The yeast Saccharomyces cerevisiae encodes two proteins, Sptl6 and Pob3, that are each highly conserved among eukaryotes, including humans. Sptl6 and Pob3 function as a heterodimer, and this factor is required for both DNA replication and RNA transcription since cells lacking normal proteins display errors in both processes. Sptl6-Pob3 influences both how often transcripts are made and the precise site chosen for initiation. It also promotes elongation of transcripts. The surprising breadth of the roles of this factor can be explained by a single simple activity: the ability to modulate the properties of nucleosomes. Since these structures affect all regions of the eukaryotic genome, they are involved in all processes that involve the genome, from establishing initiation sites for transcription and replication, to the progression of DNA and RNA polymerases, to the packaging and segregation of genomic copies. Sptl6-Pob3 is unlike its homologs from other eukaryotes in that it lacks a DNA-binding motif. Physical and genetic methods indicate that it functions together with a DNA-binding protein called Nhp6. Purified Spt 1 6-Pob3 and Nhp6 alter the structure of nucleosomes in vitro in a way that changes their electrophoretc mobility and alters the presentation of the DNA in the nucleosome. Experiments in this proposal explore the nature of the changes induced in nucleosomes by Sptl6-Pob3-Nhp6 (SPN), with the goal of understanding how SPN changes the structure of this fundamental unit of genomic packaging. The function of SPN in cells is then addressed by using genetic methods to test specific models describing steps in replication and transcription that might be promoted by SPN. The effect of diminishing SPN activity is then examined in assays that reveal the formation of transcription and replication initiation complexes in SPN mutants, and the structure of intermediates formed during elongation. Sptl6-Pob3 does not reposition nucleosomes like a standard chromatin remodeling factor, but appears to be a new type of factor that reorganizes nucleosomes. These studies will elucidate activity of this highly conserved factor in modulating the effects of a fundamental component of chromatin, and will indicate how this activity participates in two basic processes of nucleic acid metabolism: transcription and replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM064649-01
Application #
6421489
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
2002-03-01
Project End
2006-02-28
Budget Start
2002-03-01
Budget End
2003-02-28
Support Year
1
Fiscal Year
2002
Total Cost
$283,500
Indirect Cost
Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
McCullough, Laura L; Connell, Zaily; Xin, Hua et al. (2018) Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization. J Biol Chem 293:6121-6133
Shen, Zuolian; Formosa, Tim; Tantin, Dean (2018) FACT Inhibition Blocks Induction But Not Maintenance of Pluripotency. Stem Cells Dev 27:1693-1701
Sdano, Matthew A; Fulcher, James M; Palani, Sowmiya et al. (2017) A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription. Elife 6:
Valieva, Maria E; Armeev, Grigoriy A; Kudryashova, Kseniya S et al. (2016) Large-scale ATP-independent nucleosome unfolding by a histone chaperone. Nat Struct Mol Biol 23:1111-1116
McCullough, Laura; Connell, Zaily; Petersen, Charisse et al. (2015) The Abundant Histone Chaperones Spt6 and FACT Collaborate to Assemble, Inspect, and Maintain Chromatin Structure in Saccharomyces cerevisiae. Genetics 201:1031-45
Kemble, David J; McCullough, Laura L; Whitby, Frank G et al. (2015) FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs. Mol Cell 60:294-306
Voth, Warren P; Takahata, Shinya; Nishikawa, Joy L et al. (2014) A role for FACT in repopulation of nucleosomes at inducible genes. PLoS One 9:e84092
Kemble, David J; Whitby, Frank G; Robinson, Howard et al. (2013) Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT. J Biol Chem 288:10188-94
McCullough, Laura; Poe, Bryan; Connell, Zaily et al. (2013) The FACT histone chaperone guides histone H4 into its nucleosomal conformation in Saccharomyces cerevisiae. Genetics 195:101-13
Formosa, Tim (2012) The role of FACT in making and breaking nucleosomes. Biochim Biophys Acta 1819:247-55

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