PARENT SESSION
GENE REGULATION AND FUNCTION - B

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(675) FOXO1 REGULATION OF THE RAT ACTIVINA PROMOTER.

Bowen-Shauver, Jennifer¹, Unterman, Terry^{1, 2}, Gibori, Geula¹, ¹ University of Illinois at Chicago, Chicago, IL² University of Illinois at Chicago, Chicago, IL

ABSTRACT- Members of the TGF superfamily are critical regulators of a variety of reproductive processes. Activin A, a homodimer of the A inhibin chain, is expressed in both the ovary and the decidua. It functions as a positive regulator of preantral ovarian follicle development and as a negative regulator of decidual proliferation. In this study, we investigated potential regulatory elements in the activin A promoter. We previously described three Foxo response elements in a -761 bp 5′-flanking region of the activin A gene, the most distal of which was capable of strong binding to a recombinant protein containing the DNA-binding domain of Foxo1 (Fkhr). Co-transfection of an activin A promoter-reporter construct and a constitutively active Foxo1 (CA-Foxo1) expression vector in HepG2 cells resulted in a dose-dependent inhibition of the activin A promoter. Surprisingly, neither truncation of the promoter to 571 bp nor mutation of the distal Foxo-binding site prevented inhibition of the promoter by CA-Foxo1. Mutation of the two proximal Foxo response elements also failed to prevent this inhibition, although diminished basal activity was observed upon mutation of one of these sites. These results indicate that inhibition of the activin A promoter by CA-Foxo1 is mediated via transcription of an intermediate factor or protein-protein interaction rather than binding of Foxo1 to the activin A promoter. An intact DNA binding region is required for this inhibition, as it could not be induced by a Foxo1 mutant lacking DNA-binding activity. Using sequential 5′ truncations of the promoter, we determined that the inhibitory effect of Foxo1 is mediated by the region between -362 and -110 bp. Analysis of this region revealed several potentially interesting elements, including putative binding sites for BCL-6, HNF-6, HNF-1, and Pax 4. BCL-6 is a transcriptional repressor that mediates inhibition of BCL-XL by Foxo4. However, co-transfections with an expression vector for BCL-6 had no effect on activin A promoter activity. An HNF-6 expression vector also failed to affect the activin A promoter but co-transfection of this transcription factor with CA-Foxo1 did mitigate the inhibitory effect of FOXO1, indicating the potential for protein-protein interaction between these factors. Supported by NIH HD12356, U54HD40093 & F32HD043586.

KEY WORDS: forkhead, activin, promoter