PARTIAL CHARACTERIZATION OF THE HUMAN CYP1A1 NEGATIVELY ACTING TRANSCRIPTION FACTOR AND MUTATIONAL ANALYSIS OF ITS COGNATE DNA RECOGNITION SEQUENCE

Previous studies in our laboratory identified a negative regulatory do main in the 5'-flanking region of the human CYP1A1 gene containing two negative regulatory elements (NRE). Characterization of one of these elements revealed three nuclear protein binding regions: a 21-bp palin drome with a point of symmetry at -784 and two guanine- acid cytosine- rich elements that flank the palindrome, Functional studies suggested the palindrome is critical for transcriptional repression, whereas the guanine- and cytosine-rich sequences play a secondary role, In this s tudy, the interaction between nuclear proteins and the CYP1A1 NRE was further defined, Electrophoretic mobility shift assays (EMSA) indicate d that the NRE -784 palindrome alone, but not the guanine- and cytosin e-rich sequences minus the patindrome, was capable of specific nuclear protein binding, Competitive cotransfection experiments confirmed thi s observation in intact cells, Specific residues important for DNA-pro tein interactions were identified by site-directed mutagenesis and com petitive EMSA. The loss of specific protein binding was also correlate d with the loss of negative regulatory activity in a transient-express ion assay. Finally, competitive EMSA was performed with consensus olig onucleotides for known transcription factors, An NF-Y consensus sequen ce efficiently competed with the NRE probe for specific nuclear protei n binding, EMSA supershift analyses indicate that a protein immunologi cally related to NF-Y, is part of the specific nuclear protein complex binding the human CYP1A1 NRE, These studies have refined our understa nding of the sequences critical for the transcriptional repression of human CYP1A1. To our knowledge, this is also the first report implicat ing a member of the NF-Y transcription factor family in negative gene regulation.