Roles of the multifunctional protein Spn1 in transcription and chromatin biology

Abstract

Gene transcription by RNA polymerase II (RNAPII) is a tightly regulated process necessary for the development and growth of all eukaryotes. In addition to synthesizing RNA, RNAPII coordinates—through a variety of factors—nucleosome dynamics and co-transcriptional processing of pre-mRNA, which are required for normal chromatin structure, genome stability, and proper gene expression. Spn1 is a protein conserved from yeast to humans that is essential for viability, associates with RNAPII, and localizes to actively transcribed genes. Previous studies of Spn1, primarily at single genes and in vitro, suggested that it functions in transcription, co-transcriptional processing, and chromatin biology. To address the generality and conservation of Spn1 roles in these processes, we employed two strategies involving genome-wide and genetic characterization of this multifunctional factor in Saccharomyces cerevisiae. In Chapter 2 of this dissertation, we used an inducible protein degradation system to deplete Spn1 without affecting cell viability. Using RNA-seq, we observed that Spn1 is required for normal levels of more than 1,000 mRNAs. ChIP-seq analysis revealed that this is at least in part explained by changes in Rpb1 occupancy. Additionally, we found that while Spn1 is not required for association of its binding partner Spt6 to RNAPII, it is involved in optimal Spt6 recruitment to chromatin. In contrast, Spn1 strongly depends on Spt6 for binding to RNAPII. Depletion of Spn1 also resulted in drastic changes in the distributions of the histone modifications H3K36me3, H3K36me2, and H3K4me3 along protein-coding genes. Ribosomal protein genes had unique features, including significant changes in histone occupancy, a gross redistribution of H3K36me2, and increased intron retention. In Chapter 3, we constructed novel spn1 mutants based on both evolutionary and mutational conservation in human cancer cells, targeting distinct regions of the yeast Spn1 protein not previously studied. With an expanded repertoire of mutants and phenotypes analyzed, we provided additional evidence for roles of Spn1 in transcription, DNA replication, and DNA repair. Analysis of the genetic interactions between spn1 mutants and elf1∆, set2∆, or chd1∆ suggested that Spn1 and Elf1 have redundant functions, while Set2 and Chd1 act in opposition to Spn1 mostly during transcription and DNA replication, respectively.