In vivo and in vitro studies of the role of chromatin in PHO5 regulation

Abstract

The Saccharomyces cerevisiae PHO5 gene has served as an excellent model to study transcription in the chromatin environment. Transcription of PHO5 is regulated by phosphate availability through chromatin dynamics. Under activating conditions, positioned nucleosomes on the PHO5 promoter are remodeled by the coordinated function of transcriptional activators Pho2p and Pho4p. This dissertation describes the use of yeast chromatin assembly system to investigate the mechanism of PHO5 regulation. Several transcription-related activities were reconstituted on the chromatin template, including ATP-dependent chromatin remodeling and histone acetylation, which have been demonstrated to be required for activation of numerous genes. Transcriptional activators Pho2p and Pho4p by themselves were capable of binding their recognition sequences in the chromatin-assembled DNA. Chromatin reconfiguration on the PHO5 promoter required Pho2p, Pho4p and the ATP-dependent activity in the nuclear extract. However, chromatin remodeling activity alone was not sufficient to bring about PHO5 transcriptional activation. We showed that the missing activity was histone acetyltransferase. Transcriptional activator binding to chromatin, ATP-dependent chromatin remodeling and histone acetylation were separable events but all were required to activate in vitro transcription of PHO5. In vivo study on the role(s) of histone deacetylase Rpd3p in PHO5 regulation suggested that Rpd3p functions on the PHO5 promoter through controlling acetylation level of the histone tails. Rpd3p also has an indirect role in PHO5 regulation through its effect on phosphate uptake. I have demonstrated that Rpd3p regulates the turn over rate of the phosphate transporter Pho84p, which functions upstream of PHO5 in the PHO pathway. RPD3 deletion resulted in early recycling of Pho84p, resulting in a phosphate uptake defect and accelerated activation of genes involved in phosphate accumulation. Both in vitro and in vivo studies described in this dissertation have contributed significantly to the field of PHO5 regulation. The yeast chromatin reconstitution system has proved to be a very useful tool to study mechanism of transcription regulation. Chromatin reconstitution on bead-bound DNA (described in chapter 5) should provide an additional tool to investigate further details in the mechanism of transcription regulation in a chromatin context.