Reb1p-dependent DNA Bending Effects Nucleosome Positioning and Constitutive Transcription at the Yeast Profilin Promoter

Abstract

The molecular basis of constitutive gene activation is largely unknown. The yeast profilin gene (PFY1), encoding a housekeeping component of the actin cytoskeleton, is constitutively transcribed at a moderate level. The PFY1 promoter dispenses with classical transactivators and a consensus TATA box; however, it contains a canonic site for the abundant multifunctional nuclear factor rDNA enhancer-binding protein (Reb1p) combined with a dA.dT element. Reb1p binds specifically in vitro. Mutation of this site reduces PFY1 expression to about 35%. A nucleosome-free gap of about 190 bp is centered at the genomic Reb1p binding site in vivo and spans the presumptive core promoter and transcriptional initiation sites. Nucleosomes at the border of the gap are positioned. Mutation of the Reb1p motif in the genomic PFY1 promoter abolishes nucleosome positioning, fills the gap with a non-positioned nucleosome, and reduces transcription by a factor of 3. From permutation studies we conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data suggest that Reb1p plays an architectural role on DNA and that Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA.