The human genome is highly organized and regulated to express cell-type and tissue-specific genes. During interphase, chromosomes occupy distinct regions in the nucleus, known as chromosome territories, a concept proposed as early as 1885 and demonstrated in 1982. Technological developments over the last two decades have allowed changes in the three-dimensional architecture of the genome to be examined and modeled. But understanding the mechanisms that localize or mobilize chromosomal loci in the nucleus will require high- resolution studies in real time. The recent development of CRISPRainbow allows the simultaneous labeling, visualization, and real-time tracking of up to seven specific genomic loci at high resolution in live human cells. Preliminary studies show that loci on homologous and non-homologous chromosomes move with different speeds, directions, and confinement. CRISPRainbow allows quantitative categorization of the movements of various loci, detects accelerated movements at DNA double-strand breaks, and change in a chromosome's overall organization. The goal of this proposal is to answer the following questions. What is spatial range of genomic loci movements? Is the dimension of the dynamic spatial range chromosome-specific and/or dependent of its intranuclear localization? How does long-range chromatin territory relocation take place following DNA double-strand breaks? What is the interplay of transcription and the chromatin remodelers on genomic loci movements and entire chromatin organization changes? During the K99 phase, CRISPR-based single-molecule real-time microscopy will be used to quantitatively image and characterize genomic loci movements and to test the hypothesis that genomic loci movements depend on chromosome identity or on nuclear location. Chromatin territory relocation will also be tracked during DNA damage repair in single cells. During the R00 phase, chromosome conformation capture experiments will be used to molecularly characterize changes in chromosomal interactions upon DNA damage and molecular, cellular, and genetic experiments will be used to investigate chromatin dynamics in response to transcription activation/silencing, nucleosome disassembly, and the tumor suppressor p53. This proposal is highly interdisciplinary, will shed the light on human genome organization and stability, and will lead to powerful quantitative real-time methodology to investigate mechanisms of chromosome translocation in cancer cells. This study draws on expertise from mentors who are leaders in the field of single-molecule real-time microscopy, nuclear biology, and genome/nuclear architecture and who will help prepare Dr. Tu for a transition to a career as an independent investigator.
The three-dimensional organization of the human genome rearranges during cell differentiation, transcription activation, and DNA damage repair. This proposal describes a novel approach that allows chromosome dynamics to be directly visualized, tracked, and characterized at high resolution in live cells. Understanding the dynamics and mechanisms of chromosome rearrangements in response to DNA damage and transcription activation will significantly improve our understanding of human genome stability and nuclear function.
