Summary: The human genome is highly dynamic, yet the principles governing its movement are not known. Locally, chromatin undergoes constant remodeling and rearrangement associated with processes such as transcription, replication and DNA repair. At large length scales, chromatin dynamics is coherent over microns and seconds. How the local gene-level processes contribute to nucleus-wide chromatin motions remains an open question. To address this question, our overall approach is to map spatially and temporally resolved chromatin dynamics across the nucleus in mammalian cells in vivo, while connecting it with motion and transcriptional activity of specific genomic loci in real time. To do so, we will develop crosscutting tools integrating synergistically quantitative approaches derived from the physical sciences with the latest techniques from molecular biology and biochemistry. We will build an integrated experimental and analytical platform enabling simultaneous measurements of nucleus-wide and gene-specific motions in real time in vivo (Aim 1). Specifically, we will establish a data collection and analytical pipeline mapping chromatin motions across the nucleus in vivo using displacement correlation spectroscopy (DCS), while monitoring motions of genes visualized by CRISPR/dCas9 technology and tracked via new machine-learning assisted algorithms. In addition, our platform will monitor the spatiotemporal heterogeneity of chromatin across nucleus and toggle transcriptional activity of the tracked genes. Using this integrated platform, we will address the fundamental question of how gene-level transcription activity contributes to genome-wide motions (Aim 2). We will measure maps of chromatin motions and compaction across the whole genome, while simultaneously determining the local compaction and mobility of specific genes (MUC4, IL6) as a function of their transcriptional activity. Our findings will paint a new picture of the complexity and interconnectedness of gene- and genome-level dynamics and spatial heterogeneity. Finally, we will extend this approach to study interphase chromatin dynamics and compaction before and after cell differentiation of mouse embryonic stem cells (Aim 3). By linking gene-level activity to genome-wide compaction and motions, these results will have important implications for elucidating the role of chromatin dynamics in gene regulation and expression. Moreover, such knowledge will provide a framework for a mechanistic picture of chromatin dynamics in mammalian cells.