DNA-BINDING SPECIFICITY OF PAR AND C EBP LEUCINE-ZIPPER PROTEINS - A SINGLE AMINO-ACID SUBSTITUTION IN THE C/EBP DNA-BINDING DOMAIN CONFERSPAR-LIKE SPECIFICITY TO C/EBP/

PAR and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind to similar sites on the promote rs of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EB P 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, DNA se I protection experiments suggest that C/EBP recognizes a binding si te with an affinity similar to the one of the cholesterol 7 alpha hydr oxylase gene promoter every 200 to 300 bp. The frequency of PAR protei n binding sites with comparable affinities is about 20-fold lower in t he rat genome. By using a PCR-based amplification assay we selected hi gh affinity DNA-binding sites for C/EBP beta and the PAR protein DBP f rom a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequence 5'RTTAY.GTAAY3'. How ever, as expected, DBP, is considerably less tolerant to deviations fr om the consensus site. Here we have characterized a single amino acid substitution mutant of C/EBP beta that increases its target site speci ficity. This protein, C/EBP beta(V>A,) contains a valine to alanine su bstitution at position 13 of the basic domain (residue 216 of C/EBP be ta). 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 natur al contexts of the albumin and cholesterol hydroxylase promoters.