Area of Science: 07 Molecular and Cellular Biology Abstract The eukaryotic genome is present in the nucleus as a complex three-dimensional (3D) entity, the structure of which is disorganized in certain human diseases including various cancers. However, it remains unclear how 3D genome organization influences pathological processes. One impediment is the lack of established methods to investigate higher-order genome organization in three dimensions. We propose to develop a new methodology to map the 3D structure of the genome in vivo. To accomplish this goal, we will first use the chromosome conformation capture (CCC) technique to acquire vast amounts of paired DNA fragments that reflect the physical interactions among multiple genomic loci. The CCC technique involves fixation of the in vivo genome structure by paraformaldehyde (pFA), followed by restriction enzyme digestion and DNA ligation. We will follow this with a newly developed Solexa sequencing technology, which can determine sequences for several million DNA fragments in one experiment. We will create a frequency distribution table indicating the physical interactions between DNA fragments based on the large-scale sequencing results, and model the global 3D genome structure using these data. To accelerate development of this innovative system, we will use the fission yeast Schizosaccharomyces pombe as a simple test model for our technology; then, we will apply this new method to the human genome by analyzing DNA fragments containing Alu repeats, since the human genome is too large to analyze in its entirety. By comparing 3D genome structures sampled from nondiseased and diseased individuals, we will demonstrate the involvement of higher-order genome disorganization in human diseases. This innovative method has the potential to elucidate a novel but poorly understood aspect of pathogenesis, and is also applicable for disease diagnosis by detecting subtle morphological alterations of nuclear structure in diseased individuals. Thus, it has the potential to directly and profoundly affect human health.