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Chromatin Fiber and Remodeling Factor Structural Studies

Final Report Summary - CFRFSS (Chromatin Fiber and Remodeling Factor Structural Studies)

The organization and functional readout of DNA comprising the genomes of higher organisms is critically dependent on the nucleosome, the fundamental repeating unit of chromatin. Detailed knowledge of how nucleosomes interact with each other to form higher-order structures and with regulatory factors to control genomes is essential to our understanding of biology. Structural studies of the chromatin fiber (nucleosome higher-order structure) employing X-ray crystallography and cryo-electron microscopy have yielded limited information. The inadequate resolution of the resulting 3-dimensional density maps is a consequence of specimen heterogeneity generated by inherent structural degeneracy and disruption of interactions during sample preparation. The results of these studies are generally sufficient to visualize the arrangement of the nucleosomes, but insufficient to build a reliable atomic model.

Our structural studies on the chromatin fiber have used material that contains either 4 or 36 nucleosomes. In both cases, these nucleosome arrays incorporate the linker-DNA, histone-protein H1 and are imaged in the presence of magnesium ion which simulates physiological conditions. Due to the aforementioned problems, the resolution of these studies is limited at best to 18 Å, although originally, the goal was 4 Å or better. Our concurrent study of a relatively small chromatin remodeling factor bound to a nucleosome was limited by the number of intact particles that survived cryo-vitrification, such that proceeding to a 3-dimensional map was not warranted.

Future progress in chromatin structure research will likely depend on using single-particle cryo-electron microscopy. Collecting 10-1000 times more particle images and sorting them into distinct 3-dimensional classes will most probably be required.