The long-term objectives of the proposed research are to identify the proteins and mechanisms that regulate transcription by RNA polymerase II (Pol II) within the chromatin environment of eukaryotic cells. This proposal focuses on transcription elongation by Pol II and the coordination of transcription elongation with co- transcriptional events. The proposed experiments address fundamental questions related to the modification of histones on transcribed chromatin and the transition from elongation to transcription termination. A major focus of the proposal is the highly conserved, multifunctional Paf1 complex (Paf1C). Paf1C is a globally acting transcription elongation factor that associates with Pol II on most, if not all, active genes in the eukaryotic genome. In addition to regulating transcription of many genes, Paf1C is required for the establishment of histone modifications during transcription elongation and for promoting proper transcription termination.
Specific Aims 1 and 2 investigate the importance of Paf1C in establishing genomic patterns of histone H2B lysine 123 mono-ubiquitylation. This modification is of special significance because it is the first step in a conserved histone modification cascade that leads to subsequent histone methylation and acetylation events, which in turn ensure proper expression of the genome.
Specific Aim 1 investigates the mechanism by which Paf1C stimulates H2B K123 ubiquitylation. Biochemical, genetic, and protein-crosslinking approaches will be used to elucidate the physical and functional interactions of Paf1C with the ubiquitin conjugase Rad6 and the nucleosome target.
Specific Aim 2 explores the importance of Paf1C in dictating genomic patterns of H2B K123 ubiquitylation through an analysis of yeast mutant strains that disrupt two distinct pathways for Paf1C recruitment. Genomic experiments will be used to map the patterns of Paf1C occupancy and H2B K123 ubiquitylation in cells lacking Paf1C domains that tether it to components of the Pol II elongation machinery. Transcription termination marks the end of the elongation process and previous studies have implicated Paf1C and its dependent histone modifications in the regulation of termination. Therefore, a second major focus of the proposal is the role of chromatin in transcription termination.
Specific Aim 3 extends beyond previous studies of Paf1C and builds on the recent identification of a specific class of histone mutants that impair transcription termination of noncoding RNAs. The work will be performed in yeast to exploit the powerful genetic tools available in this system, including the ability to precisely replace chromosomal genes with mutant versions and to identify new regulatory factors through comprehensive genetic screens. The extensive conservation of transcription factors and histone modifications in yeast and humans strongly suggests that the outcome of the research will advance understanding of numerous human diseases, particularly multiple forms of cancer, which arise when Paf1C or the epigenetic modification of chromatin is altered.
Cancer arises from the inappropriate expression of genes that control cell division and the response of cells to extracellular signals. Normally, these genes are tightly controlled by proteins, such as the Paf1 complex (Paf1C), which regulates gene transcription and chromatin structure through mechanisms that will be elucidated in these studies. Defects in Paf1C are associated with multiple human cancers, including those of the pancreas, breast, and parathyroid, as well as developmental disorders and impaired immune responses.
Showing the most recent 10 out of 33 publications
