Characterization of the DNA-binding properties and the transactivation activity of Schistosoma mansoni nuclear receptor fushi tarazu-factor 1alpha (SmFTZ-F1alpha).
A FTZ-F1-related orphan nuclear receptor SmFTZ-F1alpha was previously identified from Schistosoma mansoni. The deduced SmFTZ-F1alpha protein contains a highly conserved DNA binding domain (DBD, C domain), a less conserved ligand binding domain (LBD, E domain) and three highly variable regions, the N-terminal A/B domain (108 aa), a large hinge region (D domain, 1027 aa) and an F domain (220 aa). Herein, we characterize the DNA binding properties and the transactivation activity of SmFTZ-F1alpha. In in vitro assays, SmFTZ-F1alpha bound as a monomer to a response element (FF1RE: TCAAGGTCA) recognized by mammalian steroidogenic factor 1 (SF-1), and to related sequences (p14: TTAAGGTCA and SmFF1a-2: CGAAGGTCA) derived from known schistosome gene promoters. Competition assays with p14 oligonucleotides containing a single mutation at each nucleotide position defined the optimum DNA sequence required for SmFTZ-F1alpha binding. The optimal consensus sequence for SmFTZ-F1alpha binding is TN(A/G)AGGTC(A/G) (N: any base). This sequence is similar but not identical to the SF-1 response element (SFRE) consensus sequence [(T/C)CAAGG(T/C)C(A/G)]. By performing yeast one-hybrid assays, the ability of SmFTZ-F1alpha to bind productively to a p14-derived 9-base pair sequence was demonstrated in vivo. The ability of the full-length SmFTZ-F1alpha to transactivate reporter gene expression was shown to be A/B domain-dependent in a yeast system. In addition, the hinge region contained an unexpected activation function (AF) domain, termed AF-3, while no transactivation activity was detected within the E/F domain. This AF-3 region (from aa 982 to aa 1110) revealed a strong autonomous transactivation activity, which was masked when it was present in the full-length SmFTZ-F1alpha. Taken together, our results suggest that SmFTZ-F1alpha possesses the characteristic DNA binding specificity of FTZ-F1 subfamily members and the capacity to transactivate a reporter gene.
Lu
Changxue
C
Department of Microbiology and Immunology, School of Medicine and Biomedical Research, State University of New York, Buffalo, NY 14214, USA.
Niles
Edward G
EG
LoVerde
Philip T
PT
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D051717:Fushi Tarazu Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012550:Schistosoma mansoni / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D051717:Fushi Tarazu Transcription Factors; D015801:Helminth Proteins; D004247:DNA