Transcription of protein-coding genes in eukaryotic cells is carried out by the protein complex RNA polymerase II. During the elongation phase of transcription, RNA polymerase II associates with transcription elongation factors which modulate the activity of the transcription complex and are needed to carry out co-transcriptional processes. Chapters 2 and 3 of this dissertation describe studies of Spt6 and Spt5, two conserved transcription elongation factors.
Spt6 is a transcription elongation factor thought to replace nucleosomes in the wake of transcription. Saccharomyces cerevisiae spt6 mutants express elevated levels of intragenic transcripts, transcripts appearing to initiate from within gene bodies. We applied high resolution genomic assays of transcription initiation to an spt6-1004 mutant, allowing us to catalog the full extent of intragenic transcription in spt6-1004 and show for the first time on a genome-wide scale that the intragenic transcripts observed in spt6-1004 are largely explained by new transcription initiation. We also assayed chromatin structure genome-wide in spt6-1004, finding a global depletion and disordering of nucleosomes. In addition to increased intragenic transcription in spt6-1004, our results also reveal an unexpected decrease in expression from most canonical genic promoters. Comparing intragenic and genic promoters, we find that intragenic promoters share some features with genic promoters. Altogether, we propose that the transcriptional changes in spt6-1004 are explained by a competition for transcription initiation factors between genic and intragenic promoters, which is made possible by a global decrease in nucleosome protection of the genome.
Spt5 is another transcription elongation factor, important for the processivity of the transcription complex and many transcription-related processes. To study the requirement for Spt5 in vivo, we applied multiple genomic assays to Schizosaccharomyces pombe cells depleted of Spt5. Our results reveal an accumulation of RNA polymerase II over the 5 ′ ends of genes upon Spt5 depletion, and a progressive decrease in transcript abundance towards the 3 ′ ends of genes. This is consistent with a model in which Spt5 depletion causes transcription elongation defects and increases early termination. We also unexpectedly discover that Spt5 depletion causes hundreds of antisense transcripts to be expressed across the genome, primarily initiating from within the first 500 base pairs of genes.
The expression of intragenic transcripts when transcription elongation factors are disrupted suggests that cells have evolved to prevent spurious intragenic transcription. However, some cases of intragenic transcription are consistently detected in wild-type cells, and some of these cases are known to be important for different biological functions. Chapter 4 of this dissertation describes our efforts to better understand the functions of intragenic transcription in wild-type cells by studying uncharacterized instances of intragenic transcription. To discover uncharacterized instances of intragenic transcription, we applied high resolution genomic assays of transcription initiation to wild-type Saccharomyces cerevisiae under three stress conditions. For the condition of oxidative stress, we show that intragenic transcripts are generally expressed at lower levels than genic transcripts, and that many intragenic transcripts are likely to be translated at some level. By comparing intragenic transcription in three yeast species, we find that most examples of oxidative-stress regulated intragenic transcription identified in S. cerevisiae are not conserved. Finally, we show that the expression of an oxidative-stress-induced intragenic transcript at the gene DSK2 is needed for S. cerevisiae to survive in conditions of oxidative stress.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/37095 |
| Date | 01 August 2019 |
| Creators | Chuang, James |
| Contributors | Winston, Fred, Khalil, Ahmad S. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-sa/4.0/ |
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