In vitro reconstitution of PHO5 promoter chromatin remodeling points to a role for activator-nucleosome competition in vivo.

The yeast PHO5 promoter is a classical model for studying the role of chromatin in gene regulation. To enable biochemical dissection of the mechanism leading to PHO5 activation, we reconstituted the process in vitro. Positioned nucleosomes corresponding to the repressed PHO5 promoter state were assembled using a yeast extract-based in vitro system. Addition of the transactivator Pho4 yielded an extensive DNase I-hypersensitive site resembling induced PHO5 promoter chromatin. Importantly, this remodeling was energy dependent. In contrast, little or no chromatin remodeling was detected at the PHO8 or PHO84 promoter in this in vitro system. Only the PHO5 promoter harbors a high-affinity intranucleosomal Pho4 binding site (UASp) where Pho4 binding can compete with nucleosome formation, prompting us to test the importance of such competition for chromatin remodeling by analysis of UASp mutants in vivo. Indeed, the intranucleosomal location of the UASp element was critical, but not essential, for complete remodeling at the PHO5 promoter in vivo. Further, binding of just the Gal4 DNA binding domain to an intranucleosomal site could increase PHO5 promoter opening. These data establish an auxiliary role for DNA binding competition between Pho4 and histones in PHO5 promoter chromatin remodeling in vivo.

Nucleosome stability at the yeast PHO5 and PHO8 promoters correlates with differential cofactor requirements for chromatin opening.

The coregulated PHO5 and PHO8 genes in Saccharomyces cerevisiae provide typical examples for the role of chromatin in promoter regulation. It has been a long-standing question why the cofactors Snf2 and Gcn5 are essential for full induction of PHO8 but dispensable for opening of the PHO5 promoter. We show that this discrepancy may result from different stabilities of the two promoter chromatin structures. To test this hypothesis, we used our recently established yeast extract in vitro chromatin assembly system, which generates the characteristic PHO5 promoter chromatin. Here we show that this system also assembles the native PHO8 promoter nucleosome pattern. Remarkably, the positioning information for both native patterns is specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract does not support proper nucleosome positioning unless supplemented with yeast extract. By competitive assemblies in the yeast extract system we show that the PHO8 promoter has greater nucleosome positioning power and that the properly positioned nucleosomes are more stable than those at the PHO5 promoter. Thus we provide evidence for the correlation of inherently more stable chromatin with stricter cofactor requirements.