Novel Flanking DNA Sequences Enhance FOXO1a DNA Binding Affinity but Do Not Alter DNA Bending

Abstract

FOXO1A, a member of the forkhead winged-helix family of proteins is a transcription factor with proapoptotic activities and plays a significant role in insulin and growth factor signaling. As such, FOXO1A is insulin responsive and binds to the insulin response element (IRE). However, multiple forkhead family members with diverse biological functions are also known to bind to the IRE. Therefore, additional DNA sequence elements may be required to provide increased binding affinity and specificity for FOXO1A. We have used the systematic evaluation of ligands by exponential enrichment (SELEX) to systematically identify additional DNA sequences important for FOXO1A binding. We demonstrate for the first time that, in addition to the IRE, two additional sequence elements are important for maximal FOXO1A binding: (1) the reverse complement (5'-GT(A/C)AACA-3') and (2) the flanking sequence (5'-ACAACA-3'). Although these additional elements do not contribute to the FOXO1A-induced DNA bending angle of 120 degrees , the presence of these additional elements does increase the affinity of FOXO1A DNA binding nearly 9-fold through a 1-to-1 binding stoichiometry. The increased binding affinity subsequently enhances the ability of FOXO1A to activate transcription from a luciferase reporter construct and from promoter regions of endogenous genes known to be direct transcriptional targets of FOXO1A.