AbstractPrecise regulation of gene expression in response to environmental changes is crucial for cell survival, adaptation and proliferation. In eukaryotic cells, extracellular signal integration is often carried out by Mitogen-Activated Protein Kinases (MAPK). Despite a robust MAPK signaling activity, downstream gene expression can display a great variability between single cells. Using a live mRNA reporter, here we monitor the dynamics of transcription in Saccharomyces cerevisiae upon hyper-osmotic shock. We find that the transient activity of the MAPK Hog1 opens a temporal window where stress-response genes can be activated. We show that the first minutes of Hog1 activity are essential to control the activation of a promoter. Chromatin repression on a locus slows down this transition and contributes to the variability in gene expression, while binding of transcription factors increases the level of transcription. However, soon after Hog1 activity peaks, negative regulators promote chromatin closure of the locus and transcription progressively stops.