title: A critical arginine residue mediates cooperativity in the contact interface between transcription factors NFAT and AP-1.
abstract: The heterologous transcription factors NFAT and AP-1 coordinately regulate cytokine gene expression through cooperative binding to precisely juxtaposed DNA recognition elements. The molecular origins of cooperativity in the binding of NFAT and AP-1 to DNA are poorly understood. Herein we have used yeast one-hybrid screening and alanine-scanning mutagenesis to identify residues in AP-1 that affect cooperative interactions with NFAT on DNA. Mutation of a single conserved Arg residue to Ala in the cJun spacer region (R285A) led to a virtually complete abolition of cooperative interactions with NFAT. The DNA-binding activity of AP-1 alone was unaffected by the cJun R285A mutation, thus indicating that this residue influences cooperative binding only. Ala-scanning mutations elsewhere in AP-1, including the cFos subunit, revealed no other strongly interacting single positions. We thus conclude that NFAT contacts AP-1 in the spacer region of the cJun subunit, making an especially important contact to R285, and that these interactions drive formation of the cooperative NFAT/AP-1/DNA complex. These results provide a general strategy for selectively ablating cooperativity between transcription factors without affecting their ability to act alone and yield insights into the structural basis for coordinate regulation of gene expression.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: B R
authors.initials: BR
authors.lastname: Peterson
authors.forename: L J
authors.initials: LJ
authors.lastname: Sun
authors.forename: G L
authors.initials: GL
authors.lastname: Verdine
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D016207:Cytokines / Q000096:biosynthesis*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018808:Transcription Factor AP-1 / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D016207:Cytokines; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA; D001120:Arginine; D001616:beta-Galactosidase; D000409:Alanine