The identification of forkhead transcription factors of the Foxo sub-family as effectors of insulin action on gene expression has filled a yawning gap in our knowledge of insulin signaling, and identified a cellular biological mechanism linking hormone signaling to regulation of the cell's transcriptional response through sub-cellular redistribution of transcription factors. Key advances of the past funding cycle include: i, identification of SIN3a as the FoxO co-repressor of glucokinase in liver; ii, discovery of FoxO1 inhibitors as selective insulin sensitizers; iii, discovery of miR205 as an integrator of insulin signaling in liver; iv, demonstration of a role of G-protein-coupled receptor Gpr17 as a FoxO1 target that modulates neuropeptide processing and energy balance. This application for competing renewal marks a conceptual break from prior work: the PI will narrow his focus on the liver, and depart from a candidate gene approach to embrace genome-wide methods of transcription factor analysis and answer three key questions relevant to diabetes pathophysiology. (i) Known FoxO1 target genes cannot account for of the entirety of its metabolic effects, prompting investigations of the unaccounted FoxO1 regulome. (ii) How does the gamut of hepatic FoxO1 targets change with insulin resistance and diabetes? (iii) Can selective hepatic insulin resistance, resulting in combined abnormalities of glucose and lipoprotein metabolism, be explained by the topology of FoxO1 DNA binding? The PI proposes to address these questions by 3 aims. In Aim 1 the PI will use a newly developed reporter mouse to determine the FoxO1 cistrome and analyze its alterations during the development of insulin-resistant diabetes, with a focus on hepatic super-enhancers as well as long-distance chromatin interactions analyzed by Chromosome Conformation Capture techniques. In Aim 2, he will use comparative cistrome analyses with CREB, GR, and Ppara to delve into the interaction of FoxO1 with other hormone-regulated transcription factors in the nutrient response. In Aim 3, the PI will integrate ChIPseq with RNAseq data in a FoxO1 regulome to study the paradox of “selective” hepatic insulin resistance of glucose vs. lipid/lipoprotein metabolism. He hypothesizes that insulin resistance results from selective modulation of different FoxO1 DNA binding sites, and proposes to use DNA affinity purification followed by mass spectrometry to isolate components of the FoxO1 regulatory complex. The combined approach should yield actionable information to design disease-modifying diabetes treatments.