Regulation of chromatin folding in space and time

Abstract Eukaryotic genomes encode genetic information in their linear sequence, but appropriate expression of their genes requires chromosomes to fold into complex and spatially distinct three-dimensional structures. Recent advances in genomic-based approaches have uncovered a hierarchy of DNA interactions, from small chromatin loops that connect genes and enhancers to larger chromosomal domains and nuclear compartments. However, despite the remarkable conservation of these organizational features and their impact on gene function, we have a very limited understanding of how chromosomes are spatially partitioned, functionally packaged, and relatively positioned in the nucleus. Technical limitations have also hindered our ability to ask questions regarding cell-to-cell variability and the relationship between chromatin folding, positioning, and function at single cell resolution. Our previous studies involved the development of two technologies that use fluorescent in situ hybridization (FISH) to interrogate chromosome positioning at single-cell resolution. Our goal is to build on this work and use these tools to elucidate how chromosomal segments find each other and then form stable interactions within cells. To this end, we have developed a rapid and precise method for identifying candidates involved in chromosome interactions. We now propose to employ this technology to isolate novel architectural proteins, which will be followed by a battery of genomic and in situ-based assays to characterize the candidates. Collectively, the studies proposed here will uncover novel molecular mechanisms underlying nuclear organization, providing a new avenue to study how chromatin folding and positioning is established and inherited, and how dysfunctional organization contributes to disease.