Binding of Gal4p and Bicoid to Nucleosomal Sites in Yeast in the Absence of Replication

Abstract

The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G₁, with α-factor, or in G₂/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites in SWI⁺ and swi1Δ yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites.

ACKNOWLEDGMENTS

We thank Kellie Cummings for initiating construction and characterization of TABic4Δ80; David Gross, Steve Hanes, Dave Burz, Stephen Johnston, Mark Johnston, and Joan Curcio for gifts of yeast strains and plasmids; Michael Kladde and Robert Simpson for helpful discussions; and the Wadsworth Center Molecular Genetics Core for oligonucleotide synthesis and DNA sequencing.

This work was supported by grant GM51993 from the National Institutes of Health.