It is becoming increasingly clear that promoter chromatin structure and the remodeling of that structure in association with gene activation are crucial facets of eukaryotic transcriptional regulation. The recent development of an in vitro MMTV-LTR system that can reconstitute the correct promoter chromatin structure and the correct remodeling of that structure in vitro presents an unprecedented opportunity to study these important facets of transcription regulation. In particular, it will now be possible to study promoter chromatin with and without bound receptor, and thus obtain information on this key first step of promoter recognition. We can then analyze the remodeled chromatin, to characterize the chromatin structure and transcription factor changes that have occurred as a result of remodeling. Because of its scale of imaging, the atomic force microscope (AFM) is well suited for studying the various structural aspects of this process that we want to analyze: the Linear Organization (nucleosome locations), the Higher-Order structure (conformations of fully hydrated chromatin and transcription factors), and Molecular Recognition Mapping (identifying specific molecules in the spreads based on antibody recognition). These approaches are a blend of established techniques and new AFM techniques that will be developed for this application but will undoubtedly prove useful for other biophysical and biological applications. The new techniques will be developed using known and defined model chromatin templates (e.g., 208-12 and known modifications thereof) and well characterized transcription factors. These techniques will then be applied to study the MMTV-LTR promoter chromatin structure and the remodeling of that structure and the results of the control, model systems used to provide a baseline to help interpret the MMTV-LTR results. The proposal brings together a physicist (Stuart Lindsay, Arizona State University, scanning probe microscopy, instrument development) and biochemists (Gordon Hager, NCI, chromatin remodeling, Dennis Lohr, ASU, transcriptional regulation/chromatin structure and Rodney Harrington, ASU, DNA-protein interactions) to develop techniques, test them on control samples and then apply them to remodeling of promoter chromatin. The collaboration will include biophysicists Hansgeorg Schindler and Peter Hinterdorfer (University of Linz, Austria) pioneers in developing nm-scale molecular recognition techniques that use an antibody attached to an AFM probe. Instrument development will be supported by Molecular Imaging Corporation.
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