The highest levels of chromatin structure, consisting of the packing of 100 and 300 angstrom chromatin fibers within mitotic and interphase chromosomes, are unknown. Understanding this level of chromatin organization, describing the structure of entire transcription or replication units, is essential to answering fundamental questions related to gene regulation, DNA recombination, cell differentiation, and cell cycle control. This project's long term objectives are to develop a model system allowing dissection of basic structure-function motifs involved in the large-scale chromatin organization of mammalian interphase chromosomes. Specifically, it will focus on the nuclear and chromosomal decondensation occurring during cell cycle progression through Gl into S phase. The strategy will be to develop an in vitro system reproducing decondensation events occurring in vivo, and to exploit this system to analyze their underlying biochemical and structural basis and functional relevance to DNA replication initiation. The grant will lay the foundation for this long term project. A detailed 3-dimensional structural analysis will compare nuclear and chromosome decondensation in vivo during Gl progression with decondensation induced in vitro using cell free extracts. Several 3-dimensional reconstruction methods, including light microscopy optical sectioning, electron microscopy serial sectioning, and electron microscopy axial tomography, will provide overlapping resolution over a complete range of size scales.
The specific aims of this grant will include the following: Interphase chromosome decondensation in vivo and in vitro will be described in terms of the intranuclear arrangement of interphase chromosomes, association of chromosomes with nuclear lamins, uncoiling of interphase chromosomes into their component large-scale chromatin structures, and packaging of 300 angstrom chromatin fibers within these individual large- scale domains. The unfolding pathway of early versus late replicating regions will be contrasted, and the architecture of actively replicating chromatin will be determined. Finally, quantitative upper limits on the experimental errors associated with these structural descriptions will be estimated.
