The overall objective of our research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to focus on an evolutionarily conserved protein complex, SWI/SNF, which is required for expression of a subset of yeast genes and for the activity of transcriptional activators. Genetic studies in yeast indicate that SWI/SNF facilitates transcription by antagonizing chromatin-mediated transcriptional repression, and our in vitro studies indicate that the ~1Mda SWI/SNF complex can use the energy derived from ATP hydrolysis to mobilize nucleosomes and disrupt nucleosome structure. SWI/SNF complexes are essential for mammalian development and inactivation SWI/SNF subunits lead to cancers in humans. This proposal continues to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the role of SWI/SNF in vivo and the biochemical mechanism by which SWI/SNF disrupts nucleosome structure in vitro.
The first aim of this proposal will test the hypothesis that the SWI/SNF and ISWI remodeling complexes function antagonistically at many inducible genes.
This aim i s addressed by chromatin immunoprecipitation assays and nucleosome mapping methods. The second objective will test the hypothesis that SWI/SNF facilitates transcription by disrupting the higher order folding of chromatin. These studies will involve sedimentation velocity analyses of remodeled nucleosomes as well as biochemical analyses of Sin-histones.
Aim 3 describes single molecule assays and SWI/SNF-DNA photo-affinity crosslinking studies to dissect the role of ATP hydrolysis in chromatin remodeling activity.
The fourth aim will address the structure of SWI/SNF bound to a mononucleosome using single particle reconstruction of cryo-EM images.
This aim also investigates the subunit-subunit interactions required for assembly of SWI/SNF. This proposal describes research that is focused on how chromosome structure affects gene expression and how the normal cellular machinery contends with this structure. Specifically, we propose continued studies on a novel protein machine, the SWI/SNF complex that facilitates gene activation by remodeling chromosome structure. This machine is essential for mammalian development and inactivation of human SWI/SNF leads to a variety of cancers. ? ? ?

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 #
5R37GM049650-16
Application #
7475056
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Carter, Anthony D
Project Start
1993-08-01
Project End
2011-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
16
Fiscal Year
2008
Total Cost
$429,383
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Watanabe, Shinya; Peterson, Craig L (2016) Response to Comment on ""A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme"". Science 353:358
Krietenstein, Nils; Wal, Megha; Watanabe, Shinya et al. (2016) Genomic Nucleosome Organization Reconstituted with Pure Proteins. Cell 167:709-721.e12
Yu, Lijian; Rege, Mayuri; Peterson, Craig L et al. (2016) RNA polymerase II depletion promotes transcription of alternative mRNA species. BMC Mol Biol 17:20
Watanabe, Shinya; Tan, Dongyan; Lakshminarasimhan, Mahadevan et al. (2015) Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C. Nat Commun 6:7108
Rege, Mayuri; Subramanian, Vidya; Zhu, Chenchen et al. (2015) Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis. Cell Rep 13:1610-22
Jeronimo, CĂ©lia; Watanabe, Shinya; Kaplan, Craig D et al. (2015) The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations. Mol Cell 58:1113-23
Xue, Yong; Van, Christopher; Pradhan, Suman K et al. (2015) The Ino80 complex prevents invasion of euchromatin into silent chromatin. Genes Dev 29:350-5
Swygert, Sarah G; Peterson, Craig L (2014) Chromatin dynamics: interplay between remodeling enzymes and histone modifications. Biochim Biophys Acta 1839:728-36
Manning, Benjamin J; Peterson, Craig L (2014) Direct interactions promote eviction of the Sir3 heterochromatin protein by the SWI/SNF chromatin remodeling enzyme. Proc Natl Acad Sci U S A 111:17827-32
Watanabe, Shinya; Peterson, Craig L (2013) Chromatin dynamics: flipping the switch on a chromatin remodeling machine. Cell Cycle 12:2337-8

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