STRUCTURE OF NUCLEOSOMES CONTAINING VARIANT HISTONES

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DESCRIPTION(adapted from applicant's abstract): The DNA of a single eukaryotic cell is over two meters in length, but compacts in the cell nucleus by a hierarchical scheme of packaging into nucleosomes and subsequent organization into higher order chromatin structures. Activation of a gene at a given time requires its identification within highly compacted chromatin. Local unpacking and remodeling of chromatin by remodeling factors then allows the binding of the transcription machinery. The central goal of this laboratory's research is to understand, at the structural level, in what manner the compacted state is altered to accommodate the transcription machinery. This was initiated by determining the three-dimensional structure of the nucleosome core particle at 2A resolution. The experiments proposed here will provide information on the changes that are imparted on nucleosome structure to make it 'transcription competent'. The specific aims are (1) To study the structure and stability of nucleosome core particles containing essential histone variants that are preferentially associated with transcriptionally active chromatin, by determining the high-resolution X-ray structure of the nucleosome core particle containing H2A.Z, and by measuring the in vitro stability of these nucleosomes using fluorescence energy transfer. (2) To investigate the structural and functional consequences of histone mutations (the SIN-variants) which bypass the requirement for an ATP-dependent chromatin-remodeling complex in yeast, by determining the X-ray structure of the yeast nucleosome core particle containing the SIN-variants of histones. The stability and dynamics of the yeast nucleosome core particle containing SIN-variants will be studied using spectroscopic methods, and the ability of wild type and mutant nucleosomes to bind transcription factors in vitro will be assayed using a variety of biochemical and spectroscopic methods. These studies will contribute directly to our knowledge of how transcription is regulated in the eukaryotic cell by modification of nucleosome structure. Because the regulation of gene expression is crucial in the control of cell growth, differentiation, and the establishment and maintenance of tissues and organs, these studies will contribute directly to the goal of understanding the molecular basis of many diseased states that are associated with aberrant gene expression.