The chromosomes of all organisms are subject to the processes of transcription, DNA replication/repair and segregation to daughter cells. Chromatin, the complex of chromosomal DNA and proteins in eukaryotes, has a profound impact on these fundamental processes, and defects in chromatin regulation are associated with a variety of human diseases including cancer. The chromatin fiber, which consists of repeating subunits of nucleosomes, is used as the foundation for the assembly of complex higher-order structures involving additional levels of condensation, looping and folding of chromosomes. The goal of this research is to contribute to an understanding of the assembly of higher-order chromatin using a combination of biochemistry and genetics in the single-celled microbe Saccharomyces cerevisiae. The focus is the formation of """"""""silenced chromatin"""""""" at a locus called HAIR that serves as a paradigm for the structure, function, and heritable nature of higher-order chromatin. HMR silencing encompasses -4 kb of chromosomal DNA and is controlled by a small DNA element called a silencer that functions by binding directly to silencer-binding proteins such as the Origin Recognition Complex (ORC) that in turn recruit specialized silencing proteins, such as Sirlp, through protein-protein interactions. This silencer-protein-complex """"""""nucleates"""""""" the formation of silenced chromatin, which includes the binding of specialized non-histone proteins, such as Sir3p to the chromatin fiber. A specific Sirlp-ORC interaction is central to formation of the silencer-protein-complex. ORC also functions in the more general and essential process of replication initiation at the hundreds of """"""""generic"""""""" (non-silencer) replication origins through out the genome. Thus some mechanism must confine a functional Sirlp-ORC interaction to silencers. We postulate that ORC-DNA interactions and additional chromatin-binding proteins insure that a stable physical interaction between Sirlp and ORC occurs only at silencers. In addition, we postulate that ORC-DNA interactions within HMR suppress ORC's function in replication initiation. Lastly, we postulate that the Sir3p binds to nucleosomes at HMR and contributes directly to """"""""remodeling"""""""" the HMR chromatin fiber into a larger domain of """"""""higher-order"""""""" chromatin. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM056890-06
Application #
6587234
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Carter, Anthony D
Project Start
1998-01-01
Project End
2006-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
6
Fiscal Year
2003
Total Cost
$280,082
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Hoggard, Timothy A; Chang, FuJung; Perry, Kelsey Rae et al. (2018) Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins. PLoS Genet 14:e1007418
Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A et al. (2018) General method for rapid purification of native chromatin fragments. J Biol Chem 293:12271-12282
Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E et al. (2017) Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development. Adv Exp Med Biol 953:49-82
Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra et al. (2016) High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation. G3 (Bethesda) 6:993-1012
Dummer, Antoinette M; Su, Zhangli; Cherney, Rachel et al. (2016) Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast. PLoS Genet 12:e1006094
Ostrow, A Zachary; Nellimoottil, Tittu; Knott, Simon R V et al. (2014) Fkh1 and Fkh2 bind multiple chromosomal elements in the S. cerevisiae genome with distinct specificities and cell cycle dynamics. PLoS One 9:e87647
Hoggard, Timothy; Shor, Erika; Müller, Carolin A et al. (2013) A Link between ORC-origin binding mechanisms and origin activation time revealed in budding yeast. PLoS Genet 9:e1003798
Shor, Erika; Fox, Catherine A; Broach, James R (2013) The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress. PLoS Genet 9:e1003680
Fox, Catherine A; Gartenberg, Marc R (2012) Palmitoylation in the nucleus: a little fat around the edges. Nucleus 3:251-5
Park, Sookhee; Patterson, Erin E; Cobb, Jenel et al. (2011) Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1. Proc Natl Acad Sci U S A 108:14572-7

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