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RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases. in vivo and in vitro elongation factor-RNA interaction data provide a missing link in understanding how processive elongation complexes are formed on active genes and disassembled at the end of genes
RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases. in vivo and in vitro elongation factor-RNA interaction data provide a missing link in understanding how processive elongation complexes are formed on active genes and disassembled at the end of genes
For transcription through chromatin, RNA Polymerase (Pol) II associates with transcription factors. Recent work revealed that transcription factors also interact with the nascent RNA to regulate gene expression. The focus of this thesis relies on the characterization of this form of interactions during the process of transcription elongation. I used our recently optimized PAR-CLIP protocol to show that many elongation factors (EFs) crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. These in vivo direct interactions were most notable for the kinases Ctk1 and Bur1 that phosphorylate the C-terminal repeat domain (CTD) of the largest Pol II subunit and for the histone H3 methyltransferases Set1 and Set2. Bioinformatic analysis indicated that most EFs crosslink preferentially to mRNAs, rather than to unstable non-coding RNAs, consistent with their recruitment to transcribed protein-coding genes. Furthermore, I developed an RNA degradation assay to test whether the observed RNA-protein interactions affect protein-chromatin binding under native conditions. Comparing protein-chromatin binding in the presence and absence of RNA revealed that RNA contributes to chromatin association in particular of the CTD serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1, Set2 and Dot1. Additionally, I confirmed the in vivo observed EF-RNA interactions for an active CTDK-I kinase complex in vitro using fluorescence anisotropy. Finally, I optimized our ChIP protocol for high-throughput sequencing and performed ChIP-Seq experiments of most Pol II EFs and histone marks involved in this study. Comparison of factor occupancies on DNA (ChIP-Seq) and on RNA (PAR-CLIP) revealed that interactions of EFs with nascent RNA are established before EFs are recruited to chromatin. Taken together, these studies argue for a role of nascent RNA in EF recruitment. In this model, EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site trigger disassembly of the elongation complex.
RNA Polymerase II, CTD kinases, PAR-CLIP, transcription elongation
Battaglia, Sofia Luciana
2017
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Battaglia, Sofia Luciana (2017): RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases: in vivo and in vitro elongation factor-RNA interaction data provide a missing link in understanding how processive elongation complexes are formed on active genes and disassembled at the end of genes. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

For transcription through chromatin, RNA Polymerase (Pol) II associates with transcription factors. Recent work revealed that transcription factors also interact with the nascent RNA to regulate gene expression. The focus of this thesis relies on the characterization of this form of interactions during the process of transcription elongation. I used our recently optimized PAR-CLIP protocol to show that many elongation factors (EFs) crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. These in vivo direct interactions were most notable for the kinases Ctk1 and Bur1 that phosphorylate the C-terminal repeat domain (CTD) of the largest Pol II subunit and for the histone H3 methyltransferases Set1 and Set2. Bioinformatic analysis indicated that most EFs crosslink preferentially to mRNAs, rather than to unstable non-coding RNAs, consistent with their recruitment to transcribed protein-coding genes. Furthermore, I developed an RNA degradation assay to test whether the observed RNA-protein interactions affect protein-chromatin binding under native conditions. Comparing protein-chromatin binding in the presence and absence of RNA revealed that RNA contributes to chromatin association in particular of the CTD serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1, Set2 and Dot1. Additionally, I confirmed the in vivo observed EF-RNA interactions for an active CTDK-I kinase complex in vitro using fluorescence anisotropy. Finally, I optimized our ChIP protocol for high-throughput sequencing and performed ChIP-Seq experiments of most Pol II EFs and histone marks involved in this study. Comparison of factor occupancies on DNA (ChIP-Seq) and on RNA (PAR-CLIP) revealed that interactions of EFs with nascent RNA are established before EFs are recruited to chromatin. Taken together, these studies argue for a role of nascent RNA in EF recruitment. In this model, EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site trigger disassembly of the elongation complex.