Uncovering the Regulatory Mechanisms of the Fork Head Transcription Factor

PROJECT SUMMARY/ABSTRACT The winged-helix DNA-binding FoxA transcription factors (TFs) play major roles in the development and homeostasis of many organs. Importantly, FoxA proteins are expressed early and continuously in many tissues and are essential both during development and postnatally for morphogenesis, glucose metabolism, and expression of multiple organ-specific genes. Mutations in FoxA proteins are linked to cancer in multiple tissue types (including the salivary gland), developmental defects that result in Parkinson-like phenotypes, and glucose dysregulation, causing hypoglycemia. However, the identity of the downstream effectors and the extent of coordination of FoxA proteins with other TFs to accomplish morphogenesis and regulate metabolic function are largely unknown. Identifying these downstream effectors and their mechanistic nuances becomes possible by studying the single Drosophila orthologue Fork head (Fkh) that represents all three FoxA members: FOXA1, FOXA2, and FOXA3. Like FoxA proteins in vertebrates, Fkh is required for morphogenesis and maintenance of organs in Drosophila, including the embryonic salivary gland (SG). In addition to its role in organ formation, Fkh is known to coordinate with two other TFs, Sage and Senseless (Sens), to maintain SG viability and to regulate the “secretome” – the collection of secreted products made by the SG. By performing ChIP-sequencing in parallel with scRNA-sequencing followed by computational analysis, we will uncover the mechanistic details of how Fkh coordinates with Sage and Sens to regulate the secretome (Aim 1). Furthermore, I will use our computational analysis to identify candidate partner TFs of Fkh to then perform a mutational screen and look for morphological phenotypes to characterize possible co-regulators of Fkh for morphogenesis (Aim 2). Through these proposed experiments in Aims 1 and 2, the mechanistic details of how Fkh coordinates with Sage and Sens to regulate the secretome can be elucidated while uncovering and characterizing co-regulators of Fkh for morphogenesis. In addition to controlling organ form and function, FoxA family proteins also play a role in metabolism. Currently, the SG is not known to have any metabolic roles but recent findings from our lab and others indicate that the SG secretome may have additional endocrine functions related to systemic growth. In Drosophila, salivary gland secreted factor (Sgsf) has been discovered as a SG secreted peptide that acts upon the AKT-MTOR pathway to ultimately affect systemic growth. By studying Fkhs role in regulating secreted factors from the SG, the novel function of the SG acting as endocrine organ can be further understood. To study this, I will utilize the biotinylating enzyme BirA*G3 in conjunction with mass spectrometry and confocal imagery to explore the potential secretion of metabolites from the SG. Candidate metabolites identified through these will then be tested for metabolic consequences within both the larval and adult stage (Aim 3). The outcomes from these experiments will provide new insights upon Fkh regulation of secretion and the SG acting as an endocrine organ.