An Investigation of the Maternal Role of Lysince Specific Histonedmethylase 1 (LSD1) and the Functional Consequences on Development Open Access

Abstract

Classically, the concept of inheritance has been restricted to the passage of DNA. However, in the past couple of decades, have come to understand that information associated with DNA independent of sequence - epigenetic information- can also be inherited and affect phenotypic outcomes across cell divisions. In this dissertation, I will discuss these epigenetic mechanisms and their influence on gene expression. I will also discuss the need to reprogram certain epigenetic information in the context of fertilization when genomic and epigenomic information is passed between generations. The main objective of this dissertation is to investigate the maternal contribution of the histone demethlyase LSD1 and to determine its function at fertilization. I argue that not only is LSD1 reprogramming essential for development but that even slight disturbances in reprogramming at fertilization can result in far-reaching consequences on phenotypes including behavioral aberrations in adult animals.

Table of Contents

Chapter 1: An introduction to epigenetic mechanisms and reprogramming of cellular identity

Abstract. 1

1.1 Epigenetic modifications and their regulatory proteins. 2

The Amine-Oxidase family of histone demethylases. 5

LSD2. 6

LSD1. 6

Figure 1: Domain Schematic of LSD1 and LSD. 7

Epigenetic memory: defining cellular identity and that of its daughter cells. 9

The (epigenetic) memory problem: a change in cell fate. 11

1.2 Natural versus artificial reprogramming. 13

Evidence for epigenetic reprogramming at fertilization. 14

Figure 2: Natural reprogramming versus artificial reprogramming. 15

A case for reprogramming epigenetic memory. 16

Figure 3: A model for H3K4me2 epigenetic memory. 18

Figure 4: Trithorax and Polycomb group protein complexes maintain epigenetic cellular memory. 20

Epigenetic regulation of pluripotency in embryonic stem cells. 21

1.3 Inheritance through gametes: maintenance and erasure. 23

Maternal effect genes: regulation from one generation to the next. 24

The maternal to zygotic transition: priming development. 26

Inheritance and reprogramming of DNA methylation. 27

Inheritance and reprogramming of histone methylation. 30

Figure 5: Summary of reprogramming events that occur at fertilization. 33

1.4 Transgenerational inheritance: evidence for errors in reprogramming?. 34

1.5 A mind at war: the functional consequences of aberrant reprogramming. 35

1.6 Outstanding questions and objectives. 37

Chapter 2: Maternally provided LSD1 enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

Abstract. 38

2.1 Introduction. 39

2.2 Results. 42

LSD1 is expressed throughout oocyte development. 42

Figure 1: Maternal Expression and Conditional Deletion of Lsd1 in Mouse Oocytes. 43

Figure 1-figure supplement 1: LSD1 Expression in Staged Oocytes. 44

Loss of maternal LSD1 results in 1-2 cell embryonic arrest. 45

Figure 1-figure supplement 2: Generation of Lsd1 mutant and control animals. 46

Figure 2: Lsd1Zp3 Embryos Arrest at the 1-2 cell Stage. 47

Figure 2-figure supplement 1: Lack of Normal Lsd1Gdf9 and Lsd1Zp3 Embryos at embryonic day 1.5 and 2.5. 48

Loss of maternal LSD1 results in a failure to undergo the MZT. 50

Figure 3: MZT is Impaired in Lsd1Zp3 Mutants. 51-52

Figure 3-figure supplement 1: Lsd1Zp3 Embryos Arrest at the 1-2 cell Stage. 53

Figure 3-figure supplement 5: Expression of Epigenetic regulators in Lsd1_/_ and Lsd1Zp3 oocytes. 54

Figure 3-figure supplement 2: Principal Component Analysis of Lsd1Zp3 2C Embryos. 56

Deletion of Lsd1 maternally with Vasa-Cre results in a hypomorphic phenotype. 57

Figure 4: Hypomorphic Phenotype in Lsd1Vasa Progeny. 58

Lsd1Vasa M-Z+ progeny exhibit abnormal behavior. 60

Figure 5: Abnormal Behaviors in Lsd1Vasa M-Z+ Adults. 61

Figure 5-figure supplement 1: Abnormal Behaviors in individual Lsd1Vasa M-Z+Adults. 63-64

Lsd1Vasa M-Z+ progeny have imprinting defects. 66

Figure 3-figure supplement 3: Expression of Epigenetic regulators in Lsd1Zp3 2C embryos. 68

Figure 3-figure supplement 4: Relative expression of epigenetic regulators in Lsd1Zp3 2C embryos. 69

Figure 6: Imprinting Defects in Lsd1Vasa Progeny. 71-72

Figure 6-figure supplement 1: Imprinting Analysis of Lsd1Vasa Progeny. 73-74

2.3 Discussion of Results. 76

Model Figure 7: Model: Loss of Maternal LSD1 Results in Defects Later in Development. 82

2.4 Appendix: Chapter 2. 83

Figure 1: Meta-analysis of Blastocyst and 2C RNA-seq data for maternally Expressed genes. 84

Figure 2: DNA methylation and expression alterations at Oct4. 85

Figure 3: Analysis of Global DNA methylation in perinatal lethality animals. 86

Chapter 3: A resource for analysis of DNA methylation status of genomically imprinted loci

3.1 Design of Resource for probing DNA methylation at imprinting control regions

Abstract. 87

Introduction. 88

Results. 93

Figure 1: Workflow for SNP verification within ICRs. 98

Figure 2: SNP verification within Grb10 ICR. 99

Figure 3: SNP verification within H19 ICR. 100

Figure 4: SNP verification within Igf2r ICR. 101

Figure 5: SNP verification within Impact ICR. 102

Figure 6: SNP verification within Lit ICR. 103

Figure 7: SNP verification within Mest ICR. 104

Figure 8: SNP verification within Peg3 ICR. 105

Figure 9: SNP verification within Peg10 ICR. 106

Figure 10: SNP verification within Snrpn ICR. 107

Figure 11: SNP verification within Zac1 ICR. 108

Concluding Remarks. 109

3.2 Methods and Materials. 110

Table 1: Allele-Specific Primers and Polymorphisms. 111-112

Table 2: Twelve-Step PCR Optimization Protocol. 115

Chapter 4: LSD1 maternal function and its effect on development

4.1 Epigenetic reprogramming at fertilization and the role of LSD1 in this process. 119

4.2 LSD1 in the oocyte. 120

4.3 LSD1and the regulation of developmental timing. 120

4.4 Implications of hypomorphic LSD1 function. 122

4.5 Potential hypomorphic effects of LSD1 defects in human patients. 123

4.6 LSD1 and DNA methylation. 125

Table 1: Expression of Known LSD1 binding partners in Mouse oocytes. 126

4.7 DNA methylation at imprinted loci: Defects and disorders. 127

4.8 DNA methylation, imprinted genes and neurological function. 129

4.9 Implications of altered LSD1 function and DNA methylation in neurological disorders: Implications of autism. 129

4.10 Conclusion. 131

Model Figure 1: Summary of reprogramming events that occur at fertilization. 132-135

References. 136-158

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Human Development Biology, Molecular Biology, Cell
Keyword	maternal effect MZT imprinted genes behavioral disorder epigenetics
Committee Chair / Thesis Advisor	Katz, David J, Emory University
Committee Members	Deal, Roger, Emory University Vertino, Paula M, Emory University Cheng, Xiaodong, Emory University Faundez, Victor, Emory University

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