Project Summary/Abstract Center for genome imaging (CGI) Objectives: Three-dimensional (3D) genome organization is a major contributor to genome func- tion, and yet, we are only at the very dawn of discovering the structural signatures that underlie that organization. Thus, the goal of the proposed studies is to develop and apply tools that will enable se- quence-specific imaging of human genomes, in their entirety, with high genomic resolution. In particu- lar, the proposed work will innovate methods for fixed and live cell imaging using diffraction-limited light microscopy and super-resolution microscopy as well as develop new tools for image analysis and ge- nome modeling. To this end, it will involve the continued collaboration of four laboratories, whose col- lective breadth of expertise covers the fields of classical and molecular genetics, chromosome dynam- ics, imaging, Hi-C analysis, convolutional neural networks, and polymer physics-based and restraint- based modeling. An equally important objective of the proposed studies is to ensure a generation of re- searchers whose personal breadth of expertise will come to match that of the entire current team. Health relatedness: Will a solid grasp of 3D genome organization have implications for under- standing human development? Will it contribute to the protection of human health? Will it contribute to strategies for early diagnostics and perhaps even the development of new therapies? The answer to all these questions is almost certainly a resounding Yes, as knowledge of 3D genome organization will en- hance our capacity to address both fundamental biological processes as well as disease. Innovation: An abundance of studies argue that genomes function as integrated units and, yet, no extant technologies enable sequence-specific imaging of entire genomes at high genomic resolution. Thus, the capacity of researchers to fathom the interplay between 3D genome organization and ge- nome function has been limited to disjointed snapshots of localized events. Accordingly, first three aims will develop the next tier of tools to put entire genomes within reach. They will advance a new method, OligoFISSEQ, and then integrate it with OligoSTORM and OligoDNA-PAINT to finally achieve high- throughput imaging at both conventional and super-resolution. They will also tackle two genomic fea- tures that have been prohibitively difficult to capture – presence of homologs in diploid cells and highly repeated sequences – as well as innovate strategies for high volume data storage, image processing and analysis, and modeling. Finally, a fourth aim will implement methods for disseminating our tools. 1. Scaling technologies toward whole genome imaging 2. Filling in gaps to visualize chromosomes end-to-end – tackling homologs and repeats 3. Probe design, image analysis, modeling, and integration of epigenetic data 4. Training, resources, and opportunities for engaging colleagues in whole genome imaging

Project Narrative/Relevance Center for genome imaging (CGI) A full understanding of the genome will require the highest resolution knowledge of how genes and chromosomes are organized in 3D space in both healthy and diseased cells. Without such knowledge, efforts to advance disease diagnosis and medical intervention will be compromised. As such, the overarching goal of the proposed studies is to develop and implement imaging and analysis strategies that can encompass entire genomes, including the distinction of homologs and the identification of highly repetitive sequences, and, thus, comprehensively elucidate how spatial organiza- tion influences genome function, stability, and inheritance.