PROJECT SUMMARY Gene and chromatin regulation are at the core of many human biological processes such as development and aging, and play an important role in disease, including during immune response and cancer progression. Much work was done over the last few decades to catalogue transcription factors and chromatin regulators, determine the gene regulatory elements where they act, and measure the chromatin states associated with them across different cell types. However, there are over a thousand transcription factors and chromatin regulators controlling gene expression in human cells. Similarly, on the DNA side, there are tens of thousands of gene regulatory elements in the human genome. Moreover, the function of these regulatory proteins (and hence of the DNA elements they bind to) can change over time in response to signals such as cell differentiation or immune responses to viral infections. These processes also show cell-to-cell heterogeneity that is important in cell-fate decisions. Consequently, our efforts to understand the general principles of gene and chromatin regulation in human cells and the type of dynamical responses they enable are severely limited by the fact that most experimental methods can only study a handful of proteins at a time, generally use methods that average across cell populations, and often only measure correlations at a given point in time. In order to address these challenges, we combine high-throughput synthetic biology, single-cell measurements of gene expression dynamics using fluorescence microscopy and flow cytometry, and mathematical modelling for both a systematic and in-depth understanding. We use these tools to answer essential question about gene and chromatin regulation in human cells: (1) How do transcription factors work: what are the biophysical rules governing their effector domains and interactions with coactivators and corepressors, and what type of dynamic responses do they enable? (2) How does the dynamics of gene silencing, activation and epigenetic memory depend on the architecture of gene regulatory elements such as enhancers, promoters, terminators and insulators? (3) What are strategies that viral proteins have evolved to interface with and perturb gene and chromatin regulation in human cells in order to increase viral gene expression and disrupt immune responses? Together, answering these questions quantitatively will help uncover the basic principles of gene regulation in human cells in the context of a dynamic chromatin environment, will provide new tools for genetic, epigenetic and cellular therapies, and will inform treatments of viral infections and immune disorders.