Single cell analysis of dynamic gene regulation

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PROJECT SUMMARY/ABSTRACT Regulatory mechanisms underlying the precise control of gene expression in normal and disease states involve multiprotein complexes such as the highly conserved Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex. Although most of the SAGA subunits have been identified, it remains essentially unknown how their functions are coordinated to precisely regulate gene expression. Thus, the SAGA complex represents an ideal paradigm to explore how multiprotein complexes regulate gene expression, and the overall goal of this project is to provide a precise mechanistic and predictive understanding for the coordination of SAGA subunit function. SAGA subunits are organized into “activity modules”. We will focus on the well-established histone acetyltransferase (HAT), TATA-binding protein (TBP), and histone deubiquitinase (DUB) activity modules in SAGA, which contain the best characterized and evolutionarily conserved SAGA subunits, and are implicated in the regulation of chromatin structure (HAT), transcription initiation (TBP) and RNA export (DUB). Our central hypothesis is that SAGA subunits and modules function together to precisely coordinate different steps in gene expression from chromatin regulation to RNA transcription to RNA export. We will investigate osmotic stress induction of high osmolarity glycerol (Hog1/p38) mitogen-activated protein kinase (MAPK) signaling and gene expression in yeast to study SAGA subunit coordination of gene expression. Importantly, we will use a newly developed detailed and integrated experimental and computational analysis of dynamic single-molecule RNA expression (FISH) in single cells to simultaneously quantify and model each of these steps in gene regulation. Excitingly, our preliminary studies have revealed that the histone acetyltransferase Gcn5p increases the dynamics of chromatin states and stochasticity in gene expression but does not regulate basal transcription, transcription initiation, or RNA degradation. We will determine how the specific HAT module subunits regulate chromatin structure and the kinetics of these processes (Aim 1). We will elucidate how transcription initiation is regulated by unique TBP module subunits (Aim 2). And we will reveal how the specific DUB module subunits differentially regulate RNA export (Aim 3). To accomplish these aims, we propose a rigorous framework of quantitative and dynamic single-cell experiments integrated with sophisticated data analysis and predictive single-cell modeling. This innovative approach will mechanistically dissect gene regulation by the medically relevant and evolutionary conserved multiprotein SAGA complex, providing the first comprehensive analysis of multiprotein gene regulatory complex coordination of gene expression within a single experiment. Furthermore, our studies will provide a blueprint to dissect how other multiprotein complexes regulate gene expression.