Dissecting Gene Regulatory Dynamics and Mechanisms with Single Cell Imaging Techniques

Abstract

Cells must be able to rapidly and specifically respond to stress to survive. In order to control RNA and protein levels in response to a signal, transcription must be tightly controlled as well. Since transcription is a highly dynamic process, studying pathways at a fine spatiotemporal resolution is essential to fully understanding signaling-induced regulation. We utilize the Saccharomyces cerevisiae Hog1 osmotic stress response pathway as a model for inducible signal response, investigating the role and dynamics of the highly conserved Spt-Ada-Gcn5-Acetyltransferase (SAGA) transcriptional cofactor complex in responsive gene regulation. As part of our larger investigation, we developed a software tool called TrueSpot to facilitate fully automated and accurate signal quantification from large batches of RNA fluorescent in situ hybridization (RNA-FISH) imaging data. We then applied TrueSpot to preliminary RNA-FISH studies of osmosensitive gene transcription profiles over time in cells with Gcn5, a catalytic subunit of SAGA, knocked out or depleted. We confirmed that perturbation of Gcn5 delayed and dampened the transcriptional response of upregulated osmosensitive genes.