DNA of eukaryotes is complexed with histones to form chromatin. There are many structural states of chromatin, beginning with repeated arrays of nucleosome cores and ending with the metaphase chromosome. While the fundamental biological roles of chromatin in the regulation of nuclear processes are well documented, very little is known at the molecular level about any of the structures of chromatin. This in turn has prevented clear understanding of the relationships between chromatin structure and nuclear functions. The research outlined in this proposal overcomes previous technical limitations, and will allow for the fiat time a systematic dissection of the structure and stability of chromatin in the solution state. Studies will utilize a novel in vitro reconstituted chromatin model system composed of arrays of positioned nucleosomes.
The specific aims of the proposed research are to (1) determine the molecular mechanism of two recently discovered features of nucleosome arrays, salt-dependent folding (in the absence of linker histones) and histone octamer dissociation, and (2) to determine how chromatin folding and dissociation influence transcription of eukaryotic 5S rRNA genes. Chromatin model system folding and dissociation will be characterized simultaneously in the analytical ultracentrifuge, using sedimentation velocity and sedimentation equilibrium approaches. Specifically, the proposed research will test four potential determinants of chromatin structure and stability: transcription factors, torsional constraint and linker DNA supercoiling, nucleosome array density, and polyvalent cations. Because each of these parameters is associated with chromatin in the intact nucleus, these studies will provide valuable insight into the structure assumed by nucleosome arrays in a simulated in vivo environment. In addition, the chromatin model systems will also be used as a substrate for in vitro transcription experiments, thus providing a unique means to analyze the functional effects of chromatin structure and stability on the process of transcription. If the specific aims of this proposal are achieved, at the completion of these studies it will be possible to tackle the long term goal of this laboratory: biophysical characterization of an active steroid hormone-regulated gene that has been reconstituted in vitro entirely from purified components.
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