Regulation of Transcription by RNA Polymerase II in C. elegans Public

Bowman, Elizabeth Anne (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/k930bx519?locale=fr
Published

Abstract

Virtually all cells within a multicellular organism contain the same DNA. Despite this, multicellular organisms are made of different cell types, and the development of different cell types from the same DNA is due to differential expression of protein coding genes. The first step of gene expression is transcription in which RNA Polymerase II (Pol II) copies a gene into an RNA transcript. Transcription involves three steps, 1) initiation, or selection of a transcriptional site, 2) elongation, or the productive generation of RNA Pol II, and 3) termination of transcription. While these steps of transcription have been well studied in single cell systems, the regulation of transcription during the development of multicellular organisms is not well characterized. Therefore, I have studied transcriptional regulation during development in the multicellular organism, C. elegans.

In this thesis, I have analyzed mutations in the large catalytic subunit of C. elegans Pol II, called AMA-1, and have mapped these onto the structure of the yeast protein to identify structural components required for normal function and regulation of this protein. In pursuing further characterization of the role of Pol II during development, I discovered a form of tissue-specific regulation of transcriptional elongation in C. elegans. The transition from Pol II initiation to elongation is accompanied by phosphorylation of serine 2 (Ser2) of the Pol II carboxy terminal domain (CTD), which was thought to be carried out by the canonical Ser2 kinase, CDK-9. However, I have uncovered CDK-9 independent Ser2 phosphorylation in the C. elegans germline. Instead, this modification is performed by the recently discovered Ser2 kinase, CDK-12. This is in contrast to somatic tissues where both CDK-9 and CDK-12 contribute to Ser2 phosphorylation. I observed that neither CDK-12 nor Ser2 phosphorylation are required in the germline for fertility. These studies are the first to show tissue-specific differences in the regulation of Ser2 phosphorylation, a marker of Pol II elongation. Overall, I have generated a novel model of tissue-specific transcriptional regulation during C. elegans development, further defining the regulatory mechanisms of this "general" process in multicellular organisms.

Table of Contents

Chapter 1: Introduction........................................................................................................................................ 1

The Pol II CTD................................................................................................................................................. 3

Positive elongation factors: Pol II CTD Serine 2 kinases.................................................................................. 5

The yeast Ser2 kinases: Ctk1/Lsk1 and Bur1/Cdk9....................................................................................... 6

Bur1/Cdk9 and Ctk1/Lsk1 regulate yeast Ser2 phosphorylation together.................................................. 7

Yeast Ser2 kinases regulate transcription-associated factor recruitment..................................................... 9

Regulation of yeast Ser2 kinases............................................................................................................. 11

The metazoan Ser2 kinases: CDK9 and CDK12......................................................................................... 12

Identification of CDK9........................................................................................................................... 12

CDK9/P-TEFb activity is regulated by its association with large complexes............................................ 14

CDK9 recruitment is regulated by gene-specific factors......................................................................... 15

Additional roles for CDK9...................................................................................................................... 16

CDK12/Cyclin K: the elusive CTD kinase complex..................................................................................... 17

Spt4/Spt5: The evolution of dual functions of a conserved transcription elongation factor............................ 19

Identification of SPT4/SPT5........................................................................................................................ 19

SPT4/SPT5 mediates PPP in higher eukaryotes by bridging NELF and Pol II binding............................... 20

SPT4/SPT5 is a conserved transcription elongation factor that enhances Pol II processivity..................... 22

Interplay of Pol II elongation factors.............................................................................................................. 23

Phosphorylation of the SPT5 CTR.............................................................................................................. 23

Spt4/Spt5 recruitment of transcription associated factors........................................................................... 25

Genome-wide model or gene-specific function?............................................................................................ 26

Germline-specific gene regulation.................................................................................................................. 26

Expression of variant basal transcription factors is a conserved hallmark of the germline........................ 27

A phase of transcriptional repression is a conserved hallmark of germline specification........................... 29

Introduction to the C. elegans germline...................................................................................................... 31

Transcriptional regulation in C. elegans......................................................................................................... 32

Characterization of transcription mutants in C. elegans............................................................................. 32

Tissue-specific transcriptional regulation in C. elegans.............................................................................. 33

Table 1.1: RNA Polymerase II elongation factor homologs............................................................................ 35

Figure 1.1: Schematic of the role of Ser2 kinases in transcriptional elongation regulation.............................. 36

Figure 2.1: Schematic of germline transcriptional regulation.......................................................................... 38

Citations......................................................................................................................................................... 39

Chapter 2: Amino acid substitutions in the C. elegans RNA Polymerase II large subunit, AMA-1/RPB-1, that result in α-amanitin resistance and/or reduced function....................................................................................................... 53

Abstract.......................................................................................................................................................... 54

Introduction................................................................................................................................................... 54

Results........................................................................................................................................................... 55

α-Amanitin Resistant Mutations.................................................................................................................. 55

Hypomorphic and Null Mutations.............................................................................................................. 57

Discussion...................................................................................................................................................... 60

Acknowledgements........................................................................................................................................ 61

Tables............................................................................................................................................................. 62

Table 2.1: Summary of α-amanitin-resistance and hypomorphic AMA-1 mutations.................................... 62

Table 2.2: DR1099 phenotype characterization......................................................................................... 64

Figures........................................................................................................................................................... 65

Figure 2.1: Sequence alignments of Saccharomyces cerevisiae (y), Caenorhabditis elegans (c), Drosophila melanogaster (d), and Homo sapiens (h) AMA-1/RPB-1 amino acid sequences.............................................. 65

Figure 2.2: Structural position of C. elegans α-amanitin resistant mutations in the homologous S. cerevisae structure.......................................................................................................................................................... 66

Figure 2.3: Positions of the C. elegans AMA-1/RPB1 mutations mapped to corresponding residues in the homologous S. cerevisae structure.................................................................................................................. 67

Citations......................................................................................................................................................... 69

Chapter 3: Temperature sensitive mutants of the RNA Polymerase II TFIID initiation factor, taf-6.2................. 71

Abstract.......................................................................................................................................................... 72

Citations......................................................................................................................................................... 74

Chapter 4: P-TEFb-independent RNA polymerase II phosphorylation and germline-soma distinction in C. elegans............................................................................................................................................................................... 75

Abstract.......................................................................................................................................................... 76

Introduction................................................................................................................................................... 76

Materials and Methods................................................................................................................................... 78

Strains Used............................................................................................................................................... 78

RNA interference........................................................................................................................................ 78

Immunofluorescence................................................................................................................................... 78

Protein Isolation and Western..................................................................................................................... 79

Transgenic Lines........................................................................................................................................ 80

Fluorescence and DNA visualization in intact worms................................................................................. 80

Results........................................................................................................................................................... 81

Tissue-specific requirements for CDK-9 and CDK-12 in Ser2 phosphorylation.......................................... 81

Embryonic Germ line-Soma conversion yields switch in kinase dependency.............................................. 83

CDK-12--dependent Ser2-P is not essential for early germ cell proliferation............................................ 83

CDK-12 is the predominant germline Ser2 kinase...................................................................................... 84

CDK-9, CDK-12, and cyclin partners are ubiquitously expressed.............................................................. 85

CDK-9, but not CDK-12, is required for proper germline development...................................................... 86

CDK-12 is required for normal H3K36me3levels in germ cells.................................................................. 88

Discussion...................................................................................................................................................... 89

Embryonic Ser2-P regulation highlights tissue-specific regulation............................................................. 89

Kinase requirements in development.......................................................................................................... 90

Mechanism behind tissue-specific kinase requirements on Ser2-P.............................................................. 92

Acknowledgements........................................................................................................................................ 95

Figures........................................................................................................................................................... 96

Figure 4.1: Kinase and cyclin dependency of Ser2-P in embryonic soma and germ cells.......................... 96

Figure 4.2: Quantification of Pol II and CTD modifications in the embryo................................................ 97

Figure 4.4: Analysis of kinase RNAi knockdown specificity and efficiency in CDK-9; CDK-12 double transgenic animals.......................................................................................................................................................... 100

Figure 4.5: FCP-1 phosphatase activity does not contribute to CDK-12-dependent Ser2-P..................... 101

Figure 4.6: PIE-1 regulates CDK-12-dependent Ser2-P........................................................................... 102

Figure 4.7: Ser2-P, but not proliferation, requires CDK-12 in early larvae.............................................. 104

Figure 4.8: Ser2-P in adult germline cells is independent of P-TEFb....................................................... 105

Figure 4.9: Quantification of Pol II and CTD modifications in the adult and gonad................................ 107

Figure 4.10: CDK-9, CDK-12, and their cyclin partners are ubiquitously expressed............................... 109

Figure 4.11: Increasing germline expression of Cyclin T (CIT-1.2) does not change germline Ser2-P kinase requirements.................................................................................................................................................. 110

Figure 4.12: SPT-5 is ubiquitously expressed............................................................................................ 111

Figure 4.13: CDK-9, but not CDK-12 or Ser2P, is essential for germline development............................ 112

Figure 4.14: CDK-12 regulates H3K36me3 levels.................................................................................... 113

Figure 4.15: CDK-9 and CDK-12 regulate transcription-dependent H3K36me3..................................... 114

Tables........................................................................................................................................................... 115

Table 4.1: Cyclin T RNAi Embryonic Lethality.......................................................................................... 115

Table 4.2: New Transgenic Lines.............................................................................................................. 116

Table 4.3: Additional Strains Made.......................................................................................................... 117

Table 4.4: Cloning Methods..................................................................................................................... 118

Table 4.5: Primer sequences.................................................................................................................... 120

Citations....................................................................................................................................................... 121

Chapter 5: Towards the purification of C. elegans primordial germ cell nuclei by INTACT for genomic analysis 125

Introduction................................................................................................................................................. 126

Methods....................................................................................................................................................... 128

Strains used.............................................................................................................................................. 128

Generation of transgenic lines................................................................................................................. 128

Visualization of transgenic animals and immunofluorescence.................................................................. 129

Results......................................................................................................................................................... 130

Expression of NPP-9:BLRP in embryonic germline precursors................................................................. 130

Expression of BirA in the germline........................................................................................................... 131

Discussion.................................................................................................................................................... 132

Acknowledgments........................................................................................................................................ 133

Tables........................................................................................................................................................... 134

Table 5.1: Transgenic lines....................................................................................................................... 134

Table 5.2: Cloning Methods..................................................................................................................... 135

Table 5.3: Primers.................................................................................................................................... 136

Figures......................................................................................................................................................... 137

Figure 5.1: Addition of a destabilizing PEST sequences causes selective expression of npp-9:BLRP in P4/Z2/Z3....................................................................................................................................................................... 137

Figure 5.2: ceBirA expresses in the adult germline and is maternally loaded into embryos..................... 138

Citations................................................................................................................................................... 139

Chapter 6: Discussion...................................................................................................................................... 141

New tools for the analysis of gene expression in C elegans......................................................................... 142

Identification of germline-specific Ser2 phosphorylation in C. elegans....................................................... 142

Comparison of Ser2 phosphorylation models........................................................................................... 144

Ser2-P is not required for germline development...................................................................................... 145

A possible role for a 5' transcriptional checkpoint in C. elegans.............................................................. 146

Conservation of germline-specific transcriptional elongation control....................................................... 148

Figure 6.1: Summary of thesis and schematic of model............................................................................... 150

Citations....................................................................................................................................................... 152

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research Field
Mot-clé
Committee Chair / Thesis Advisor
Committee Members
Dernière modification

Primary PDF

Thumbnail Title Date Uploaded Actions
Regulation of Transcription by RNA Polymerase II in C. elegans () 2018-08-28 13:26:55 -0400

Supplemental Files

Thumbnail Title Date Uploaded Actions