DNA-binding specificity of PAR and C/EBP leucine zipper proteins: a single amino acid substitution in the C/EBP DNA-binding domain confers PAR-like specificity to C/EBP.
PAR and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind similar sites on the promoters of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a more relaxed binding specificity than PAR proteins, in that they recognize many sites within promoter or randomly selected rat genomic DNA sequences that are ignored by PAR proteins. Thus, DNAse I protection experiments suggest that C/EBP recognizes a binding site with an affinity similar to the one of the cholesterol 7 alpha hydroxylase gene promoter every 200 to 300 bp. The frequency of PAR protein binding sites with comparable affinities is about 20-fold lower in the rat genome. By using a PCR-based amplification assay we selected high affinity DNA-binding sites for C/EBP beta and the PAR protein DBP from a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, is considerably less tolerant to deviations from the consensus site. Here we have characterized a single amino acid substitution mutant of C/EBP beta that increases its target site specificity. This protein, C/EBP beta V > A, contains a valine to alanine substitution at position 13 of the basic domain (residue 216 of C/EBP beta). C/EBP beta V > A selectively binds only the subset of C/EBP sites that are also DBP sites, both as oligonucleotides and within the natural contexts of the albumin and cholesterol hydroxylase promoters.
Falvey
E
E
Department of Molecular Biology, University of Geneva, Switzerland.
Marcacci
L
L
Schibler
U
U
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; C485975:DBP protein, rat; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors