The DNA of eukaryotes is packaged with core histone octamers to form nucleosomal arrays. In certain regions of the genome (e.g., telomeres), nucleosomal arrays are assembled into specialized structures termed heterochromatin. Genes packaged within heterochromatin tend to be transcriptionally silenced, distinguishing them from the genes which either are regulated or constitutively expressed. In S. cerevisiae, telomeric heterochromatin is associated with formation of a specific heritable nucleoprotein structure assembled from nucleosomal arrays and the silent information regulator (Sir) proteins, Sir2p, Sir3p, and Sir4p. It has been proposed that Sir proteins function in a multi-subunit SIR complex to help create a transcriptionally silenced heterochromatin domain. In the present proposal, I plan to use quantitative biochemical and biophysical approaches to characterize the macromolecular interactions of Sir proteins, both in the absence and presence of nucleosomal arrays. Specifically, I propose to: (1) characterize the self-association and heteromeric interactions of yeast telomeric silencing proteins in solution in vitro, (2) characterize the interaction of yeast telomeric silencing proteins with nucleosomal arrays in solution in vitro, (3) characterize the structural features of the supramolecular nucleoprotein complexes assembled in vitro from yeast silencing proteins and nucleosomal arrays using analytical hydrodynamic, electrophoretic, and electron microscopic techniques. The proposed studies will be the first to characterize the assembly, stability and higher order structures of the nucleoprotein complexes formed in vitro from defined nucleosomal arrays and pure yeast telomeric silencing proteins. The information obtained from these studies will provide a framework for understanding how chromatin fibers are assembled into functionally specialized heterochromatin domains. Additionally, the innovative approach developed to quantitatively study telomeric silencing protein complexes will be generally applicable for rigorous solution analysis of macromolecular assemblages in the giga-dalton size range.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066834-03
Application #
6743746
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Chin, Jean
Project Start
2003-05-01
Project End
2007-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
3
Fiscal Year
2004
Total Cost
$283,838
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
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
Hansen, Jeffrey C; Wexler, Brian B; Rogers, Danielle J et al. (2011) DNA binding restricts the intrinsic conformational flexibility of methyl CpG binding protein 2 (MeCP2). J Biol Chem 286:18938-48
Yang, Chenghua; van der Woerd, Mark J; Muthurajan, Uma M et al. (2011) Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes. Nucleic Acids Res 39:4122-35
Ghosh, Rajarshi P; Nikitina, Tatiana; Horowitz-Scherer, Rachel A et al. (2010) Unique physical properties and interactions of the domains of methylated DNA binding protein 2. Biochemistry 49:4395-410
Hansen, Jeffrey C; Ghosh, Rajarshi P; Woodcock, Christopher L (2010) Binding of the Rett syndrome protein, MeCP2, to methylated and unmethylated DNA and chromatin. IUBMB Life 62:732-8
Hite, Kristopher C; Adams, Valerie H; Hansen, Jeffrey C (2009) Recent advances in MeCP2 structure and function. Biochem Cell Biol 87:219-27
Adkins, Nicholas L; McBryant, Steve J; Johnson, Cotteka N et al. (2009) Role of nucleic acid binding in Sir3p-dependent interactions with chromatin fibers. Biochemistry 48:276-88

Showing the most recent 10 out of 15 publications