The goal of this project is to define the interactions between RNA polymerase II, the basal transcription factors, and the chromatin template that lead to accurate transcription initiation and productive elongation. Using the yeast Saccharomyces cerevisiae as a model, several fundamental aspects of gene expression will be studied. This project period will focus on the mechanisms for targeting co-transcriptional histone methylation of histone H3K4 and H3K36. It is clear that the Bur1 kinase promotes transcription through chromatin, as the requirement for Bur1 can be bypassed by mutating H3K36, or by deleting the H3K36 methyltransferase Set2 or several other chromatin-related factors. The first specific aim is to further probe the substrates and functions of Bur1. Experiments in the second aim will explore the interactions between the methylations at H3K4 and H3K36. Although these modifications have typically been considered as separate events, preliminary data indicates they are not independent. It is still not clear exactly how these modifications affect transcription, so their role in affecting transcription elongation will be probed by a series of genetic and molecular experiments. H3K4 tri-methylation is localized near promoters and it has been proposed that the Set1/COMPASS complex is recruited to the Serine 5 phosphorylated form of the RNA polymerase II C-terminal domain (CTD). However, there is no experimental evidence for a direct interaction between COMPASS and the CTD. The third specific aim will be to test whether COMPASS can directly bind specific phosphorylated forms of the CTD, and if so, which subunits are responsible. The fourth specific aim will follow up on preliminary data suggesting that the Rpb4 subunit of RNA polymerase II is involved in mediating interactions between the transcription complex and the elongation factor Spt6. Protein interaction experiments will test for direct interactions between Spt6 and the Rpb4/7 heterodimer, while chromatin immunoprecipitation experiments will explore the effect of Rpb4 deletion on histone deposition and modification. Affinity chromatography and mass spectrometry will be used to identify other Rpb4/7 binding proteins to see if other chromatin-related complexes interact with RNA polymerase II through this subcomplex. In the last specific aim, initiation and elongation complexes formed under various conditions will be purified on immobilized templates and then analyzed by mass spectometry. Although it is possible some new factors associated with transcription complexes will be identified, what is of greater interest is the exchange of factors that are likely to occur at various stages of transcription. The experiments in these five specific aims will significantly increase our understanding of the RNA polymerase II transcription reaction and its interactions with the chromatin template. This fundamental knowledge is essential for understanding how mutations in transcription factors and histone modifying enzymes lead to diseases such as cancer and developmental defects. ? ? ?
| Joo, Yoo Jin; Ficarro, Scott B; Soares, Luis M et al. (2017) Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation. Genes Dev 31:2162-2174 |
| Woo, Hyeonju; Dam Ha, So; Lee, Sung Bae et al. (2017) Modulation of gene expression dynamics by co-transcriptional histone methylations. Exp Mol Med 49:e326 |
| Soares, Luis M; He, P Cody; Chun, Yujin et al. (2017) Determinants of Histone H3K4 Methylation Patterns. Mol Cell 68:773-785.e6 |
| Kim, Ji Hyun; Lee, Bo Bae; Oh, Young Mi et al. (2016) Modulation of mRNA and lncRNA expression dynamics by the Set2-Rpd3S pathway. Nat Commun 7:13534 |
| Hahn, Steven; Buratowski, Stephen (2016) Structural biology: Snapshots of transcription initiation. Nature 533:331-2 |
| Soares, Luis M; Radman-Livaja, Marta; Lin, Sherry G et al. (2014) Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns. Cell Rep 6:961-972 |
| Marquardt, Sebastian; Escalante-Chong, Renan; Pho, Nam et al. (2014) A chromatin-based mechanism for limiting divergent noncoding transcription. Cell 157:1712-23 |
| Suh, Hyunsuk; Hazelbaker, Dane Z; Soares, Luis M et al. (2013) The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits. Mol Cell 51:850-8 |
| Soares, Luis M; Buratowski, Stephen (2013) Histone Crosstalk: H2Bub and H3K4 Methylation. Mol Cell 49:1019-20 |
| Kim, TaeSoo; Xu, Zhenyu; Clauder-Munster, Sandra et al. (2012) Set3 HDAC mediates effects of overlapping noncoding transcription on gene induction kinetics. Cell 150:1158-69 |
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