This proposal addresses mechanisms used by the histone chaperones FACT and Spt6 to promote assembly, disassembly, and reconstruction of chromatin. The structure of chromatin strongly influences transcription, replication, and DNA repair in all eukaryotes, so understanding how chromatin is formed and maintained is central to understanding each of these core processes. The yeast Saccharomyces cerevisiae is used as a powerful model system that has genetic, biochemical, and molecular tools available to study fundamental processes common to all eukaryotes. The highly collaborative approach proposed here takes full advantage of these tools by using a broad range of methods simultaneously. FACT can convert nucleosomes into an alternative structural form (the reorganized nucleosome) in which both the DNA and the histones are more accessible than normal, which is important for opening chromatin for processes that read information from the DNA. This reorganization activity is reversible, so FACT can also assemble nucleosomes out of loosely associated DNA and histones to construct chromatin, which is important for limiting access to the DNA to protect its physical integrity and also to optimize expression of genes at the appropriate level. It is not clear how the decision to make nucleosomes or take them apart is made, how FACT is recruited to specific substrates, or how it is regulated to perform the correct function at the correct time.
Aim 1 examines these issues with purified components in vitro, using different versions of FACT and histones to characterize reorganized nucleosomes, to determine how FACT influences the formation and resolution of this state, and how FACT substrates are chosen. Spt6 is another essential histone chaperone with many biochemical properties in common with FACT, but Spt6 cannot reorganize nucleosomes and FACT cannot perform the distinct physiological functions of Spt6. Comparing the activities and functions of these two chaperones will therefore provide insight into how similar biochemical activities are used to perform distinct functions. Histone chaperones have an important role in tuning the local properties of chromatin, but mechanisms used are poorly understood.
Aim 2 addresses this using MNase-Seq and RNA- Seq to assess chromatin quality and establishment of appropriate repression in strains with mutations in FACT, Spt6, and histones. Specific models that have been proposed by others are also tested, as well as exploration of the effects of curaxins on FACT, probing reports suggesting this class of potential chemotherapeutic drugs act by inhibiting FACT activity. An unexpected and previously undescribed system for preventing the accumulation of histone mRNAs beyond their normal levels has been discovered, revealing a novel mechanism for regulation.
Aim 3 proposes initial investigation of this system and the roles of histone chaperones in regulating the rate of turnover of histone proteins and chromatin.
Maintaining health requires each cell in the body to choose properly from among the large number of genes it has available, expressing only those needed for that particular kind of cell to perform its functions at that particular time. The pattern of gene expression must be flexible enough to allow one kind of cell to turn into another kind during development or to respond if circumstances change, but inappropriate patterns of expression must be avoided as these can lead to the unrestrained growth associated with cancers. A key mechanism for regulating gene expression this way is to limit access to the DNA until that access is considered appropriate. This proposal examines how two core factors (FACT and Spt6) participate in constructing and revising this accessibility barrier (called chromatin). The studies are conducted using a type of yeast because this simple model organism is easier and less expensive to study than human cells, but it performs core functions like chromatin formation in similar ways, making this a powerful way to study broadly conserved processes.
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