The long-term objectives of this project are to increase our understanding of eukaryotic transcription elongation, with a focus on histone chaperones. Histone chaperones control the assembly and disassembly of nucleosomes during transcription, replication, and repair. The proposed experiments address the conserved histone chaperone Spt6, using the yeast Saccharomyces cerevisiae, as a model system. Spt6 is conserved and its human counterpart has been implicated in developmental control and in cancer. Previous analysis of Spt6 has demonstrated that it is broadly required for transcription and chromatin structure in both yeast and mammalian cells. While it is established that Spt6 interacts with histones, RNA polymerase II, and other proteins, the mechanisms by which it functions are unknown. The proposed experiments in Specific Aim 1 will address the interactions of Spt6 with two other essential and conserved histone chaperones, Spn1/Iws1 and FACT. Preliminary studies have shown that an spt6 mutant, spt6-YW, that impairs the physical interaction of Spt6 with Spn1, has changes in growth, transcription and chromatin structure. Additional studies identified suppressor mutations that compensate for spt6-YW mutant defects. Several of these suppressor mutations cause clustered changes in a conserved surface of FACT.
Aim 1. 1 tests the model that spt6-YW and its suppressors control transcription and chromatin structure via alterations of the transcription elongation complex and histone modifications. This will be assayed by a set of ChIP-seq experiments.
Aim 1. 2 studies the changes in FACT that compensate for the Spt6 defect. These results will provide new understanding of the functional relationships among histone chaperones and provide insights into why so many of them are vital during transcription.
Specific Aim 2 focuses on Spt6 binding to histones, an essential function for all of histone chaperones.
This aim tests the model that Spt6 has multiple histone binding sites in its highly acidic and disordered N-terminal domain.
Aim 2. 1 will isolate spt6 mutants in the N-terminal region that are defective for function.
Aim 2. 2 will use these mutants to define Spt6-histone binding in vitro.
Aim 2. 3 will address key issues regarding Spt6-histone interactions during transcription. Together, these experiments will elucidate an essential function of Spt6.
Specific Aim 3 addresses a related but distinct role for Spt6, in the control of genome integrity, as spt6 mutants display genome instability phenotypes. The proposed experiments will test whether Spt6 is required for genome stability by the control of chromatin structure, transcription, or resolving transcription-replication conflicts. Experiments will assay RNA:DNA hybrids, which contribute to genome instability, double-strand DNA breaks, and will test the model that Spt6 is required for DNA replication as well as transcription. The results will provide new understanding of the control of genome stability, a fundamental and conserved process important for human health.
All organisms must regulate the expression of their genetic information for normal growth and development. This application proposes to study this regulation by the analysis of proteins that are essential for normal gene expression. The information learned from these studies will improve our understanding of human biology.
