AML3/CBFalpha1 is required for androgen-specific activation of the enhancer of the mouse sex-limited protein (Slp) gene.

A complex 120-base pair enhancer, derived from the mouse sex-limited protein (Slp) gene, is activated solely by the androgen receptor (AR) in specific tissues, although it contains a hormone response element recognized by several steroid receptors. The generation of this transcriptional specificity has been ascribed to the interactions of the receptor with tissue-specific nonreceptor factors bound to accessory sites within the enhancer. Protein-DNA interaction assays revealed two factors binding the 5' part of the enhancer that differ widely in abundance between cells showing AR-specific activation of the Slp element compared with those that also permit activation by glucocorticoid receptor (GR). The factor designated B formed a complex centered on the sequence TGTGGT, a core motif recognized by members of the AML/CBFalpha transcription factor family. This complex was competed by a high affinity binding site specific for AML/CBFalpha and was specifically supershifted by an antibody to AML3/CBFalpha1, placing factor B within the AML3/CBFalpha1 subclass. Interestingly, this factor was shown to bind to a second site in the 3' part of the enhancer, positioned between the two critical AR binding sites. Transfection studies revealed that AML1-ETO, a dominant-negative AML/CBFalpha construct, abrogated AR induction of the enhancer, but not of simple hormone response elements. Furthermore, overexpression of AML3/CBFalpha1 could rescue the AML1-ETO repression. Finally, glutathione S-transferase-AML/CBFalpha fusion proteins demonstrated direct interaction between AML/CBFalpha and steroid receptors. Although this interaction was equivalent between AML1/CBFalpha2 and AR or GR, AML3/CBFalpha1 showed stronger interaction with AR than with GR. These data demonstrate that AML3/CBFalpha1 is functionally required for hormonal induction of the Slp enhancer and that direct, preferential protein-protein interactions may contribute to AR-specific activation. These results demonstrate an intriguing role of AML3/CBFalpha1 in steroid- as well as tissue-specific activation of target genes.