PROJECT SUMMARY Cells must regulate their genome so that genes are expressed in the appropriate cell type, and at the correct time. Further, as cells go through the cell cycle chromosomes must be replicated, compacted and then faithfully segregated. All these processes require correct folding of the genome. Defects in folding, e.g., the looping of genes to incorrect distal enhancers, or incorrect chromosome compaction during mitosis, can lead to genes being expressed in the wrong place and at the wrong time, or to genome instability. Such defects can lead to diseases including cancer. There has been tremendous progress in identifying folding principles of chromosomes, how folding changes during the cell cycle, roles of cis elements in folding chromosomes and long-range gene regulation, and the molecular machines and mechanisms that fold chromosomes. In interphase chromosomes fold into topologically associating domains and cohesin-mediated loops. Chromosomes also compartmentalize to form active and inactive chromatin domains through a phase separation process. In mitosis, chromosomes refold into linearly compressed arrays of condensin-mediated loops. We and others showed that alternation of cohesin-mediated loop formation and condensin-mediated loop formation drives cell cycle stage-dependent chromosome folding. However, three critical aspects of chromosome organization have remained largely unexplored, mostly due to lack of experimental approaches. First, little is known about the identity of cis elements that mediate interactions between sister chromatids, yet these interactions are critical for faithful chromosome segregation. Second, the topological state of chromosomes, i.e., the presence of intra- and inter-chromosomal catenations is almost entirely unexplored. Catenations form during replication and transcription and these create impediments to correct gene expression and chromosome segregation and therefore the cell must constantly resolve these. Cis elements and trans factors involved in controlling the topological state of the genome are largely uncharacterized. Third, factors and cis elements driving chromosome compartmentalization are poorly characterized. We recently developed three new genomic technologies, SisterC, Multi-Contact 3C, and Liquid Chromatin Hi-C that allow studying each of these three outstanding questions respectively. Here, we will employ these methods to identify genomic DNA elements, and their mode of action, that control chromosome compartmentalization, catenation and decatenation, and that play roles in disentangling and segregating sister chromatids.