could help in understanding epigenetics of human diseases such as cancer. The results from the proposed research may help to transform our understanding of endocrinology of disease vectors.