Activation of the weakly regulated PHO8 promoter inS.cerevisiae:chromatin transition and binding sites for the positive regulatory protein PHO4

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Abstract
PHO8 encodes an alkaline phosphatase in Saccharomyces cerevisiae whose transcription is regulated by the phosphate concentration in the medium. This occurs through the action of several positive and negative regulatory proteins, also involved in the regulation of other members of the phosphatase gene family. A central role is played by PHO4, the gene encoding a DNA binding regulatory protein. Digestion experiments with DNasel, micrococcal nuclease and 20 different restriction nucleases show that under conditions of PHO8 repression, there is a highly ordered chromatin structure at the promoter consisting of three hypersensitive regions, approximately 820 to 690, 540 to 510, and 230 to 160 bp upstream of the initiation codon. These hypersensitive sites are surrounded by DNA organized in nucleosomes. Gel shift analysis and in vitro footprinting revealed the presence of two PHO4 binding sites at the PHO8 promoter: a low affinity site at -728 and a high affinity site at -532. Each one is located within a hypersensitive site. Upon derepression of PHO8, the chromatin structure changes significantly: The two upstream hypersensitive sites containing the PHO4 binding sites merge, resulting in a long region of hypersensitivity. This transition is PHO4 dependent. However, not all of the promoter becomes nucleosome free. Instead, as a novel feature, regions of intermediate accessibility are generated upstream and downstream of the third hypersensitive site, the latter region encompassing the TATA-box. The available data fit best into a concept that these regions are organized in unstable or partly unfolded nucleosomes.