Identification of the DNA binding site for NGFI-B by genetic selection in yeast.
An in vivo selection system for isolating targets of DNA binding proteins in yeast was developed and used to identify the DNA binding site for the NGFI-B protein, a member of the steroid-thyroid hormone receptor superfamily. The feasibility of the technique was verified by selecting DNA fragments that contained binding sites for GCN4, a well-characterized yeast transcriptional activator. The DNA binding domain of NGFI-B, expressed as part of a LexA-NGFI-B-GAL4 chimeric activator, was then used to isolate a rat genomic DNA fragment that contained an NGFI-B binding site. The NGFI-B response element (NBRE) is similar to but functionally distinct from elements recognized by the estrogen and thyroid hormone receptors and the hormone receptor-like proteins COUP-TF, CF1, and H-2RIIBP. Cotransfection experiments in mammalian cells demonstrated that NGFI-B can activate transcription from the NBRE with or without its putative ligand binding domain.
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
T E
TE
Wilson
T J
TJ
Fahrner
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M
Johnston
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J
Milbrandt
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010957:Plasmids; D011494:Protein Kinases; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011494:Protein Kinases; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C052130:GAL4 protein, S cerevisiae; C539820:Nr4a1 protein, rat