SAT-032 FOXO1 Regulation of Neurod4 Enhancer Activity in Somatotrope Differentiation

Abstract Disclosure: R.A. Abubakar: None. B.S. Ellsworth: None. Growth hormone (GH) plays a critical role in growth and metabolism. GH deficiency (GHD), sometimes arising from impaired somatotrope differentiation, results in metabolic and cardiovascular complications. NEUROD4, a transcription factor essential for brain development and somatotrope differentiation, may be regulated by the forkhead factor, FOXO1. FOXO1 is also known to promote somatotrope differentiation, however the molecular mechanisms underlying this regulation remain unclear. This study investigates FOXO1’s direct role in modulating an intronic Neurod4 enhancer and its implications for somatotrope differentiation and function. Constructs containing either a wild-type (WT) Neurod4 enhancer (Nd4PRLpGL3) or a FOXO consensus site mutant enhancer (Nd4FOXOmtPRLpGL3) coupled with a prolactin minimal promoter were transfected into mouse somatotrope-like GH3 cells. Mutation of the FOXO consensus site within the Neurod4 enhancer reduces luciferase activity by approximately 50% compared to the WT enhancer, suggesting that FOXO1 directly activates the enhancer. The Neurod4 enhancer was characterized in vivo using transgenic mice expressing tdTomato under the control of the Neurod4 enhancer with a prolactin minimal promoter as described earlier. RNAscope in situ hybridization was performed to visualize tdTomato expression in mice at embryonic day 18.5 when somatotropes are differentiated. Expression of tdTomato is apparent in the pituitary gland, including somatotropes. Expression is also seen in brain regions including the hippocampus confirming the possibility of a role for this enhancer in driving Neurod4 expression in somatotropes. Together these data suggest that FOXO1 directly regulates Neurod4 expression by binding to its intronic enhancer. These findings provide insights into the transcriptional network governing somatotrope development. Presentation: Saturday, July 12, 2025