Characterization of Transcription Regulation in C. elegans: The Role of SIG-7 in Coordinating RNA Polymerase Elongation and mRNA Processing Open Access

Ahn, Jeong Hyun (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/4j03d0249?locale=en
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Abstract

The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with the elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Much of the coordinated events are mediated by their interactions with the C-terminal domain (CTD) of Pol II. The specificity of the interaction can be obtained by modulating the structure of the CTD by numerous post-translational modifications (PTMs). One class of enzymes that can modulate the CTD is the peptidyl prolyl isomerases (PPIase). Here I characterize a member of the cyclophilin family of PPIases that interacts with Pol II to coordinately regulate transcription elongation and splicing in C. elegans. SIG-7 contains multiple functional domains, including the PPI domain and RNA-interacting domains. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II elongation is defective, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation.

Table of Contents

CHAPTER1: INTRODUCTION.......................................................................................... 1

An overview of transcription regulation............................................................................... 2

Transcription Initiation/Elongation regulation............................................................ 4

Factors affecting Pol II pausing................................................................................... 6

The release of Pol II pausing by P-TEFb...................................................................... 8

Factors alleviating the nucleosome barrier during transcription elongation.............. 9

Regulation of Co-Transcriptional Splicing................................................................ 10

Chromatin and splicing.............................................................................................. 11

The Role of the CTD in Splicing................................................................................. 14

The Roles of Peptidyl Prolyl Isomerases During Transcription.............................................. 16

PPIases and the Pol II CTD........................................................................................ 18

The Cyclophilin PPIases............................................................................................. 21

Further Investigations into Transcriptional Memory........................................................... 23

Figures............................................................................................................................. 25

Figure 1. The features of pre-mRNA recognized by spliceosome and step-wise assembly of spliceosome complex 25

CHAPTER 2: A Conserved Nuclear Cyclophilin is Required for Both RNA Polymerase II Elongation and Co-Transcriptional Splicing in Caenorhabditis elegans................................................................................................................................ 26

Abstract........................................................................................................................... 27

Introduction.................................................................................................................... 28

Result.............................................................................................................................. 32

Mapping and characterization of sig-7 mutants........................................................ 32

sig-7 encodes a conserved nuclear cyclophilin that is essential for development....... 34

SIG-7 Localizes to Transcriptionally Active Chromatin........................................... 36

SIG-7 is essential for normal gastrulation during embryonic development.............. 37

SIG-7 physically interacts with Pol II in vivo............................................................. 38

sig-7 RNAi causes a global decrease in embryonic transcript levels.......................... 39

sig-7 RNAi causes changes in RNA processing........................................................... 41

sig-7 RNAi causes a global change in Pol II occupancy and distribution within gene bodies 44

Depletion of SIG-7 causes a reduction in Pol II isoforms and histone modifications associated with transcription elongation 46

Revisiting transgene desilencing in sig-7(RNAi) or sig-7(cc629) mutant................... 48

nrde assay reveals that the presence of the genetic balancer chromosome is somehow mimicking the NRDE function via unknown mechanism.................................................................................................................................... 50

Discussion........................................................................................................................ 53

Material and Methods....................................................................................................... 57

Worm strains and maintenance.................................................................................. 57

sig-7::GFP::3XFLAG transgenic strain...................................................................... 57

RNAi-mediated Depletion........................................................................................... 57

Immunofluorescence................................................................................................... 58

Immunoprecipitation assays....................................................................................... 59

Protein isolation and Western blot analysis............................................................... 60

RNA purification and qRT-PCR................................................................................ 61

Library preparation and RNA sequencing................................................................. 62

Analysis of RNA-seq................................................................................................... 62

Library preparation from ChIP material for sequencing.......................................... 63

Analysis of ChIP-seq data.......................................................................................... 63

Protein sequence alignment........................................................................................ 65

Transgene desilencing assay....................................................................................... 65

Acknowledgments............................................................................................................ 65

Figures............................................................................................................................. 67

Figure 1. Repetitive transgenes are desilenced in both somatic and germ cells after the sig-7 RNAi. 67

Figure 2. The sig-7 gene, protein, mutant alleles, and orthologs in other species...... 68

Figure 3. Pleiotropic defects in sig-7 mutants............................................................ 69

Figure 4. SIG-7 is a ubiquitously expressed nuclear protein...................................... 70

Figure 5. Sequence alignment of SIG-7 orthologs..................................................... 71

Figure 6. SIG-7 is associated with transcriptionally active chromatin...................... 72

Figure 7. sig-7 is required for hallmarks of zygotic transcription............................. 73

Figure 8. SIG-7 associates with RNA Pol II in vivo................................................... 74

Figure 9. SIG-7 interacts with RNA Pol II in vivo..................................................... 75

Figure 10. RNA-seq analysis of sig-7(RNAi) embryos reveals a global transcription defect. 76

Figure 11. SIG-7 is required for germline transcription............................................ 77

Figure 12. Comparison of embryonic stages present in control vs sig-7(RNAi)........ 78

Figure 13. sig-7(RNAi) predominantly affects zygotic gene expression.................... 79

Figure 14. SIG-7 is required for efficient splicing of nascent transcripts.................. 80

Figure 15. qRT-PCR analysis of the effect of sig-7(RNAi) on splicing...................... 81

Figure 16. sig-7(RNAi)-dependent changes in RNA Pol II occupancy correlate with expression changes. 82

Figure 17. sig-7(RNAi)-dependent changes in RNA Pol II occupancy among different gene classes are consistent with defects observed by RNA-seq...................................................................................................................... 83

Figure 18. RNA Pol II distribution within genes is altered by sig-7(RNAi)................ 84

Figure 19. RNA Pol II phosphoepitopes and histone H3 modifications associated with transcription elongation are altered by sig-7(RNAi)..................................................................................................................................... 85

Figure 20. H3K36me3 is more significantly affected than H3K4me3 during embryonic development in sig-7(RNAi). 86

Figure 21. ccEx7271 repetitive array gets de novo H3K4 di-methylation mark when it is desilenced in adult oocyte. 87

Figure 22. The increased GFP reporter expression is accompanied by decreased expression of RNAi pathway genes in sig-7(RNAi) animals..................................................................................................................................... 88

Figure 23. Repetitive transgene array can be desilenced by knocking down RNA Pol II by RNAi 89

Figure 24. The presence of genetic balancer, not the defects of SIG-7 function, is responsible for suppression of Muv phenotype in eri-1 background................................................................................................................. 90

CHAPTER3: H3K4 and H3K36 Methylation in Germline stem cells and Their Roles in Transgenerational Maintenance of Germline Function 91

Introduction.................................................................................................................... 92

A brief history of the emergence of the field of epigenetics....................................... 92

A rationale for the need of maintaining cellular memory......................................... 93

The role of DNA methylation in epigenetic memory.................................................. 94

The Role of Histone Methylation in Epigenetic Memory........................................... 97

Result............................................................................................................................ 104

GFP tagged WDR-5 protein is expressed in adult gonads, and the maternally inherited WDR-5 protein shows a persistence over a few cell divisions during early embryogenesis....................................................................... 104

1XFLAG::GFP::WDR-5 can restore H3K4 methylation in the wdr-5.1(ok1417) mutant. 105

Both the transcription independent H3K4 methylation and the transcription dependent H3K36 methylation is required for the proper establishment of H3K36 methylation by MES-4 in the GSCs.................................. 105

The met-1(n4337); wdr-5.1(ok1417) double mutant causes a transgenerational sterility due to various germline defects 106

The wdr-5.1(RNAi) causes a precocious sterility in the M+Z- mes-4(ok2326) mutant 107

WDR-5 males do not produce cross progeny........................................................... 108

Loss of set-17 or set-30 does not noticeably affect transcription dependent H3K4 methylation in the adult germline 109

Discussion...................................................................................................................... 110

Material and Method...................................................................................................... 114

Worm strains and maintenance................................................................................ 114

1XFLAG::GFP::WDR-5.1 transgenic strain generation........................................ 114

RNAi-mediated transcript knockdown..................................................................... 115

Immunofluorescence................................................................................................. 115

Protein isolation and Western blot analysis............................................................. 116

Brood size assay........................................................................................................ 117

Figures........................................................................................................................... 118

Figure1. Summary of the methylation dynamics of H3K4 and H3K36 during embryonic and an adult germline development 118

Figure 2. Live WDR-5:GFP expression (ckSi35)..................................................... 119

Figure 3. Western blot for GFP confirms the expression of WDR-5.1.................... 120

Figure 4. The expression of FLAG::GFP::WDR-5.1 is sufficient to restore the loss of H3K4me3 from the GSCs in the wdr-5.1 mutant. 121

Figure 5. An increase in H3K36me3 is observed in the GSCs of met-1;wdr-5.1 double mutants. 122

Figure 6. Synergistic defect in the fertility of the met-1;wdr-5.1 double mutant..... 123

Figure 7. The loss of both wdr-5.1 and met-1 causes sterility due to various defects during germline development 124

Figure 8. The wdr-5.1(RNAi) causes a precocious appearance of the maternal effect sterile (mes) phenotype of the mes-4 mutant (M+Z-) 125

Figure 9. Nether set-17 nor set-30 is the major transcription dependent H3K4 methyltransferase in the adult gonad of C. elegans 126

CHAPTER 4: The significance of my studies and future directions................................ 127

The significance of SIG-7 studies.............................................................................. 128

The significance of WDR-5 project.......................................................................... 129

Figure............................................................................................................................ 132

Figure 1. A potential model of SIG-7's function...................................................... 132

References...................................................................................................................... 134

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