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.
Ning
Y M
YM
Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618, USA.
Robins
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DM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D003181:Complement C4; D050659:Core Binding Factor Alpha 1 Subunit; D004742:Enhancer Elements, Genetic; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D009363:Neoplasm Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011944:Receptors, Androgen / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D001798:Blood Proteins; C018386:C4a protein, mouse; D003181:Complement C4; D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase