Epigenetic Regulation of Hormone Action in Tribolium and Aedes

SPECIFIC AIMS Insect hormones similar to those in humans, other animals and plants regulate multiple processes involved in growth, development, metamorphosis and reproduction. Some insects compete with us for food and transmit human and livestock diseases, but others are beneficial. Therefore, there has been a continuous demand for insect control methods that are target-specific. Juvenile hormones (JH) and ecdysteroids (20- hybroxyecdysone, 20E, is the most active form) are among the major hormones that regulate almost every aspect of insect life. Since these hormones are not present in vertebrates, they represent attractive targets for the development of environmentally-friendly insect control methods. During the last two decades, there has been excellent progress in understanding the molecular basis of ecdysteroid and JH action [1-4]. The ecdysone receptor (EcR) binds to its heterodimeric partner utraspiracle (USP), ligand 20E and ecdysone response elements (EcRE) and regulates expression of ecdysone-response genes such as E75, E74, E93 and Broad complex (BR-C) and the products of these genes regulate many physiological, developmental and reproductive processes [1-3]. A bHLH transcription factor, methoprene tolerant (Met), has been identified as a JH receptor [5-7]. The Met binds to JH and steroid receptor co-activator homolog (SRC/Taiman/FISC) [15-18] and JHRE present in the promoters of JH-response genes, Krüppel homolog (Kr-h1) and Hairy [8-11] and directly regulate their expression. The Kr-h1 regulates expression of E93, a HTH transcription factor plays an important role in both 20E and JH action [12, 13] and BR-C [14] and suppresses metamorphosis. Non-steroidal ecdysone agonists and JH analogs have been discovered, and commercialized for the control of pests and disease vectors [15-17]. 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 in regulation of postembryonic development in Tribolium castaneum and Aedes aegypti [18-23]. Hormone action requires multiple layers of regulation because the same circulating levels of hormones exert diverse functions in different tissues and under various physiological conditions. Based on the published literature and preliminary data included in this application, we hypothesize that epigenetic regulators HDACs, HATs, histone methyltransferase (HMT) and histone demethylases (HDM) modulate 20E and JH action in regulation of postembryonic development and reproduction. The main goal of this proposal to test this hypothesis by studying the mechanisms of action of epigenetic regulators in JH and 20E regulation of postembryonic development and reproduction using T. castaneum, TcA cells, Aedes aegypti and Aag-2 cells as model systems. Well annotated genome sequence, functioning of systemic RNA interference (RNAi) and excellent response to 20E and JH make T. castaneum an excellent model insect for proposed studies. Recent advances in RNAi, CRISPR/Cas9 mediated genome editing methods, and well-studied 20E and JH action in adult females allow us to conduct proposed research in Ae. aegypti as a model insect for disease vectors. Specific Aim 1: Identification and characterization of epigenetic modifiers required for 20E and JH regulation of postembryonic development and female reproduction. We will employ RNAi and CRISPR/Cas9 genome editing methods to identify HATs, HDACs, HMTs and HDMs required for postembryonic development and female reproduction in T. castaneum and Ae. aegypti. RNA sequencing, bioinformatics analysis, RT-qPCR, enzyme assays and cell culture approaches will be employed to study developmental expression, 20E and JH regulation, enzyme activity, target genes and pathways regulated by identified epigenetic modifiers. Specific Aim 2: Uncover mechanisms of action of epigenetic modifiers required for 20E and JH regulation of postembryonic development and female reproduction. The mechanisms of action of 6-8 epigenetic modifiers identified by experiments proposed under Specific Aim 1 will be studied. Quantification of levels of acetylation, deacetylation, methylation and demethylation of target proteins and their enrichment at the promoters of target genes will be performed by employing Western blots, ChIP and ELISA assays using antibodies that recognize modified proteins. Reporter, Two-hybrid and pull-down assays and mass spectrometry approaches will be employed to identify interactions of epigenetic modifiers with partner proteins at the promoters of target genes. Overall Impact: Results from the proposed research will impact medicine by providing information for the development of vector control methods. The results from these studies will also advance our knowledge in epigenetic modification of histones and other proteins, especially on their functions and mechanisms of action. Most studies on epigenetics focus on the identification of epigenetic marks. The proposed studies will reveal functions and mechanisms of action of epigenetic marks and help in understanding epigenetics of human diseases such as cancer. The interdisciplinary and complementary approaches proposed in two Specific aims will ensure success because they are based on the methods that are currently being used in the PI’s laboratory. Not much is known on epigenetic regulation of hormone action. Therefore, the proposed research will lead to novel findings that may transform our understanding of endocrinology of the disease vectors. 01