Molecular cloning and characterization of Schistosoma mansoni Ftz-F1 interacting protein-1 (SmFIP-1), a novel corepressor of the nuclear receptor SmFtz-F1.
In order to characterize protein cofactors of the Schistosoma mansoni nuclear receptor SmFtz-F1, we have screened a yeast two-hybrid adult worm cDNA library using a construct expressing the D, E and F domains of SmFtz-F1 as bait. One of the selected clones encoded a sequence without homologues in any other species, apart from Schistosoma japonicum. The complete sequence was obtained by 5' and 3' rapid amplification of cDNA ends (RACE) and comprised 3660 nucleotides with an open reading frame of 788 amino acids. The gene is expressed at all schistosome life cycle stages at a 5-11-fold higher level than SmFtz-f1. The protein, named SmFIP-1, interacted with SmFtz-F1 in a GST pull-down assay and in a mammalian two-hybrid assay in CV-1 cells. Although SmFIP-1 contains a consensus NR box (LXXLL) this was not involved in the interaction with SmFtz-F1. However, interaction did depend on the AF2-AD motif in the nuclear receptor ligand binding domain. Deletion analysis showed that the C-terminal moiety of SmFIP-1 was involved in the binding, but this could not be localized to a particular motif, suggesting that the binding may be conformation-dependent. Finally, SmFIP-1 markedly repressed SmFtz-F1-mediated transcription in a dose-dependent manner from the SmFtz-f1 gene promoter demonstrating that SmFIP-1 is a schistosome-specific transcriptional corepressor.
Oger
Frédérik
F
INSERM U547, Institut Pasteur de Lille, France.
Bertin
Benjamin
B
Caby
Stéphanie
S
Dalia-Cornette
Jocelyne
J
Adams
Martin
M
Vicogne
Jérome
J
Capron
Monique
M
Pierce
Raymond J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D005260:Female; D051717:Fushi Tarazu Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015801:Helminth Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008018:Life Cycle Stages; D008297:Male; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012550:Schistosoma mansoni / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D051717:Fushi Tarazu Transcription Factors; D015801:Helminth Proteins