During the elongation phase of the transcription cycle many protein factors are recruited to RNA polymerase II (Pol II). One such factor is the polymerase associated factor 1 complex (Paf1C). Paf1C associates with Pol II during transcription elongation and promotes co-transcriptional histone modifications. Co- transcriptionally placed histone modifications are important to transcription regulation and loss or aberrant patterning of these marks can contribute to cancer. The Arndt lab recently found an interaction between Cdc73, a member of Paf1C, and Spt6, a conserved histone chaperone. I hypothesize that this previously unrecognized interaction is contributing to the recruitment of Paf1C to actively transcribed genes. I will use Saccharomyces cerevisiae as model system in this study, which is made possible by the high degree of conservation between these transcription elongation factors in yeast and humans. I will determine if this interaction is involved in recruitment of Paf1C by rapidly depleting Spt6 from cells and testing Paf1C occupancy by genome-wide chromatin immunoprecipitation followed by exonuclease digestion and deep sequencing (ChIP-exo). I will also identify the residues that mediate the interaction between Cdc73 and Spt6 by in vitro crosslinking and mass spectrometry. Once identified these residues will be mutated and pulldown and gel shift assays will be performed to validate that the mutations disrupt the interaction. Additionally, I will test histone modifications after depletion of Spt6 and in strains where point mutations that abolish the interaction have been generated. Together these methods will reveal the effects of Spt6 loss on Paf1C recruitment and Paf1C dependent histone modifications. While executing this research plan I will receive training on collection and analysis of ChIP-exo data and mass spectrometry data. I will also receive instruction on genetic manipulation of yeast, molecular cloning and biochemistry. I plan to publish at least two papers sharing the findings of these experiments with the scientific community and providing an opportunity for me to continue my training on scientific writing. Completing this project will inform the scientific community of an interaction between two critical transcription regulators that has not yet been characterized and provide me with the training and publications necessary to obtain an excellent post-doctoral appointment. Thus, this project will put me on track to achieve my goal of managing my own research program.
Cancer is a collection of diseases caused by misregulation of cellular processes, like gene expression, that when altered can lead to aberrant cell growth. In healthy cells, the Paf1 complex (Paf1C) physically associates with RNA polymerase II and helps to regulate gene expression by promoting co-transcriptional histone modifications; if this complex is unable to perform these critical functions cancer and developmental defects occur. The goal of this project is to investigate the recruitment of Paf1C to Pol II by another conserved transcription elongation factor (Spt6) and to determine how loss of this recruitment pathway affects Paf1C dependent histone modifications.
