The ISWI type chromatin remodeling complexes have been shown to be involved in transcription repression, and the regulation of transcription elongation and termination. Reorganization of chromatin and regions in chromosomes are fundamental in cellular development and differentiation. We propose to further our understanding of these processes by a two-prong approach of (1) extensive biochemical characterization of the ISWI family of proteins and (2) the molecular genetic analysis of mutant versus wild type ISWI complexes in vivo.
(Health Relevance): Our objective is to study the mechanism of regulating gene expression and cellular development by the manner in which DNA is packaged DNA inside the cell by the ISWI type of ATP-dependent chromatin remodeling complexes. These chromatin remodeling complexes are known to regulate many different aspects of cell development and differentiation, and as such are also involved in oncogenesis. The long term significance of this project is the elucidation of a fundamental biological process that influences and controls those processes in the cell that involves reading, repairing, copying, or changing of DNA. Important key regulatory steps of cells developing from stem cells to terminally differentiated cell types have been shown to involve reorganization of DNA in the form of chromatin and in particular to involve the type of transcription repression mediated by this type of chromatin remodeling complexes. ISWI type complexes also have a role in siRNA mediated silencing and in the synthesis of noncoding RNA. We will incorporate both the strength of yeast genetics and that of the techniques that we have developed for investigating the structure and dynamics of large macromolecular assemblies to narrow in on the structural and functional properties of the ISW2, ISW1a, and ISW1b chromatin remodeling complexes in combination with high through put techniques to afford a genomic view of these properties in the cell. All the deletions and mutations that are made will be examined extensively both in vivo and in vitro such that it will be possible to reveal important mechanistic details of chromatin remodeling by ISW2, ISW1a, and ISW1b that are physiologically important and yet well understood because of the underlying biochemical analysis that has been done.
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