Spt6 is a conserved eukaryotic transcription factor, known to interact with both nucleosomes and RNA polymerase II (RNAPII) to control transcription. We have initiated study of Spt6 in S. pombe in order to identify both novel and conserved roles in regulation of transcription and chromatin. We first constructed and analyzed spt6 mutants by several approaches. As Spt6 is known to be required for histone H3K36 methylation in both Saccharomyces cerevisiae and human cells, we examined the global levels of several histone modifications; we found that in S. pombe, Spt6 is required for both H3K4 and H3K36 trimethylation. We examined the chromatin state at two highly expressed genes, \(act1^+\) and \(pma1^+\), and found that there is a defect in recruitment of the methyltransferases responsible for those marks, Set1 and Set2, respectively. We also observed loss of nucleosomes, as well as a decrease in histone H2B monoubiquitylation. These results suggest that Spt6 plays an important role in chromatin regulation during transcription. We also conducted transcriptional analysis of an spt6 mutant by both microarray and high-throughput sequencing (RNA-seq) and discovered that Spt6 plays a critical role in maintaining the integrity of transcription genome-wide. We found that Spt6 is required to repress antisense transcription, with nearly 70% of genes having antisense transcripts increased by at least two-fold in an spt6 mutant. We also found that transcription of most long terminal repeats (LTRs) is derepressed. Finally, we found that a major class of transcripts elevated in the spt6 mutant is derived from heterochromatin, which is normally silenced. To study the heterochromatic silencing defect in greater detail, we analyzed the chromatin state of the pericentric repeats and found a decrease in H3K9 trimethylation, elevated levels of H3K14 acetylation, reduced recruitment of several known silencing factors and a loss of siRNA production. We also see a very modest increase in RNAPII recruitment. Based on this combination of phenotypes, Spt6 is likely to contribute to both transcriptional and post-transcriptional silencing mechanisms. Taken together, we have found that Spt6 plays several important roles to control transcription in both euchromatin and heterochromatin in S. pombe.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10336973 |
| Date | January 2011 |
| Creators | Kiely, Christine M. |
| Contributors | Winston, Fred Marshall |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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