Nucleosomal arrays consist of core histone octamer-DNA complexes spaced at ~200 bp intervals along a DNA molecule. They are the fundamental building blocks of chromosomal superstructures, and the substrates for all functional processes in the eukaryotic nucleus; e.g., transciption, replication, repair. Nucleosomal arrays in solution exist in equilibrium between unfolded, moderately folded, extensively folded and oligomerized conformational states. Formation of each of these states is mediated by the core histone N-termini, acting through multiple molecular mechanisms. Incorporation of linker histones into nucleosomal arrays to form chromatin arrays shifts the equilibrium towards the extensively folded and oligomerized states, and in the process dramatically alters the structure of the linker DNA that connects adjacent nucleosomes in the array. The objectives of the proposed research are to delineate and characterize the mechanisms by which the core histone N-termini and linker histones exert their effects on chromatin folding and transcription by eukaryotic RNA polymerases. Specifically, using length- and compositionally-defined nucleosomal and chromatin arrays assembled from pure DNA and histone components, in combination with an innovative technical approach that integrates analytical ultracentrifugation, quanititative agarose gel electrophoresis, and electrophoresis, and electron cryo-microscopy, Dr. Hansen proposes to: (1) characterize how selective removal and targeted acetylation of the core histone N-termini influence nucleosomal array folding and oligomerization, and to determine whether the N-termini form homotypic or heterotypic complexes with each other, (2) characterize how different linker histone domains and sequence isotypes influence chromatin folding and linker DNA structure, and (3) perform correlative in vitro transcription studies to determine the extent to which chromatin folding represses transcription by RNA polymerases II and III. The proposed experiments are strongly hypothesis driven, and directly address several long-standing paradigms relating to chromatin structure and function. These studies will help provide the foundation necessary to achieve the long-term goal of assembling functionally important genetic loci in vitro entirely from pure histone and non-histone components.
| Kalashnikova, Anna A; Rogge, Ryan A; Hansen, Jeffrey C (2016) Linker histone H1 and protein-protein interactions. Biochim Biophys Acta 1859:455-61 |
| Maeshima, Kazuhiro; Rogge, Ryan; Tamura, Sachiko et al. (2016) Nucleosomal arrays self-assemble into supramolecular globular structures lacking 30-nm fibers. EMBO J 35:1115-32 |
| Szerlong, Heather J; Herman, Jacob A; Krause, Christine M et al. (2015) Proteomic characterization of the nucleolar linker histone H1 interaction network. J Mol Biol 427:2056-71 |
| Kalashnikova, Anna A; Porter-Goff, Mary E; Muthurajan, Uma M et al. (2013) The role of the nucleosome acidic patch in modulating higher order chromatin structure. J R Soc Interface 10:20121022 |
| Rogge, Ryan A; Kalashnikova, Anna A; Muthurajan, Uma M et al. (2013) Assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning DNA. J Vis Exp : |
| Kalashnikova, Anna A; Winkler, Duane D; McBryant, Steven J et al. (2013) Linker histone H1.0 interacts with an extensive network of proteins found in the nucleolus. Nucleic Acids Res 41:4026-35 |
| Szerlong, Heather J; Hansen, Jeffrey C (2012) Activator-dependent acetylation of chromatin model systems. Methods Mol Biol 833:289-310 |
| McBryant, Steven J; Hansen, Jeffrey C (2012) Dynamic fuzziness during linker histone action. Adv Exp Med Biol 725:15-26 |
| Panchenko, Tanya; Sorensen, Troy C; Woodcock, Christopher L et al. (2011) Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical termini. Proc Natl Acad Sci U S A 108:16588-93 |
| Muthurajan, Uma M; McBryant, Steven J; Lu, Xu et al. (2011) The linker region of macroH2A promotes self-association of nucleosomal arrays. J Biol Chem 286:23852-64 |
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