FACT is a complex of two proteins that are found in all eukaryotes, including humans, and is essential for viability in all organisms tested. Nucleosomes are the fundamental subunit of chromatin, which is the DNA packaging in eukaryotes. Their properties govern the flow of information from the genome. The goals of this project are to determine how FACT causes nucleosomes to adopt an alternative structure and to learn when and where the ability to form and resolve such structures is useful in living cells. FACT is unique among factors that regulate chromatin because it does not modify the histones that make up nucleosomes, it does not use an energy source to move the histone cores along the DNA, and it works through a mechanism that has not been observed with any other histone chaperone. FACT has been identified in a wide range of systems biology studies seeking factors that regulate cellular physiology, promote normal embryonic development, and allow stem cells to maintain pluripotency. Both the mechanism of FACT action and the circumstances in which it is used are therefore of high importance for understanding basic eukaryotic physiology. An integrated approach using a yeast model system is proposed for these studies to take advantage of the range of tools available in these organisms.
The first aim of the proposal is to analyze the properties of nucleosomes that have been altered by FACT to produce what is called a "reorganized" form. This structure is significantly different from canonical nucleosomes, but its nature remains poorly characterized. Purified histones and DNA fragments labeled with fluorescent dyes will be used to probe the properties of reorganized nucleosomes, and mutant histone and FACT proteins will be used in these assays to allow the results to be compared with the properties of FACT in living cells.
The second aim i s to characterize the roles of FACT by identifying mutations that make it easier or harder to tolerate FACT defects using whole-genome sequencing, and by asking how distinct mutations in FACT and other histone chaperones affect the ability to maintain repression of cryptic promoters and promote stability of distinct chromatin states using quantitative PCR measurements of transcripts and chromatin immunoprecipitations (ChIPs).
The third aim i s to ask how FACT affects chromatin structure near origins of DNA replication, how this contributes to regulating origin firing, and to test a hypothetical role for FACT in depositing new chromatin after the genome is duplicated during replication.
This aim uses some established ChIP technology but also requires development of some novel methods.

Public Health Relevance

Project Narrative Understanding and treating pathological conditions (poor health) requires understanding of normal physiology (good health). FACT is a protein complex that is needed for two fundamental processes in human cells, DNA replication and RNA transcription, so learning about how FACT functions is a basic part of learning about how human cells work. This proposal takes advantage of powerful experimental methods that are easy to use with simple yeast cells but are difficult or impossible to use with more complex human cells, and the fundamental mechanisms under study here have been found to be the same in both kinds of cells in many cases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064649-10
Application #
8241952
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
2002-03-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
10
Fiscal Year
2012
Total Cost
$311,455
Indirect Cost
$103,124
Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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
McCullough, Laura; Rawlins, Robert; Olsen, Aileen et al. (2011) Insight into the mechanism of nucleosome reorganization from histone mutants that suppress defects in the FACT histone chaperone. Genetics 188:835-46
Stillman, David J (2010) Nhp6: a small but powerful effector of chromatin structure in Saccharomyces cerevisiae. Biochim Biophys Acta 1799:175-80
Han, Junhong; Li, Qing; McCullough, Laura et al. (2010) Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication. Genes Dev 24:1485-90
Xin, Hua; Takahata, Shinya; Blanksma, Mary et al. (2009) yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement. Mol Cell 35:365-76

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