Gene expression is a tightly regulated process to ensure that genes are activated in the right cell at the right time. In the last half century, our knowledge of gene regulation has greatly advanced, but the majority of measurements come from large populations of cells. However, individual cells in a population can exhibit considerable variability in transcriptional responses, arising from the random collision of molecules. This stochastic gene expression variation can influence important cell fate decisions and can also contribute to heterogeneity in tumors. To understand the mechanisms and dynamics of gene expression in single cells, our group employs and develops cutting-edge single-molecule microscopy techniques to visualize the behavior of individual protein and RNA molecules in living cells. Our imaging assays are combined with novel gene-specific targeting approaches to modulate transcription of specific genes. By utilizing a combination of single-molecule microscopy, biophysical, genetic and molecular biology approaches in both yeast and mammalian model systems, we aim to understand the molecular mechanisms of transcription regulation, and how stochasticity in transcription modulates cell-to-cell variability and contributes to cancer progression.