Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization. Together these yield greater than a 10,000:1 linear condensation of DNA. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 250:1 packing ratio and involve the organization of entire transcription and replication functional domains, their compaction within mitotic and interphase chromatids, and their architecture within interphase nuclei. Essentially uncharacterized at this time, it is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure and chromosome architecture, the functional significance of specific conformational changes associated with transcription and replication, and the underlying mechanisms which regulate these conformational transitions.
Our specific aims are to answer the following questions: 1) What structural motifs underlie large-scale chromatin condensation/decondensation during the mitotic cell cycle? 2) Which steps in large-scale chromatin condensation during formation of mitotic chromosomes are dependent on SMC protein function? 3) What changes in large scale chromatin structure and intranuclear positioning are associated with DNA replication initiation? 4) What are the DNA sequence requirements for condensed versus extended interphase large-scale chromosome structures? In addition we will explore several experimental approaches to test the role of specific proteins in maintaining specialized large-scale chromatin structures within interphase chromosomes and to search for new proteins involved in large- scale chromatin organization and nuclear architecture. This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on integrating structural models of large-scale chromatin organization with the underlying biochemistry and molecular biology. In particular we are interested in exploring the mechanistic links between transcription, replication, and large- scale chromatin and nuclear structure. Knowledge gained from this research should provide valuable insight into regulation of transcription and improved design of gene therapy vectors and stable transgene expression.
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