The goal of this project is to develop live-cell single-molecule imaging technologies to visualize and manipulate Post-Translation Marks (PTMs) deposited on chromatin. Chromatin PTMs regulate which genes are expressed or silenced, which ensures correct establishment and maintenance cell types. PTM alterations result in aberrant expression and are involved for instance in cancer progression. In light of the role played by chromatin PTMs in health and disease, we need a better understanding of when, how and by whom these epigenetic marks are deposited, and whether they play a causal or accessory role. The tool of choice to address mechanistic questions is live-cell single-molecule imaging, because it captures sequences of events in individual cells. Yet current imaging techniques remain unable to quantify PTM deposition at individual genomic loci or their impact on transcription dynamics. To solve these issues, we will develop a technology that combines innovative PTM reporters and actuators with subcellular optogenetics and quantitative analysis. Spatial selectivity will allow background-free imaging at a locus of interest while increasing the chances of capturing transient interactions between biological partners. We will demonstrate the scalability of the technique to multiple loci imaged simultaneously. We will complement the imaging component with novel perturbation methods to induce targeted PTMs at a locus. When combined, the technology will allow controlling PTM levels while imaging subsequent chromatin and transcription in real time. Our lab has a long track record in advancing technologies that image gene expression. In addition, the strategy is backed by extensive preliminary data, indicating high chances of success. The tools we will develop provide means to interrogate the role of PTMs at an unprecedented resolution. Combined with adequate quantitative kinetic models, single-cell and single molecule data hold the promise to understand how robust epigenetic landscapes emerge from transient individual interactions.