Abstract Type 2 Diabetes (T2D) is a disease affecting more than 20 million people in the United States alone. T2D results from the proliferative and functional failure of the insulin-producing β-cells within the Islets of Langerhans. Successful long-term treatment of T2D may require improvement in both performance and replication of β-cells. FoxM1 is required for β-cell replication postnatally and in the adult pancreas. I have previously shown male- specific restoration of aged β-cell proliferative potential and enhanced insulin secretion in mice that inducibly express a constitutively active FoxM1. Although the mechanisms of these gender-specific effects in β-cells are unknown, FoxM1 interacts with the estrogen receptor α (ERα) at transcriptional sites in other cell types. Moreover, the functional targets as FoxM1 as well as its co-regulators and cooperative transcription factor binding partners at both proliferative and functional targets are relatively unknown and completely unexplored in the β-cells. The experiments proposed here will remedy this dearth of knowledge. In Aim 1a, I will determine if FoxM1 and ERα are cooperative binding partners using MOW-ChIP in sorted proliferating and quiescent β-cells from both male and female islets. I will then investigate the role of ERα during FoxM1-mediated enhanced insulin secretion using genetic and pharmacological inhibition of estrogen signaling. In Aim 1b, I will study whether FOXM1 is required for normal insulin secretion in EndoC-βH3 cells. I will identify functional targets of FoxM1 using RNA-Seq on β-cells FACS-sorted from wild-type and FoxM1- deficient islets. I will then examine the potential roles of these targets downstream of activated FoxM1 in cultured mouse islets using pharmacological or genetic ablation. In Aim 2, I will explore how FoxM1 distinguishes between proliferative targets, which are expressed in all tissues, and β-cell-specific functional targets. To accomplish this Aim, I will implement de novo motif analysis by mining the MOW-ChIP data collected in Aim 1. I will also perform immunoprecipitation against FOXM1 in human islets to identify FOXM1 co-factors. Predicted and immunoprecipitated factors will be tested in human islets and pancreatic sections by Re-ChIP, proximity-mediated ligation assays, and assays to identify synergistic effects with FOXM1 on β-cell function. The experiments proposed in this grant will test the overall hypothesis that FoxM1 upregulates genes that promote glucose-stimulated insulin secretion and that these functional genes are controlled differently than the cell-cycle progression genes controlled by FoxM1. These Aims collectively will help identify potential druggable targets for future therapies in treating T2D.