The overall objective of this research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Dr. Peterson's 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 several transcriptional activators. Genetic studies in yeast suggest that SWI/SNF functions by antagonizing chromatin-mediated transcriptional repression. Over the past budget period he has made progress in understanding how SWI/SNF functions. In particular, he purified SWI/SNF complex from yeast; demonstrated that SWI/SNF can use the energy of ATP hydrolysis to disrupt nucleosome structure in vitro; evaluated the catalytic remodeling activity of SWI/SNF on reconstituted nucleosome arrays; and determined that SWI/SNF facilitates the binding of a transcriptional activator to a nucleosomal site in vivo. Over the next budget period the investigator plans to continue 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 the proposal will investigate the role of SWI/SNF in transitions in chromatin structure of the yeast HO gene in vivo.
This aim will be addressed by high resolution mapping of nucleosome positioning and a chromatin immunoprecipitation assay that uses antibodies to acetylated histones. The second objective is to test the hypothesis that SWI/SNF is targeted to chromosomal locations by gene-specific activators. These studies will use a chromatin immunoprecipitation method, as well as an in vitro nucleosome array assay.
The third aim will use a quantitative nucleosome array assay to investigate the relationship between SWI/SNF action, the histone N-termini, and ADA/GCN5 histone acetyltransferases.
This aim will also test the hypothesis that disruption of histone-histone interactions is required for SWI/SNF catalysis. The fourth objective will use a battery of SWI2/SNF2 ATPase motif mutants and a photoaffinity crosslinking method to define the role of ATP binding and hydrolysis in SWI/SNF remodeling.
This aim will also use fluorescence methods to test the hypothesis that SWI/SNF action causes movements of nucleosomal DNA. The fifth aim will use in vivo labeling and in vitro association assays to investigate the subunit organization of SWI/SNF.
| Clapier, Cedric R; Iwasa, Janet; Cairns, Bradley R et al. (2017) Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nat Rev Mol Cell Biol 18:407-422 |
| Gioacchini, Nathan; Peterson, Craig L (2017) Chromatin remodeling: a complex affair. EMBO Rep 18:1673-1674 |
| 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 |
| Echtenkamp, Frank J; Gvozdenov, Zlata; Adkins, Nicholas L et al. (2016) Hsp90 and p23 Molecular Chaperones Control Chromatin Architecture by Maintaining the Functional Pool of the RSC Chromatin Remodeler. Mol Cell 64:888-899 |
| 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, Célia; 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 |
| 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; Radman-Livaja, Marta; Rando, Oliver J et al. (2013) A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme. Science 340:195-9 |
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