The Design and Use of a High throughput Microscopy Screen to Monitor Transcription Factor Localization in Saccharomyces cerevisiae
Year: 2014
Type: dissertation
Abstract: Cellular signalling networks control gene expression by modulating the activities of transcription factors through regulatory mechanisms that affect their transactivation or transrepression potential, DNA-binding affinity, localization, or abundance. This regulation often requires the site-specific phosphorylation of the transcription factor by a kinase. I have developed a high throughput microscopy screen and used it to monitor the nucleo-cytoplasmic distribution and abundance of 122 different GFP-tagged transcription factors in 84 different Saccharomyces cerevisae kinase mutant backgrounds and in wild type cells after exposure to eight different environmental stress conditions that modulate kinase signalling pathways. Thirty one GFP-tagged transcription factors exhibited changes in localization or abundance, mostly in response to an acute exposure to environmental stress rather than to the chronic loss of a non-essential kinase. These screens revealed that the Yero88c transcriptional repressor, which was not previously known to undergo changes in localization, translocates from the cytoplasm to the nucleus in response to multiple environmental stresses; its paralog, Yblo54w, displayed similar behavior. Yero88c and Yblo54w are known to repress the transcription of ribosome biogenesis (Ribi) genes in response to environmental stress and the downregulation of the TORC1 or PKA signalling pathways (Lippman Broach, 2009). I show that the nuclear accumulation of Yero88c and Yblo54w is inhibited by TORC1 and PKA signalling. Further analysis implicates the kinase Sch9 and the Tap42-associated phosphatase complexes in the regulation of Yero88c localization by TORC1. Serine-to-alanine substitutions at six putative PKA/Sch9 consensus phosphoacceptor sites in its sequence causes Yero88c to localize to the nucleus constitutively. These data suggest that under optimal growth conditions PKA and Sch9 phosphorylate Yero88c causing it to localize to the cytosol, whereas the activation of TORC1-regulated phosphatases in response to stress induces the dephosphorylation and nuclear accumulation of Yero88c resulting in the repression of Ribi genes and the attenuation of cell growth.
Source: TSpace (University of Toronto)
Author: Michael John Cox
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2588083687