The study of transcription in yeast has led to the identification and characterization of many conserved genes that have general roles as transcriptional regulators. The genetic selection known as the Spt selection has been particularly productive, identifying many transcriptional regulators including histones, chromatin modifiers, TATA binding protein (TBP), regulators of TBP, and transcription elongation factors. Because the Spt phenotype has been indicative of proteins with roles during transcription, we used a newly developed systematic yeast overexpression library to screen for genes that cause the Spt- phenotype when overexpressed. We recovered essentially all of the factors that were previously identified using random genomic libraries, but additionally recovered an uncharacterized gene, PSH1, which encodes a RING domain protein that was reported to co-purify with the FACT histone chaperone complex and histones in large-scale proteomic studies. We propose the following Specific Aims to characterize PSH1 and its potential role in transcription regulation.
For specific aim 1 we will determine the domains of Psh1 that are required for its normal function and for its overexpression phenotype. Psh1 has two recognizable motifs that suggest functional roles for this protein;a RING finger domain, and a highly acidic stretch of amino acids. To test this, point mutations in crucial amino acids and deletion derivatives that include the identified domains of Psh1 will be created. These mutants will also serve to identify domains that are important for the biochemical activity of Psh1 as proposed in Specific Aims 2 and 3.
For specific aim 2 we will determine if Psh1 interacts with the FACT complex and histones and whether it is associated with chromatin. The identification of PSH1 in the Spt overexpression screen suggests that it has a role in transcription. The goal of this Specific Aim is to gain a biochemical understanding of its specific functions. We will determine whether Psh1 co-IPs with FACT and histones, and chromatin-IPs will be used to evaluate the association of Psh1 with chromatin. Combined, these approaches will establish whether Psh1 is associated with FACT, histones and transcribed regions in vivo.
For specific aim 3, we will determine whether Psh1 ubiquitinates the FACT complex or other SPT genes. Our very recent preliminary data shows recombinant GST-Psh1 to possess ubiquitin-protein ligase activity in vitro. Using missense and deletion mutants constructed in Specific Aim 1, we will determine the domains required for ubiquitin-protein ligase activity in vitro. Having detected E3 ubiquitin-protein ligase activity, the next goal is to identify the in vivo substrate(s) of Psh1. We will take a candidate approach by testing in vivo substrates such as the FACT subunits Spt16 and Pob3.
