INTERACTION OF AN NF-KAPPA-B-LIKE FACTOR WITH A SITE UPSTREAM OF THE C-MYC PROMOTER

The c-myc protooncogene has been implicated in control of growth and differentiation of mammalian cells. For instance, growth arrest is often preceded by reduction in c-myc mRNA and gene transcription. To elucidate the mechanisms of control of c-myc gene transcription, we have begun to characterize the interaction of nuclear factors with the 719-base-pair (bp) c-myc regulatory domain, located 1139-421 bp upstream of the P1 start site of the mouse gene. Nuclear extracts from exponentially growing WEHI 231 murine B-lymphoma cells formed multiple complexes in mobility-shift assays. Changes in complex distribution were observed in growth-arrested WEHI 231 cells, and a major site of this interaction mapped to a 21-bp sequence that is similar to the sequences recognized by the NF-κB family of proteins. Binding of NF-κB-like factors was demonstrated by oligonucleotide competition. Induction of complex formation upon 70Z/3 pre-B- to B-cell differentiation, enhancement of binding by GTP, and detergent-induced release of inhibitor protein suggested that NF-κB itself is one member of the family that can bind. Transfection of thymidine kinase-chloramphenicol acetyltransferase constructs containing the 21-bp c-myc sequence into Jurkat cells demonstrated increased chloramphenicol acetyltransferase activity upon phorbol ester and phytohemagglutinin treatment. These results suggest the involvement of NF-κB-like factors in the regulation of c-myc transcription.