It is now abundantly clear that the expression pattern of a cell's genetic information is greatly influenced by the organization of DNA in chromatin structures. Modulations of chromatin structure, which range from modifications of individual histone proteins to large scale chromatin remodeling events, play key roles in gene expression, and disruption of these structures results in aberrant development and differentiation, including oncogenicity. This proposal aims to provide unique new information, by applying electron cryo-microscopy (EC-M) to native and fully defined chromatin assemblies to reveal their 3D solution conformation. Studies will also include the conformational changes that result when chromatin is modified in ways known to be important in gene expression. Direct imaging provides key visual information that can be used to place biochemical and genetic data in the context of the living nucleus. 1. Specific histone domains which are known to play crucial roles in chromatin compaction and organization will be localized within chromatin arrays using a novel approach in which gold beads are bound to cysteine residues. 2. Chromatin conformation will be studied under the ionic conditions thought to be present in the nucleus, and which cause extreme compaction and self- association in vitro, will be studied using a time-resolved adaptation of EC-M whereby the effects of altering salt conditions can be manipulated in the millisecond time range. 3. The conformational effects of core histone acetylation and linker histone phosphorylation, both known to have a major functional impact on chromatin, will be determined. 4. The conformation and molecular determinants of large scale chromatin compaction (heterochromatin) will be studied in lymphocytes. When lymphocytes are stimulated to proliferate, either in vivo or in cell culture, their heterochromatin disperses, thus allowing a controlled study of the changes in conformation and composition that occur during chromatin dispersion. These studies will also be relevant to the chromatin dispersion that occurs in lymphomas and leukemic lymphocytes. 5. The process of transcription on nucleosomal templates will be studied using EC-M, providing new information on the manner in which RNA polymerase molecules are able to transcribe through nucleosomes.
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