Chromatin, the physical packaging of eukaryotic chromosomes, plays a major role in determining the patterns of gene silencing and expression across the genome. The active reorganization of chromatin structure, critical for gene regulation, is achieved by ATP-dependent machines called chromatin remodelers, which disassemble, slide, and reassemble nucleosomes on DNA. Disruptions in chromatin remodeler function perturb gene expression and have been directly linked with a number of cancers and developmental disorders. At present, it is not understood at a molecular level how remodelers reposition and reorganize nucleosomes, and what factors give rise to particular biochemical characteristics of remodelers. This proposal aims to uncover how different domains of the Chd1 remodeler participate in the nucleosome sliding reaction. X-ray crystallography will be used to visualize both the central ATPase motor as well as the C-terminal DNA-binding domain in complex with DNA substrates, which will reveal how remodelers recognize and distort duplex DNA. The contributions of the Chd1 DNA-binding domain to the speed, spacing, and direction of nucleosome sliding will be determined with Chd1 variants that have modified binding domains and/or variations in the linking segment to the ATPase motor. The ATPase motor is regulated by a pair of N-terminal chromodomains, which appear to enhance substrate specificity of the remodeler. Rapid kinetic analyses of nucleosome sliding reactions using stopped flow FRET will be used to identify stage(s) in the nucleosome sliding cycle affected by ATPase regulation. The results of this research will advance our understanding of how chromatin remodelers work and select their substrates, which are essential steps for interpreting changes in chromatin landscapes between healthy and diseased cells.

Public Health Relevance

The proper packaging of DNA into chromosomes is vitally important for normal cell growth and survival, and disruption of factors that organize chromosomes lead to many types of developmental diseases and cancer. Understanding how these organizing factors carry out their tasks is essential for identifying how cells are transformed to diseased states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084192-07
Application #
8727583
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
2008-04-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
7
Fiscal Year
2014
Total Cost
$294,003
Indirect Cost
$104,003
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Nodelman, Ilana M; Horvath, Kyle C; Levendosky, Robert F et al. (2016) The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome. Nucleic Acids Res 44:7580-91
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Hauk, Glenn; Bowman, Gregory D (2015) Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1. PLoS One 10:e0131690
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Nodelman, Ilana M; Bowman, Gregory D (2013) Nucleosome sliding by Chd1 does not require rigid coupling between DNA-binding and ATPase domains. EMBO Rep 14:1098-103

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