PROJECT SUMMARY Each human nucleus contains two meters of DNA that is packaged into an organelle a mere ~10 µm diameter. Despite this dramatic difference in scale, the three-dimensional organization of the genome is non-random and plays a critical functional role in health and disease. My research group is focused on the building robust and scalable methods to study the organization of chromosomes in 3D space, the interactions they participate in with at the inter- and intra-chromosomal level, and the associated RNAs and proteins that occupy functionally relevant sites. The motivation for this work is to better understand the mechanisms by the organization and composition of genomic intervals relevant for health and disease faithfully conduct the essential DNA transactions of transcription, replication, and repair. Our previous studies focused on the development of multiplexed fluorescent in situ hybridization (FISH) methods to map chromosome structure in individual cells and the creation of biochemical screening methods to identify the molecular factors present at the anchor sites for 3D chromosome loops. To this end, we have introduced new multiplexed FISH technologies and adapted these to facilitate proximity labeling and affinity purification of factors from target genomic intervals and chromatin associated RNAs. We now propose to build on these efforts by further developing FISH technologies to support a broader range of questions relevant to chromosome biology and making efforts to speed the adoption of advanced FISH technologies, by extending our proximity labeling approach to more challenging genomic targets important for the regulation of gene expression, and by investigating the position and composition of highly repetitive DNA sequences, which we hypothesize play an integral role in maintaining the structure and stability of the genome. Collectively, the studies proposed here will produce enabling new genomic technologies, produce rich datasets that detai