Biological Validation Development

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PROJECT SUMMARY / ABSTRACT – BIOLOGICAL VALIDATION DEVELOPMENT The ultimate goals of this Module are to demonstrate that we are able to predict chromosome locations and trajectories relative to different nuclear compartments from genome-wide, DamID and TSA-Seq mapping data AND to identify the biological significance of this chromosome positioning. We will accomplish these goals through three major aims. Each will involve iterative cycles of experimentation and computer modeling. The first is to validate predictions for chromosome positioning and dynamics with respect to major nuclear compartments from genome-wide DamID and TSA-Seq data. This involves initially calibrating the output of our genome maps, such that we can estimate contact frequencies and distance distributions relative to each of the major nuclear compartments considered separately. More ambitiously, this calibration data will be used as input for computer modeling in the DAM Module: mapping data for multiple nuclear compartments will be combined with other genomic data to yield more accurate predictions of chromosome location and trajectories. These predictions will then be tested by direct microscopy observations and used to refine our predictive modeling. Second, we aim to identify DNA regions, and ultimately cis elements, responsible for targeting chromosome regions to specific nuclear compartments. Our third and final goal is to predict and test the functional consequences of chromosome loci localization near different nuclear compartments. We will do this through a novel combination of live-cell readout of functional assays as a function of chromosome position AND through a deliberate, rewiring of the trajectories of endogenous chromosomes, followed by readouts using molecular methods. More specifically, we will focus on the following Specific Aims: 1: Validate intranuclear chromosome position as a function of DamID and TSA compartment genome-wide mapping data; 2: Test predicted mechanisms by which intranuclear chromosome compartmentalization is established; 3: Test predicted functional consequences of chromosome localization to different nuclear compartments Completion of these Aims should provide us with the capability and confidence for interpreting the biological significance of changes in genome organization relative to nuclear compartments that we observe in different cell and tissue types. We anticipate that this level of nuclear organization plays a critical but previously unrealized role in establishing and maintaining tissue-specific patterns of gene expression. Therefore the work in this Module will be critical for the future application of our new mapping technologies towards the improved understanding of cell function in normal development and human disease.