The eukaryotic genome is organized into chromatin, a complex nucleoprotein assembly that regulates access to the DNA. Chromatin consists of nucleosomes, disk-shaped octamers composed of two copies each of the four core histone proteins that wrap 147 bp of DNA around their outer surface. Nucleosomes compact into higher order assemblies that are stabilized by non-histone proteins such as linker histone H1. Our objective is to develop a quantitative understanding of how chromatin assembles and switches between inaccessible and accessible states, thereby regulating essential DNA metabolic processes. The incorporation of different histone variants (non-allelic versions of core histones with distinct sequence and expression pattern) at specific locations in the chromosome has specific and profound effects on many cellular functions. Histone chaperones also influence genome accessibility, and are a structurally diverse group of proteins that promote ATP-independent histone exchange, nucleosome assembly and disassembly. Histone chaperones and histone variants are functionally connected. Here we test the hypothesis that the incorporation of specific histone variants affects nucleosome stability and dynamics. This would result in altered interactions of variant nucleosomes with linker histones, and in differences in higher order structures (Aim 1). A change in thermodynamic and dynamic properties of variant nucleosomes also likely affects their ability to be acted upon by the various histone chaperones (Aim 2). We will quantify the specificity of several histone chaperones for major-type and variant histones, and test novel hypotheses regarding their ability to stabilize a folded state of histones and to prevent noncanonical histone-DNA interactions both in vitro using our battery of analytical approaches and in vivo on a genome-wide scale. Single-particle fluorescence resonance energy transfer and small angle x-ray scattering will be used to characterize in-solution states of variant nucleosomes, while the effect of histone chaperones on the folded state of histone variant complexes will be assayed by hydrogen-deuterium exchange coupled to mass spectrometry. The strength of our approach lies in the combination of rigorous analytical approaches with in vivo studies to investigate the effect of chaperone deletion on histone distribution. The proposed studies are highly significant as they systematically and quantitatively test interconnected hypotheses regarding the biological activities of histone variants and histone chaperones in the context of both nucleosomes and higher order nucleosome assemblies. We challenge current views of nucleosome conformation, and describe structural states that have enormous potential to impact genome organization and accessibility.

Public Health Relevance

The machinery that transcribes and replicates the information encoded in the human genome is prevented access due to the packaging of all genomic DNA with an equal amount of protein to form chromatin. Here we study two functionally related activities that regulate DNA accessibility and thus vital cellular processes through the modulation of chromatin structure. We are using quantitative and structural approaches to gain insight into how structural transitions within chromatin allow vital biological processes to occur.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM067777-09
Application #
8321362
Study Section
Special Emphasis Panel (ZRG1-BCMB-S (02))
Program Officer
Preusch, Peter C
Project Start
2003-05-01
Project End
2016-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
9
Fiscal Year
2012
Total Cost
$358,532
Indirect Cost
$113,309
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
Chen, Xu; D'Arcy, Sheena; Radebaugh, Catherine A et al. (2016) Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the Inducible GAL Locus. Mol Cell Biol 36:1287-96
White, Alison E; Hieb, Aaron R; Luger, Karolin (2016) A quantitative investigation of linker histone interactions with nucleosomes and chromatin. Sci Rep 6:19122
Prasad, Rashmi; D'Arcy, Sheena; Hada, Arjan et al. (2016) Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes. Mol Cell Biol 36:2262-71
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D'Arcy, Sheena; Martin, Kyle W; Panchenko, Tanya et al. (2013) Chaperone Nap1 shields histone surfaces used in a nucleosome and can put H2A-H2B in an unconventional tetrameric form. Mol Cell 51:662-77
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