Epigenetic and post-translational modifier regulation of juvenile hormone action

 描述（由申请人提供）：一直存在开发靶标特异性昆虫控制方法的持续需求。保幼激素和蜕皮激素是调节昆虫发育和生殖的主要激素。由于这些激素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目标。阻碍这一努力的是缺乏对JH作用的分子基础的理解。在过去的几年里，在分子水平上理解JH的作用已经取得了巨大的进展。一种bHLH转录因子，耐甲氧普烯（Met），已被确定为JH受体。类固醇受体共激活剂同源物（SRC/Taiman/FISC）和周期已被确定为参与JH作用的重要辅助因子。Kruppel同源物（Kr-h1）和bHLH转录因子、Hairy和E93已被鉴定为介导JH作用的重要转录因子。初步实验表明，环AMP反应元件结合蛋白（CBP）是赤拟谷盗组织、TcA和Aag-2细胞中组蛋白H3乙酰化以及JH诱导JH反应基因所必需的。此外，组蛋白去乙酰化酶（HDAC）抑制剂曲古抑菌素A（TSA）也诱导TcA和Aag-2细胞中JH反应基因的表达。染色质修饰物Brahma、Snr 1和CHD 1是调节T.栗色。JH在不同的组织和不同的生理条件下发挥不同的功能。我们推测，表观遗传和翻译后修饰的蛋白质参与JH行动发挥关键作用，在多维度基因调控策略采用JH。因此，本提案的主要目标是利用这些最新的发展，以确定表观遗传修饰剂，并确定其作用机制，在调制JH反应使用T。castaneum、TcA和Aag-2细胞。该提案的两个具体目标是1。确定参与JH作用的表观遗传和翻译后修饰调控的关键参与者;确定JH作用的表观遗传和翻译后修饰调控机制。在第一个具体目标中，我们将采用RNAi，细胞培养和报告基因测定来鉴定和表征关键蛋白，包括组蛋白乙酰基转移酶（HAT），HDAC和参与JH作用调节的染色质修饰剂。时间，空间和激素调控的基因，以及它们的功能，JH行动将被确定。在第二个具体目标中，我们将采用质谱、RNAi、双杂交、下拉、芯片和报告基因测定来鉴定Met中的乙酰化赖氨酸、JH作用所需的HAT、HDAC和Brahma、Snr 1和CHD 1的结构域，将HAT、HDAC和Brahma、Snr 1和CHD 1定位在Kr-h1、Hairy和E93启动子上，并鉴定HAT、HDAC、Brahma、Snr 1和CHD 1的相互作用伴侣。本研究的预期结果，JH行动的分子基础的理解和参与JH行动的关键蛋白质的鉴定将有助于控制昆虫媒介的方法的发展。这些研究也将促进我们对组蛋白和其他核蛋白的表观遗传和翻译后修饰的认识，特别是对修饰剂的功能和作用机制的认识。

 DESCRIPTION (provided by applicant): There has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids are the major hormones that regulate development and reproduction in insects. Since these hormones are not present in vertebrates, they represent attractive targets for the development of insect control methods. Hindering this effort is the lack of understanding on the molecular basis of JH action. Tremendous progress has been made in understanding JH action at the molecular level during the past few years. A bHLH transcription factor, methoprene tolerant (Met), has been identified as a JH receptor. A steroid receptor co-activator homologue (SRC/Taiman/FISC) and Cycle have been identified as important co-factors involved in JH action. Kruppel homologue (Kr-h1) and bHLH transcription factor, Hairy and E93 have been identified as important transcription factors that mediate JH action. Preliminary experiments showed that Cyclic AMP response-element binding protein (CBP) is required for acetylation of histone H3 as well as for JH induction of JH-response genes in Tribolium castaneum tissues, TcA and Aag-2 cells. In addition, histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA) also induces expression of JH-response genes in TcA and Aag-2 cells. Chromatin modifiers Brahma, Snr1 and CHD1 but not DNA methyl transferase are required for regulation of metamorphosis and reproduction in T. castaneum. JH exerts diverse functions in different tissues and under various physiological conditions. We hypothesize that epigenetic and post- translational modification of proteins involved in JH action play critical roles in multi-dimensionl gene regulation strategies employed by JH. Therefore, the main objective of this proposal is to capitalize on these latest developments to identify epigenetic modifiers and determine their mechanisms of action in modulation of JH response using T. castaneum, TcA and Aag-2 cells. The two Specific Aims of the proposal are 1. To identify key players involved in epigenetic and post-translational modifier regulation of JH action and 2. To determine the mechanisms involved in epigenetic and post-translational modifier regulation of JH action. In the first Specific Aim, w will employ RNAi, cell culture and reporter assays to identify and characterize key proteins including histone acetyl transferases (HAT), HDACs and chromatic modifiers involved in modulation of JH action. Temporal, spatial and hormonal regulation of identified genes as well as their function in JH action will be determined. In the second Specific Aim, we will employ mass spectrometry, RNAi, two-hybrid, pull- down, Chip and reporter assays to identify acetylated lysines in Met, domains of HATs, HDACs and Brahma, Snr1 and CHD1 required for JH action, localize HATs, HDACs and Brahma, Snr1 and CHD1 on Kr-h1, Hairy and E93 promoters and identify interacting partners of HATs, HDACs, Brahma, Snr1 and CHD1. The expected outcome of this study, an understanding of the molecular basis of JH action and identification of critical proteins involved in JH action will help in development of methods for control of insect vectors. The proposed studies will also advance our knowledge on epigenetic and post-translational modification of histones and other nuclear proteins especially on the functions and mechanisms of action of the modifiers.