Project Summary/Abstract A key tenet of the 4DN project is that nuclear functions (e.g., gene expression) is directly influenced by the spatial organization of the genome, not only in relation to the 1D sequence distance between chromosomal regions but also with respect to their three-dimensional spatial organization in the nucleus. While there has been substantial progress in our understanding of the functional implications of the relative proximity of genomic loci in terms of their three-dimensional spatial distance through sequencing-based (e.g., Hi-C) and imaging-based (e.g., Multiplexed FISH) omics data, the functional implications of the dynamic positioning of loci within the nuclear topography (i.e., the volumetric mapping of loci with respect to the nuclear microenvironment) remains a more challenging task. In particular, because systematic evaluation of the functional role of the nuclear topography in health and disease requires the effective integration of sequencing-based and imaging-based omics data from multiple different tissue types, biological statuses, conditions, and laboratories. This goal requires the development of standardized nuclear reference coordinate systems in which spatial data from different sources, often varying largely in nuclear shape, is first mapped against common nuclear landmarks and then integrated into one Common Coordinate Framework (CCF). Here we propose to establish guidelines for the 4DN Nuclear CCF and define methods for mapping the location of nuclear boundaries across different experimental systems and assess the opportunity and feasibility of different experimental methods. We will produce benchmarking Multiplexed FISH datasets to evaluate different nuclear boundary visualization approaches identified on the basis of current user practices and will suggest methods that are minimally detrimental to the productivity of experimental laboratories. Our proposal will expedite the development of a cross-consortium 4DN nuclear CCF capable of univocally mapping the location of FISH Omics imaged target genomic segments with respect to the NE, nuclear bodies, the center of the nucleus, and each other in a manner that can then be used to integrate data across different experimental contexts and will allow incorporation in integrated predictive modeling frameworks. In summary, by directly addressing the functional significance of nuclear topography on nuclear function, a CCF would make 4DN-produced imaging data more valuable for computational scientists and for the community at large.