PROJECT SUMMARY The eukaryotic genome is compacted in a basic repeating structure called chromatin, the anomalous regulation of which is characteristic of many diseases. In recent years, the technologies to study chromatin have leapt forward, allowing us to investigate chromosome interactions in three dimensions. However, these methods rely on measurements taken at a baseline state, thus, a barrier remains to understanding the biological functions of chromatin in a dynamic system. It has become increasingly evident that viruses manipulate the nuclear environment to generate viral progeny, necessarily hijacking host chromatin resources for viral benefit. Therefore, virus infection provides an ideal dynamic system in which to investigate chromatin function. Moreover, interrogating virus-host interactions has led to advances in virus biology and the discovery of some of the most profound areas of molecular biology ranging from p53 to splicing. With the recent advances in chromatin methodologies, we are now in an ideal position to take the next step in understanding chromosome biology in the three-dimensional context of dynamic biological systems. In this proposal, we aim to employ cutting-edge chromatin technology combined with virus infection to reveal fundamental chromatin functions. We focus on nuclear replicating viruses, adenoviruses and multiple herpesviruses, as test cases for the virus to pinpoint vulnerabilities in chromatin function exploited by pathogens. We have successfully identified three scenarios in which cellular chromatin is distinctly reorganized for viral benefit: 1) adenovirus protein VII causes global chromatin reorganization through linker histone displacement to disrupt the cell cycle and impact transcription; 2) herpes simplex virus infection causes marginalization of host chromatin by generating new regions of heterochromatin that promote egress of viral progeny; and 3) human cytomegalovirus polarizes cellular chromatin to generate a functional viral assembly center. We will systematically investigate the mechanisms by which these dramatic nuclear rearrangements occur in three dimensions using a combination of high-resolution immunofluorescence microscopy, electron microscopy, and chromosome capture techniques. In the previous funding period, our approaches defined multiple previously unknown vulnerabilities of chromatin, positioning us to take the next step into understanding the mechanisms of chromatin organization in the nucleus in 3D. Completion of these studies will identify new chromatin targets and assist development of innovative therapies for cancer, inflammation, and viral diseases.