Epigenetic and posttranslational modifier regulation of Juvenile hormone action

There has been a continuous demand for the 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 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 of 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) mediated acetylation of histone H3 as well as 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 posttranslational modification of proteins involved in JH action play critical roles in multi-dimensional 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 posttranslational 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, we will employ RNAi, cell culture and reporter assays to identify and characterize key proteins including histone acetyl transferases (HAT), histone deacetylases (HDAC) and chromatic modifiers involved in modulation 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 that transmit deadly diseases. The proposed studies will also advance our knowledge in epigenetic and post-translational modification of histones and other nuclear proteins especially on their functions and mechanisms of action.