Mechanisms of Transcription Elongation on Chromatin and Gene Silecing
- Author(s): Kuryan, Benjamin George
- Advisor(s): Carey, Michael F
- et al.
In the nucleus, the genomes of eukaryotes are packaged with histone proteins to form nucleosomes. Nucleosomes are the biological substrate for all of the processes that require access to the genomic DNA sequence. The first half of my research sought to address questions related to how one of these processes, transcription by RNA pol II, occurs on chromatin and how this process affects chromatin structure. The second half of my research focused on yeast silent chromatin and how it is affected by histone modifications and other chromatin factors. Chapter 1 of this dissertation is a general introduction to transcription and chromatin biology. This chapter should give the reader a general introduction to the importance of these topics, how the two intersect, and the key concepts required to understand the fields today. Chapter 2 of the dissertation describes published work demonstrating the cooperation between the histone chaperone NAP1 and the chromatin remodeling complex RSC. The data shows, in a reconstituted biochemical system, that these enzymes coordinate to evict one H2A-H2B dimer from the nucleosome and allow elongation through a nucleosome template. Chapter 3 of the dissertation describes work involving the function of a core module of the Rpd3 histone deacetylase complex in stabilizing chromatin structure independent of histone deacetylase activity. This core complex has both chromatin assembly activity and the capability to block nucleosome eviction by the RSC complex. Chapter 4 is a study of telomere position effect variegation and heterochromatin in yeast. A key result from the study is that methylation of H3K79 blocks the ability of the Sir complex to silence chromatin in vivo and in vitro without disrupting the ability of the complex to bind to nucleosomes. Chapter 5, the final chapter, is a study that started with a proteomic screen to identify proteins that interact with yeast heterochromatin. This screen identified the Ino80 complex as an interactor with heterochromatin. This interaction was confirmed in a reconstituted system and subsequent in vivo analysis showed that key subunits of the complex are important for silencing the hidden mating locus HML.