FOXO (Forkhead box O1 transcription factors) factors interact with and modify the activity of other transcription factors, including nuclear hormone receptors. However, not all of the structural domains within the FOXO proteins that mediate these functional interactions have been clearly defined. To address this issue, we used a constitutively active (nuclear) mutant of FOXO1 a (designated FCXOA3) and within FOXOA3 made additional mutations to alter the putative nuclear hormone interacting domain (NID), minimal activation domain (MAD), DNA-binding domain (DBD), and the N terminus. We document that FOXOA3 enhanced the hormone-dependent transcriptional activity of liganded progesterone receptors A (PGRA) on a glucocorticoid response ele me nt- responsive promoter, PGRA on the insulin-like growth factor-binding protein 1 promoter, and estrogen receptor a on an estrogen response element-responsive promoter. The effects of FOXOA3 on PGRA were dependent, in part, on an intact NID, the MAD, and N-terminal domain. In striking contrast, a FOXOA3 DNA-binding mutant (FOXOA3-mDBD) modulated PGRA, PGRB, and ESR1 activities by distinctly different mechanisms, markedly elevating ligand-independent activity of these nuclear hormone receptors even in the double mutant lacking the MAD. Furthermore, both FOXOA3 and FOXOA3-mDBD enhanced the activity of a transcriptionally defective PGRA lacking its AF1 transactivation domain, indicating that this region of the receptor is not essential in this context. Since FOXOA3, FOXOA3-mDBD, and FOXOA3-mNID all bound PGRA in a GST pull-down assay, it appears that the LXXLL (leucine-X-X-leucine-leucine) motif within the NID is not critical for FOXOA3 interactions with PGRA, but may modify the recruitment of other co-regulatory molecules. Collectively, the results show that FOXOA3 exerts co-regulatory functions independent of DNA binding and that the DNA-binding defective form of FOXO1a is transcriptionally active as a co-regulator of these nuclear hormone receptors.
