Understanding the role of MRG-1 in C. elegans germline and somatic development Público

Abstract

The chromatin structure formed by the packaging of DNA into nucleosomes greatly influences the transcriptional regulation of genes. Histone modifications like acetylation and methylation alter how tightly DNA interacts with histones therefore changing gene accessibility to transcriptional machinery. Variations to histone modification patterns and chromatin structure can be detrimental to gene regulation and development. Therefore, it is necessary to understand the chromatin related proteins that modify and bind to histones because of their contribution to the overall chromatin landscape.

Human MRG15 (MORF4-Related Gene on Chromosome 15) is a chromatin associated protein that binds to methylated lysine 36 on histone H3 (H3K36me) through a conserved chromodomain. Similar to other chromatin proteins, MRG15 interacts with chromatin modifying proteins such as histone acetyltransferases (HAT) and histone deacetylases (HDACs) that contribute to transcriptional activation and repression, respectively. MRG-1, the C. elegans ortholog of MRG15, contains the conserved chromodomain and was recently shown to interact with similar HDAC complexes. However, the function of the MRG-1 chromodomain and its role in chromatin complexes has never been analyzed.

In this work, I mutated key residues in the conserved MRG-1 chromodomain and discovered novel phenotypes in mrg-1 mutants. Null mutations of mrg-1 lead to meiotic germline defects that produce sterile, but viable, phenotypically normal offspring. However, I demonstrate that point mutations expected to disrupt the conserved chromodomain (mrg-1 CD) cause embryonic lethality, suggesting a role for the MRG-1 chromodomain in somatic development. Additionally, the mrg-1 CD mutation produces a dominant RNAi resistance phenotype. The MRG-1 CD protein properly localizes in the germline but is excluded in somatic tissues at later stages. These distinct phenotypes in mrg-1 null versus chromodomain mutants indicate separate roles for MRG-1 in the germline and soma. Interestingly, both mrg-1 deletion and chromodomain mutants show signs of improper histone acetylation regulation in the germline, consistent with MRG-1’s proposed role in a larger histone modifying complex. Through this research I identified novel phenotypes and distinct contributions of the MRG-1 chromodomain in germline and somatic tissue that will aid in understanding the influence of overall chromatin structure in development.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1. INTRODUCTION 1

1.1 INTRODUCTION TO EPIGENETICS 1

1.2 HISTONE MODIFICATIONS AND CHROMATIN     3

1.3 CHROMATIN MODIFIERS: WRITERS, ERASERS AND READERS    4

1.4 H3K36ME 6

1.5 HISTONE MODIFICATION CROSS TALK    8

1.6 CHROMATIN’S CONTRIBUTION TO DEVELOPMENT        9

1.7 THE MRG FAMILY OF PROTEINS  10

1.8 MRG15 CHROMODOMAIN IN CHROMATIN REGULATION          11

1.9 CONSERVATION OF THE MRG15 CHROMODOMAIN      11

1.10 C. ELEGANS GERMLINE AND MATERNALLY LOADED PROTEINS 13

1.11 SCOPE OF DISSERTATION          14

1.12 FIGURES 17

CHAPTER 2. MRG-1 FUNCTIONS IN THE SOMA AND THE GERMLINE           22

2.1 ABSTRACT 22

2.2 INTRODUCTION   23

2.3 RESULTS   27

2.3.1 Zygotic Requirements for MRG-1 in Meiosis   27

2.3.2 MRG-1 is required for the regulation of crossovers during meiosis    29

2.3.3 H3K36me3 is Required for Normal Meiotic progression         29

2.3.4 M-Z- mrg-1 animals Exhibit Zygotic Defects in Soma   30

2.3.5 Mutations in MRG-1’s Chromodomain Cause Embryonic Lethality    31

2.3.6 Maternally provided CD mutant MRG-1 is not sufficient for normal germline development. 32

2.3.7 The MRG-1 Chromodomain is not required for normal meiotic progression 35

2.3.8 Zygotic MRG-1 combined with Maternal MRG-1CD partially rescues germline development           37

2.3.9 The MRG-1 CD Mutant Exhibits a Dominant RNAi Defect       37

2.4 DISCUSSION         39

2.4.1 Both maternal and zygotic MRG-1 are required for normal germline development and function    39

2.4.2 Zygotic expression of mrg-1 is necessary for normal somatic development  41

2.4.3 The MRG-1 Chromodomain Mutations Cause Novel Phenotypes       42

2.4.4 A Requirement for the MRG-1 Chromodomain in RNAi.         43

2.5 MATERIALS AND METHODS        45

2.6 FIGURES   50

CHAPTER 3. MRG-1 IN A CHROMATIN INTERACTING COMPLEX      79

3.1 ABSTRACT 79

3.2 INTRODUCTION   80

3.3 RESULTS   82

3.3.1 Neither Wildtype MRG-1 or MRG-1 CD form detectable dimers in vivo         82

3.3.2 The MRG-1 Chromodomain does not interact with H3K36me3 peptide        84

3.3.3MRG-1 CD colocalizes with MRG-1 WT in the germline but is lost in the post-larval soma     85

3.3.4 mrg-1 CD and mrg-1 null mutants have an increased level of H3K9 acetylation in the germline       86

3.4 DISCUSSION         87

3.4.1 MRG-1 does not dimerize with itself    87

3.4.2 Wild Type MRG-1 Chromodomain does not interact with H3K36me3 peptide in vitro          89

3.4.3 The MRG-1 chromodomain is necessary for MRG-1's persistence in the soma          89

3.4.4 MRG-1 contributes to histone acetylation regulation 90

3.5 MATERIALS AND METHODS        91

3.6 FIGURES   95

CHAPTER 4. CONCLUSIONS AND FUTURE DIRECTIONS         103

4.1 SUMMARY OF DISSERTATION     103

4.2 MRG-1 POTENTIALLY FUNCTIONS IN VARIOUS ROLES AND COMPLEXES           104

4.3 IDENTIFYING SPECIFIC HISTONE RESIDUES THAT INTERACT WITH MRG-1 CHROMODOMAIN  107

4.4 FINAL THOUGHTS AND ADDITIONAL EXPERIMENTS       109

REFERENCES  111

LIST OF FIGURES

FIGURE 1.1 HISTONE MODIFICATIONS AND CHROMATIN STRUCTURE.       17

FIGURE 1.2 HISTONE MODIFICATION COORDINATION.        18

FIGURE 1.3 CHROMATIN MODIFYING PROTEINS.     19

FIGURE 1.4 SCHEMATIC OF THE STRUCTURAL MOTIFS IN THE MRG FAMILY OF PROTEINS.           20

FIGURE 1.5 MRG15 CONSERVED CHROMODOMAIN ALIGNMENT.  21

FIGURE 2.1. MRG-1 M+Z- MUTANTS EXHIBIT SYNAPTIC DELAY       51

FIGURE 2.2. MRG-1 M+Z- KNOCKDOWN CAUSES DEFECTIVE CROSSOVER REGULATION    52

FIGURE 2.4. DEPLETION OF H3K36ME3 CAUSES SYNAPTIC DELAY.  54

FIGURE 2.5. MRG-1 CHROMODOMAIN MUTATION CAUSES EMBRYONIC ARREST. 56

FIGURE 2.6. MATERNAL MRG-1 CHROMODOMAIN MUTANT ALONE IS NOT SUFFICIENT FOR GERMLINE DEVELOPMENT.          57

FIGURE 2.7. MRG-1 CHROMODOMAIN MUTANT SUPPORTS NORMAL MEIOSIS.    58

FIGURE 2.8. ZYGOTIC EXPRESSION OF WT MRG-1 IN COMBINATION WITH MATERNAL MRG-1 CD CAN PARTIALLY RESCUE FERTILITY.  59

FIGURE 2.9. MRG-1 CD MUTATION CAUSES A DOMINANT RNAI RESISTANCE PHENOTYPE.           60

SUPPLEMENTAL FIGURE 2.1. MRG-1 (TM1227) MUTANTS HAVE DECREASED BROOD SIZE AND INCREASED EMBRYONIC LETHALITY.    62

SUPPLEMENTAL FIGURE 2.2 MET-1; MES-4 RNAI MUTANTS HAVE A DECREASED BROOD SIZE.     64

SUPPLEMENTAL 2.3. MRG-1 M-Z- MUTANTS EXHIBIT SOMATIC DEFECTS.  65

SUPPLEMENTAL FIGURE 2.4. MATERNALLY LOADED MRG-1 CD AND MRG-1 WT ARE PRESENT IN EARLY EMBRYOS.       67

SUPPLEMENTAL 2.5. MRG-1 CHROMODOMAIN MUTANTS ARREST DURING EMBRYOGENESIS.    68

SUPPLEMENTAL FIGURE 2.6. MRG-1CD IS MATERNALLY LOADED INTO THE GERMLINE.   69

SUPPLEMENTAL 2.7. ZYGOTIC EXPRESSION OF WT MRG-1 PARTIALLY RESCUES GERMLINE DEVELOPMENT.       70

SUPPLEMENTAL FIGURE 2.8.FERTILITY OF MRG-1CD/WT::GFP WORMS ARE NOT AFFECTED BY GFP RNAI.          71

SUPPLEMENTAL FIGURE 2.9. MRG-1CD/+ L1 LARVAE DO NOT ARREST AFTER AMA-1 RNAI.          72

SUPPLEMENTAL FIGURE 2.10 MRG-1 CD/+ HERMAPHRODITES EXHIBIT SOME SYP-1 AGGREGATION AFTER HIM-3 RNAI.           73

SUPPLEMENTAL FIGURE 2.11. MRG-1 CD MUTANTS ARE RESISTANT TO AMA-1 RNAI.     74

SUPPLEMENTAL FIGURE 2.12 SUMMARY OF GENETIC AND PROTEIN PHENOTYPES OF THE MRG-1(TM1227) AND MRG-1CD MUTANT. 75

FIGURE 3.1. MRG-1 WT DOES NOT DIRECTLY INTERACT WITH ITSELF OR MRG-1 CD.         96

FIGURE 3.2. WILDTYPE MRG-1 CHROMODOMAIN PEPTIDE DOES NOT INTERACT WITH H3K4ME3 OR H3K36ME3.         97

FIGURE 3.3 MRG-1CD::MCHERRY COLOCALIZES WITH MRG-1WT::DEGRON::GFP  IN THE GERMLINE.     98

FIGURE 3.4 MRG-1CD::MCHERRY IS EXCLUDED IN GUT SOMATIC TISSUE.  99

FIGURE 3.5. MRG-1 CD AND MRG-1 NULL MUTANTS HAVE INCREASED LEVELS OF H3K9 ACETYLATION. 100

SUPPLEMENTAL FIGURE 3.1 PURIFICATION OF N-TERMINALLY TAGGED GST MRG-1 CHROMODOMAIN PEPTIDE.           101

SUPPLEMENTAL FIGURE 3.2 WILDTYPE MRG-1::MCHERRY IS FOUND IN THE GERMLINE AND SOMA.       102

LIST OF TABLES

TABLE 2.1. LIST OF STRAINS   47

SUPPLEMENTARY TABLE 2.1. MRG-1 M-Z- SOMATIC DEFECT          65

S TABLE 2.2. GUIDE RNA SEQUENCES.          76

S TABLE 2.3 OLIGONUCLEOTIDE REPAIR SEQUENCES.          79

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular
Palavra-chave	epigenetics chromatin mrg-1
Committee Chair / Thesis Advisor	William Kelly, Emory University
Committee Members	Paula Vertino, University of Rochester David Katz, Emory University Roger Deal, Emory University Leila Rieder, Emory University

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