Chromatin was first defined by Flemming in 1882 as the `substance in the cell nucleus which is readily stained'that `is refractile to digestion'. The term stuck, and now is taken to refer to DNA (the readily stained component) and the associated proteins that make it refractile to digestion. It has become clear over the past twenty years that chromatin structure is vastly more dynamic than previously suspected. Regulation of chromatin structure frequently regulates the ability of proteins to access DNA, and in this manner plays a key role in regulating transcription, replication and recombination of the genome. The basic structural component of chromatin, the nucleosome, plays a central role in this regulation. The position of nucleosomes on the DNA can be changed, nucleosomes can be evicted, and nucleosomes can contain various histone variants and can be covalently modified in different ways, allowing them to have differential abilities to interact with the regulatory machinery. The dynamic nature of chromatin has been intensively studied for the past fifteen years, with much of the resultant information pertaining to the type and extent of covalent modification of the nucleosome. Information on the location of nucleosomes and on the full spectrum of proteins that might be involved in regulating a specific genomic locus has lagged behind this flood of information on covalent alterations to chromatin. The purpose of the experiments described in this application is to investigate how the location of nucleosomes can be altered in vitro and in vivo and to determine the full spectrum of proteins that might be involved in these types of regulation on a given genomic locus.
Three Aims are proposed:
Aim 1 will characterize nucleosome remodeling in vitro by examining individual remodeled products and through structural approaches;
Aim 2 will characterize structural changes to chromatin during regulation of a set of genes in cultured human cells;
and Aim 3 will develop technology to catalog proteins and RNA that bind to specific loci.

Public Health Relevance

Epigenetic mechanisms impact the expression of the human genome by altering the ability of individual genes to be expressed in different cell types, and are known to contribute strongly to normal development and to many disease processes including cancer. These mechanisms rely in large part upon mechanisms to modify chromatin structure, the focus of this grant application. This application proposes experiments to understand how human chromatin structure might be regulated to effect changes in gene expression and as such is directly relevant to an understanding of numerous disease processes from cancer to infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM048405-21
Application #
8401895
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Carter, Anthony D
Project Start
1993-01-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
21
Fiscal Year
2013
Total Cost
$380,849
Indirect Cost
$165,680
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
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Mieczkowski, Jakub; Cook, April; Bowman, Sarah K et al. (2016) MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat Commun 7:11485
West, Jason A; Mito, Mari; Kurosaka, Satoshi et al. (2016) Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization. J Cell Biol 214:817-30
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Lei, Ienglam; West, Jason; Yan, Zhijiang et al. (2015) BAF250a Protein Regulates Nucleosome Occupancy and Histone Modifications in Priming Embryonic Stem Cell Differentiation. J Biol Chem 290:19343-52
Bowman, Sarah K; Deaton, Aimee M; Domingues, Heber et al. (2014) H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex. Elife 3:e02833
West, Jason A; Davis, Christopher P; Sunwoo, Hongjae et al. (2014) The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol Cell 55:791-802
West, Jason A; Cook, April; Alver, Burak H et al. (2014) Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming. Nat Commun 5:4719

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