Project Summary The nuclear package that comprises the eukaryotic genome not only stores genetic information but also mediates cell-type-specific gene expression. The hierarchical genome organization is tightly regulated to precisely control cell functions. Interphase chromosomes occupy distinct nuclear spaces, a conserved genome architecture known as chromosome territories. Technological advances over the last two decades have revealed many new aspects of the three-dimensional architecture of the genome. However, understanding the mechanisms that localize and mobilize chromosomal loci and territories in the nucleus requires high-resolution studies in real time under physiological conditions. In the past five years, we have developed CRISPR-based high-resolution live-cell imaging techniques using multiple colors to localize and track up to seven genomic loci simultaneously. Recently, we have replaced fluorescent proteins with small cell-permeable RNA-interacting molecules that improve brightness and reduce the size of tags by >100-fold. Our preliminary data revealed surprising dynamic and structural aspects of the chromatin: (1) homologous and non-homologous chromosomal loci moved at different speeds and in different directions; (2) large-scale chromosomal domains continuously rearranged in minutes in non-stressed conditions, termed chromosome morphological dynamics; (3) chromosome conformations were temperature-sensitive; and (4) transformed and non-transformed cells had distinct chromosome conformations. In mouse embryonic stem cells, the mobility of promoters and enhancers correlates with transcriptional activity for specific genes; however, how chromatin mobility correlates with transcriptional activity is poorly understood and controversial. Building upon our preliminary results, we propose to investigate four key concepts: (i) how chromosomal DNA is organized in individual chromosome territories, (ii) what factors drive chromosome morphological dynamics, (iii) how active genes are positioned relative to non- transcribed DNA regions to craft the landscape of the genome, and (iv) how chromatin movements correlate with transcriptional activities in the nucleus. We will perturb transcription, temperature, and microtubule polymerization to identify factors that govern chromosome dynamics. Integration of non-invasive imaging approaches with biophysical models and RNA-seq data will provide new information on the mechanistic and functional foundations of real-time chromatin dynamics and gene positioning at the single chromosome level in the nucleus.