Modulation of c-myc transcription by triple helix formation

Abstract

The human c-myc oncogene promoter was used as a model with which to study the mechanism of action of oligodeoxyribonucleotides targeted to a gene regulatory region. The nuclease-hypersensitive element, NHE, lying -115 bp from the P1 promoter of the human c-myc gene, is known to be required in cis for transcription of the gene from both P1 and P2 promoters (Fig. 1). Inhibition of c-myc transcription by an oligonucleotide designed to bind to NHE by triplex formation has been observed in a cell-free transcription assay. Using a reconstituted transcription system with the semipurified PuF transcription factor whose site of interaction resides within the NHE, it is shown here that the oligonucleotide inhibits PuF-mediated transcription. These findings, together with data presented elsewhere showing that: (1) PU1 binds to cloned DNA fragments to form a colinear triplex; (2) PU1 inhibits transcription in nuclear extracts; (3) triple helix formation inhibits the binding of PuF to its target NHE element in an in vitro binding competition assay (E. Postel, R. Durland, and M. Hogan, submitted); (4) triplex formation at the NHE target site can occur in living HeLa cells treated with the triplex-forming PU1 oligomer, and (5) c-myc mRNA synthesis in these treated cells is repressed, clearly support the proposed model in which the oligonucleotide targeted against the c-myc NHE promoter region binds to form a triplex, thereby blocking access to the regulatory protein PuF. This results in promoter-sensitive repression of transcriptional activation of the c-myc gene. The potential for manipulation of gene expression by oligonucleotides targeted to a DNA sequence of the c-myc oncogene promoter and other gene promoters is clear.