We are investigating mechanisms by which different activities in the cell nucleus are connected to the transcription apparatus via interactions with the hyper-phosphorylated C-terminal repeat domain (PCTD) of elongating RNA polymerase II. Because differential phosphorylation of the CTD determines which proteins bind to it, we are characterizing the activities and chromosomal distribution of Ctk1-kinase, the principal elongation-phase CTD kinase in yeast. Excitingly, we just identified the Ctk1 functional counterparts in Drosophila and human cells as CDK12 and CDK13, and we are beginning intensive investigations of these metazoan "Ctk1 kinases." We are also characterizing numerous factors that interact with Ctk1-generated forms of the PCTD (PCTD-associating proteins, or "PCAPs"). We recently discovered a novel set of CTD-associated DNA repair / recombination proteins and events ("CARR" proteins / events), and we have begun experiments delving into the components and mechanisms at play in this new area of investigation. To continue our investigations, we propose the following SPECIFIC AIMS:
AIM 1 : Analyze "Ctk1-kinases" and CTD Phosphorylation, from Yeast to Man. Many important features of CTD phosphorylation and function are poorly understood;our goal is to illuminate these features. Toward this end we have engineered constructs and developed approaches that will facilitate more in-depth analysis of yeast Ctk1-kinase (CTDK-I) and better evaluation of PCTD functions in RNA processing and chromatin remodeling. In addition, our demonstration that metazoan CDK12 and CDK13 are CTD kinases orthologous to yCTK1 enables us to extend our investigations into D. melanogaster and H. sapiens. Given the virtual absence of knowledge about these CTD kinases in animals, we foresee that our results will provide a host of novel insights into CTD phosphorylation and transcription elongation-related events in multicellular eukaryotes.
AIM 2 : Investigate CTD-Associated Repair &Recombination, from Yeast to Man. We will follow up vigorously our recent demonstration that PCTD-associating proteins play critical roles in responding to DNA damage and suppressing inappropriate recombination in transcribed genes. Because these insights are so new, there is only rudimentary knowledge about mechanisms and macromolecular components involved. To help fill this huge knowledge void, we will investigate "Ctk1 kinase"-dependent CARR inside of transcribed genes in yeast, fruit fly and man. These studies will delineate molecular players and mechanisms of PCTD- dependent responses to DNA damage, and they will provide insights into events and mechanisms of PCTD- dependent modulation of transcription-associated recombination. Because mutations affecting the human versions of CARR proteins can lead to human disease, including cancer, we know already that many of our insights will be medically relevant.

Public Health Relevance

Overall, the proteins and processes we study in yeast are conserved in human cells and are critical to proper gene expression and genome stability. Mutations affecting these proteins can lead to a large number of human diseases, including cancers. Thus, what we learn is directly relevant to human biology and medicine, and our results will accelerate the discovery of new treatments and cures for human diseases, including several cancers.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM040505-23
Application #
8681459
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Sledjeski, Darren D
Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Durham
State
NC
Country
United States
Zip Code
27705
Liu, Jiangxin; Fan, Shilong; Lee, Chul-Jin et al. (2013) Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat. J Biol Chem 288:10890-901
Winsor, Tiffany Sabin; Bartkowiak, Bartlomiej; Bennett, Craig B et al. (2013) A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II. PLoS One 8:e60909
MacKellar, April L; Greenleaf, Arno L (2011) Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II. J Biol Chem 286:36385-95
Werner-Allen, Jon W; Lee, Chul-Jin; Liu, Pengda et al. (2011) cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72. J Biol Chem 286:5717-26
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Bartkowiak, Bartlomiej; Liu, Pengda; Phatnani, Hemali P et al. (2010) CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev 24:2303-16
Kanagaraj, Radhakrishnan; Huehn, Daniela; MacKellar, April et al. (2010) RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription. Nucleic Acids Res 38:8131-40
Li, Ming; Phatnani, Hemali P; Guan, Ziqiang et al. (2005) Solution structure of the Set2-Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1. Proc Natl Acad Sci U S A 102:17636-41
Kizer, Kelby O; Phatnani, Hemali P; Shibata, Yoichiro et al. (2005) A novel domain in Set2 mediates RNA polymerase II interaction and couples histone H3 K36 methylation with transcript elongation. Mol Cell Biol 25:3305-16

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