A NUCLEOSOME PRECLUDES BINDING OF THE TRANSCRIPTION FACTOR PHO4 IN-VIVO TO A CRITICAL TARGET SITE IN THE PHO5 PROMOTER

Activation of the Saccharomyces cerevisiae PHO5 gene by phosphate star vation is accompanied by the disappearance of two pairs of positioned nucleosomes that flank a short hypersensitive region in the promoter. The transcription factor Pho4 is the key regulator of this transition. By in vitro footprinting it was previously shown that there is a low affinity site (UAS(p)1) which is contained in the short hypersensitive region in the inactive promoter, and a high affinity site (UAS(p)2) w hich is located in the adjacent nucleosome. To investigate the interpl ay between nucleosomes and Pho4, we have performed in vivo footprintin g experiments with dimethylsulfate. Pho4 was found to bind to both sit es in the active promoter. In contrast, it binds to neither site in th e repressed promoter. Lack of binding under repressing conditions is l argely due to the low affinity of Pho4 for its binding sites under the se conditions. Despite the increased affinity of Pho4 for its target s ites under activating conditions, binding to UAS(p)2 is prevented by t he presence of the nucleosome and can only occur after prior disruptio n of this nucleosome in a process that requires UAS(p)1. Protection of the PHO5 UAS(p)2 by the nucleosome is not absolute, however, since ov erexpression of Pho4 can disrupt this nucleosome even when UAS(p)1 is deleted. Also under these conditions, with only UAS(p)2 present, ail f our nucleosomes at the PHO5 promoter are disrupted, whereas no chromat in change at all is observed when both UAS elements are destroyed.