Abstract 19003: The Molecular Basis of Cooperative Transcriptional Regulation by Foxo1 and Ets1 on a Conserved Endothelial Cell-Specific Enhancer Site Determined by the Ternary Complex Crystal Structure
Establishment and maintenance of the vasculature is mediated by a highly organized transcriptional program requiring the correct spatial and temporal expression of a large number of genes. Evidence suggests that endothelial-specific gene expression may be regulated combinatorially by transcription factor partnerships. However, no understanding of the molecular basis for mechanisms governing endothelial-specific synergistic gene expression yet exists. Here, we show the molecular basis for a transcription factor partnership on an enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This cis-acting enhancer element is bound and activated by Forkhead (FOX) and ETS transcription factors and also plays a role in the expression of genes important for adult neovascularization and vascular integrity. We have determined the 2.7Å resolution crystal structure of the DNA-binding domain of FOXO1 (FOXO1-DBD) and the DNA-binding domain of ETS1 (ETS1-DBD) bound to a 21-bp DNA duplex of the composite FOX:ETS endothelial-specific site. The ternary complex structure reveals the ETS1-DBD α/β architecture with three α-helices packed against four antiparallel β-strands to form a ‘winged helix-turn-helix’ topology and with helix-α3 embedded in the major groove. FOXO1-DBD in the complex displays a ‘winged-helix’ architecture with helix-α3 binding the major groove base sequence AATAA. Loop region H2-H3 of FOXO1-DBD is stabilized into a unique conformation to make salt-bridge mediated contact with ETS1 in the minor groove. The ternary complex structure reveals the molecular details of selective recruitment of FOXO1 by ETS1 to this non-canonical FOX:ETS site. These findings are supported by EMSA studies showing FOXO1-DBD binds poorly to FOX:ETS DNA in the absence of ETS1-DBD and underscores the cooperativity of ETS and FOX activities on the cis-acting sequence. This important ternary complex provides understanding of how FOX and ETS synergistically mediate transcriptional activity on an enhancer directing expression specifically and exclusively to the developing vasculature, as well as cooperatively regulate gene expression in adult neovascularization and vascular hemostasis.
- © 2013 by American Heart Association, Inc.