This thesis investigates the transcriptional regulation and signalling dynamics of
the High Osmolarity Glycerol (HOG) pathway in yeast, focusing on its impact on gene
expression under stress conditions. Using single-cell analysis methods, including phage
coat protein reporters, we explore how Mitogen-Activated Protein Kinase (MAPK)
pathways influence transcriptional dynamics. By integrating experimental and compu-
tational approaches, we provide quantitative insights into transcriptional processes with
high temporal resolution. Key contributions include the development of single cell anal-
ysis tools, expanding the capabilities of the phage coat reporter system. This enables
the measurement of RNA pol II elongation rate and absolute quantification of mRNA
numbers using Genetically Encoded Multimeric nanoparticles (GEMs). Furthermore,
we developed a pulse-width modulation-based microfluidics platform that enabled pre-
cise control of stress inputs, revealing distinct transcriptional responses under dynamic
conditions.
Our findings challenge the conventional usage of Hog1 nuclear relocalization as a
proxy for MAPK influence on gene expression, showing weak correlations between nu-
clear residency and MAPK driven gene expression. By investigating a diverse set of
promoter, both stress-responsive and constitutive, we show that global cellular param-
eters, the innate ability of a given cell to transcribe, are a good predictor of observed
heterogeneity.
By advancing methods and techniques for single-cell transcriptional analysis, this
work lays the foundation for future investigations into MAPK-dependent gene regu-
lation and its broader implications in cellular adaptation to environmental changes.
These findings contribute to a deeper understanding of gene expression heterogeneity.