Identification of genes related to transcriptional regulation using a novel systematic yeast library
Jones, Grace Marie
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The study of transcription in yeast has led to the identification and characterization of many conserved genes that have general roles in transcriptional regulation. The genetic selections known as the Bur selection and the Spt selection have been particularly productive, identifying many of these factors including histones, chromatin modifiers, TATA binding protein (TBP), regulators of TBP, and transcription elongation factors. The Bur selection uses a deletion of the Upstream Activating Sequence (UAS) in the promoter region of SUC2 resulting in the cells being unable to grow on media containing sucrose, a Bur+ phenotype. The Spt selection uses a transposon element insertion in the promoter regions of both HIS4 and LYS2, causing the cells to be unable to grow on media lacking histidine and lysine, a Spt+ phenotype. While most of these factors were identified through genomic mutations, the use of random overexpression libraries has also played a role. Random libraries however, do not facilitate systematic screens and often genes are missed that might be of interest for a specific screen.;Here we describe the construction of a systematic yeast overexpression library and its validation using the Bur and Spt selections. Based on these phenotypes we screened for genes, that upon overexpression caused a Bur - phenotype or a Spt- phenotype. We recovered essentially all of the factors that were previously identified using random libraries, but additionally recovered other unknown high-copy genes one new BUR, YLR247c and three new SPT genes; RAD26, POB3 and PSH1. Of these four genes, RAD26 and POB3 are well-characterized transcription factors. However, the remaining two genes, YLR247c and PSH1 are uncharacterized. Here we focus on the one of those genes, PSH1..;PSH1 causes the Spt- phenotype when overexpressed, encodes for a C3HC4 RING finger. Psh1 was previously shown to interact with the FACT elongation factor complex and histones. Here we show that recombinant GST-Psh1 has ubiquitin E3 ligase activity in vitro and that a functional RING domain and the ubiquitin-conjugating enzymes, Rad6 and Ubc8, are required for the high-copy Spt- phenotypes. The physical interaction between Psh1 and several transcription factors led us to detect specific poly-ubiquitination of Spt16, a subunit of the FACT elongation complex, upon PSH1 overexpression, which is dependent on Rad6. The poly-ubiquitination of Spt16 does not lead to its degradation. Taken together these results suggest that Spt16 is a substrate of Psh1 and the E2, Rad6, resulting in the poly-ubiquitination of Spt16, possibly disrupting its function leading to the Spt- phenotype. Furthermore we present a model that describes Psh1 as being involved in regulating normal cryptic transcripts through the ubiquitination of Spt16. Finally, additional research has been proposed to verify the role of Psh1 in regulating cryptic transcription.
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