Epigenetic regulation of hormone action in Tribolium and Aedes

项目摘要 一直存在对开发靶标特异性的昆虫控制方法的持续需求。 保幼激素（JH）和蜕皮激素（20-羟基蜕皮激素，20 E是最活跃的形式）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 不存在于脊椎动物中，它们代表了开发昆虫控制方法的有吸引力的靶标。 阻碍这一努力的是对这些激素作用的分子基础缺乏了解。一直 最近在理解20 E和JH作用的分子基础方面取得了很好的进展。关键人物， 包括受体、共激活因子、应答元件和参与这些作用的靶基因。 激素已经被确认。已经发现了非甾体蜕皮激素激动剂和JH类似物， 商业化用于控制害虫和疾病媒介。该计划支持的研究表明， 表观遗传修饰，特别是由组蛋白乙酰转移酶调节的组蛋白乙酰化水平 (HAT)和组蛋白脱乙酰酶（HDAC）调节JH和20 E的作用。激素的作用需要多个层次 因为相同的激素循环水平在不同的组织中发挥不同的功能， 在不同的生理条件下。根据已发表的文献和本报告中包含的初步数据， 应用中，我们假设，表观遗传调节HDACs，HATs，组蛋白赖氨酸甲基转移酶， （HKMT）和组蛋白去甲基化酶（HDM）调节JH和20 E在调节胚后发育中的作用。 发育和繁殖。本提案的主要目标是通过研究 表观遗传修饰剂在JH和20 E作用中的作用机制使用T. castaneum、TcA细胞、Ae. aegypti 和Aag 2细胞作为模型系统。该提案的两个具体目标是1。以鉴定和表征 20 E和JH调节胚后发育和雌性所需的表观遗传修饰剂 繁殖和2。研究20 E和JH调节所需的表观遗传修饰剂的作用机制 胚胎后发育和雌性生殖的过程。在第一个特定目标，RNAi，CRISPR/Cas9 基因组编辑方法、RNA测序、生物信息学、RT-qPCR、酶测定和细胞培养 将使用各种方法来识别和表征HAT、HDAC、HKMT和HDM， 胚后发育和雌性生殖。castaneum和Ae.埃及人。在第二个具体 目的，将使用蛋白质印迹和ChIP研究10-12表观遗传修饰剂的作用机制， 报告基因、双杂交和下拉测定以及质谱法。拟议的结果 研究将通过为制定病媒控制方法提供信息来影响医学。这 这些信息有助于理解癌症等人类疾病的表观遗传学。的结果 拟议中的研究可能有助于改变我们对疾病媒介的内分泌学的理解。

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.