DNA CONFORMATION DEPENDENT REGULATOR OF GENE EXPRESSION

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A novel transactivator of c-myc has been cloned. This protein binds a far upstream element (FUSE) of c-myc to stimulate promoter activity. Since FBP binds only the non-coding strand (NCS) of a far upstream elements (FUSE) of c-myc in a sequence-specific manner, and not double- stranded (ds) DNA, formation of the protein-DNA complex, in vivo, first requires unwinding of the DNA helix. FBP manifests a distinct dsDNA melting activity. In negatively supercoiled DNA, the targeted strand separation of DNA by FBP, in the NCS FBP binding site that is next to an A + T rich region of helical instability, enables this trans-acting protein to selectively expose and bind its NCS cognate sequence. These findings suggest a model in which FBP 1) scans dsDNA 2) melts single stranded DNA in regions of helical instability, 3) exposes and binds the single-stranded cognate sequence in FUSE and 4) stimulates promoter activity. In transfection studies with mutated FUSE-reporter and FBP expression vectors, the impact of helical instability and supercoiling on 1) FBP targeting to FUSE and 2) promoter transactivation will be tested. To achieve this, binding of FUSE containing topoisomers which bind a FBP affinity column will be characterized. In addition, a series of mutations that alter the degree of helical instability in the A +T rich region, but that do not alter NCS binding to FBP, will be performed. The presence/absence of non-B conformational changes at this site will be determined by probing with MBN and/or bromoacetaldehyde (BAA) and correlated with susceptibility to targeting by FBP. To ascertain whether FUSE targeting by FBP is linked to transcriptionally induced torsion in assemble chromatin, two promoters of opposite polarity will be linked to a common c-myc upstream segment containing the regulatory element and stably integrated into genomic DNA. By nuclease and chemical cleavage analysis, a hierarchy of other non FUSE sites in the upstream sequences of c-myc which form helix to coil transitions in the presence of supercoiling will also be identified. The impact of FBP expression on the regulation of c-myc expression and cellular differentiation will be determined, by selective removal with ribozymes and oligonucleotides from undifferentiated cells. These studies will elucidate the function(s) of an interesting new class of regulators of gene expression.