Analysis of Promoter-Specific Repression by Triple-Helical DNA Complexes in a Eukaryotic Cell-Free Transcription System

A site-specific triple-helical DNA complex has previously been shown to inhibit DNA binding by eukaryotic transcription factor Spl. To examine the functional consequences of such inhibition, homopurine target sequences for oligonucleotide-directed triple-helix formation were inserted in various configurations relative to Spl transcription activator binding sites, upstream of the TATA element of recombinant eukaryotic promoters. The resulting promoters were tested for activity in the presence or absence of recombinant human Spl in a Drosophila in vitro transcription system lacking endogenous Spl. When triple-helical complexes were assembled on the promoters by incubation with specific oligodeoxyribonucleotides, promoter-specific repression of basal transcription was observed in the absence of Spl. Transcriptional repression required the preassembly of triple-helical complexes before addition of nuclear extract. The degree of basal repression was a function of the number and proximity of triple-helical complexes relative to the basal promoter complex. Repression did not result from triple-helix-induced template degradation. Addition of recombinant Spl did not cause derepression. These results suggest that triple-helical complexes can repress transcription primarily by blocking promoter DNA assembly into initiation complexes rather than by occluding Spl binding. One of several plausible mechanisms for triple-helix-induced repression involves changes in DNA flexibility. Evidence in favor of this model is provided by a permutation-dependent gel mobility assay in which formation of site-specific triple-helical complexes is shown to stiffen double-helical DNA.