Abstract 1707 Elucidating the Role of Juvenile Hormones in Doublesex Regulation using Drosophila

Specification of ovaries and testes from a bipotential gonad is essential for animal reproduction. Across vertebrate and invertebrate animals, ovary and testis development requires the expression of Doublesex and Mab3-Related 1 (DMRT1), known simply as Doublesex (Dsx) in Drosophila where it was discovered. Dsx/DMRT1 is a zinc finger-like transcription factor that has sex-specific functions in a restricted number of tissues, including the gonads and brain. In many species, restricted function appears to be imparted by restricted expression, yet how dsx expression is regulated is unclear. Several studies in diverse invertebrate species implicate a family of isoprenoid small molecules called Juvenile Hormones (JHs). JHs are produced by a neuroendocrine gland and enter the cytoplasm of responding cells, where they bind to the bHLH-PAS transcription factor, Methoprene tolerant (Met). Upon ligand binding, Met translocates to the nucleus and binds to its co-factor Taiman (Tai) to influence transcription of target genes. Using publicly available modENCODE data, we have identified five Met/Tai-occupied sites upstream of the dsx locus in Drosophila. One of these sites contains two Met binding motifs approximately 600-800bp upstream of the dsx transcription start site, suggestive of an enhancer region for the dsx gene. We hypothesize that JHs regulates Dsx expression through direct transcriptional control. To test this hypothesis, we are investigating the transcriptional requirements of JH in Dsx expression by disrupting the JH receptor binding motifs upstream of the dsx gene using site-directed mutagenesis and CRISPR-mediated homology directed repair. We expect that disrupting dsx-proximal Met/Tai binding sites will result in a loss of dsx transcription and development of an intersexual phenotype. From this data, we hope to gain insight into the longstanding mystery of how Dsx expression is regulated, as the factors which contribute to its expression remain unclear. This work was supported by R00HD097306 from NICHD and start-up funds from UTSA for LJB.