THE ROLE OF GENOMIC IMPRINTS IN PLACENTAL BIOLOGY by Erik Alexander Koppes BS ...

297 Pages·2016·5.31 MB·English

by  Erik Koppes

THE ROLE OF GENOMIC IMPRINTS IN PLACENTAL BIOLOGY

by
Erik Alexander Koppes
B.S., Purchase College, 2007
M.A., Columbia University, 2009

Submitted to the Graduate Faculty of
the School of Medicine in partial fulfillment
of the requirements for the degree of
PhD in Integrative Molecular Biology

University of Pittsburgh
2016
UNIVERSITY OF PITTSBURGH
SCHOOL OF MEDICINE

This dissertation was presented

by

Erik Alexander Koppes

It was defended on
April 19, 2016
and approved by
Gerard Apodaca, PhD, Professor
Yaacov Barak, PhD, Associate Professor
Deborah Chapman, PhD, Associate Professor
Jeffrey Hildebrand, PhD, Associate Professor
Dissertation Advisor: J. Richard Chaillet, MD PhD, Associate Professor

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Copyright © by Erik Alexander Koppes
2016
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THE ROLE OF GENOMIC IMPRINTS IN PLACENTAL BIOLOGY
Erik Alexander Koppes, PhD
University of Pittsburgh, 2016

Genomic imprinting is a process by which heritable epigenetic marks at a subset of genomic loci
are established in a sex-specific manner in parental gametes and then maintained in nascent
offspring. This study probes the poorly understood function of genomic imprints in placental
biology. Genomic imprints are responsible for the regulation of parent-of-origin specific
monoallelic expression of clusters of imprinted genes. The primary epigenetic mark that
distinguishes parental alleles at imprinted loci is 5-methylcytosine in the context of cytosine-
guanine (CpG) dinucleotides within differentially methylated domains (DMDs). The Dnmt1 gene
encodes the maintenance DNA methyltransferase, an enzyme responsible for replicating CpG
methylation that is critical throughout the process of genomic imprinting. Genetic disruption of
the oocyte specific isoform of Dnmt1 (Dnmt1o) results in partial and wide-spread loss of DMD
methylation during preimplantation development and has strong effects on embryonic and
extraembryonic development. In this dissertation the morphology of DNMT1o-deficient
placentas is examined and their abnormal phenotypes correlated with loss of methylation at
specific DMDs. A strong association between loss of methylation at the Kcnq1 DMD and
accumulation of trophoblast giant cells was made. In addition, an association between loss of
methylation at the Peg10 DMD and loss of fetal viability and placental labyrinthine volume was
made. In conjunction with my study of the Dnmt1Δ1o model, I have engineered a novel targeted
deletion of the imprinted Klf14 gene and found it has an effect on placental growth. My thesis
unambiguously shows that genomic imprints are essential for placental development.
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TABLE OF CONTENTS
1.0 INTRODUCTION .............................................................................................................................................. 1
1.1 GENOMIC IMPRINTING ............................................................................................................................................ 1
1.1.1 Discovery of genomic imprints ................................................................................................................... 1
1.1.2 Establishment and maintenance of imprinted DNA methylation ................................................. 4
1.1.3 DNA methyltransferase mouse models ................................................................................................. 10
1.2 GENOMIC IMPRINTS IN HUMAN DEVELOPMENTAL DISORDERS ....................................................... 14
1.2.1 Beckwith Wiedemann Syndrome ............................................................................................................. 15
1.2.2 Silver-Russell syndrome .............................................................................................................................. 16
1.2.3 Prader-Willi syndrome and Angelman syndrome ............................................................................ 18
1.2.4 Pseudohypothyroidism type I ................................................................................................................... 20
1.2.5 Transient neonatal diabetes mellitus Type 1 ...................................................................................... 20
1.3 PLACENTAL INFLUENCE ON PREGNANCY OUTCOME ............................................................................. 23
1.3.1 Requirements for prenatal development.............................................................................................. 23
1.3.2 Intrauterine growth restriction ................................................................................................................ 24
1.3.3 Preeclampsia ..................................................................................................................................................... 25
1.3.4 Gestational diabetes mellitus ..................................................................................................................... 25
1.3.5 Fetal origins of adult diseases ................................................................................................................... 26
1.3.6 Maternal influences on placental function ........................................................................................... 26
1.4 PLACENTAL DEVELOPMENT AND FUNCTION ............................................................................................ 27
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1.4.1 Comparison of human and mouse placental structure and development .............................. 27
1.4.2 Preimplantation development .................................................................................................................. 29
1.4.3 Implantation ..................................................................................................................................................... 31
1.4.4 Labyrinthine zone development ............................................................................................................... 32
1.4.5 Junctional zone development .................................................................................................................... 35
1.4.6 Trophoblast giant cells ................................................................................................................................. 36
1.5 GENOMIC IMPRINTS WITH POSSIBLE PLACENTAL FUNCTIONS ........................................................ 39
1.5.1 Nnat ...................................................................................................................................................................... 39
1.5.2 Gnas....................................................................................................................................................................... 40
1.5.3 Mest ....................................................................................................................................................................... 43
1.5.4 Nap1l5 .................................................................................................................................................................. 45
1.5.5 Peg10 .................................................................................................................................................................... 46
1.5.6 H19 ........................................................................................................................................................................ 47
1.5.7 Kcnq1 .................................................................................................................................................................... 49
1.5.8 Snrpn .................................................................................................................................................................... 50
1.5.9 Peg3 ...................................................................................................................................................................... 51
1.5.10 Plagl1 ................................................................................................................................................................. 53
1.5.11 Grb10 ................................................................................................................................................................. 54
1.5.12 Zrsr1 ................................................................................................................................................................... 56
1.5.13 Dlk1 .................................................................................................................................................................... 57
1.5.14 Igf2r .................................................................................................................................................................... 61
1.5.15 Impact ............................................................................................................................................................... 62
1.6 SPECIFIC AIMS ............................................................................................................................................................ 69
2.0 PLACENTAL PHENOTYPES OBSERVED IN THE DNMT1Δ1O MOUSE MODEL ............................. 71
2.1 SUMMARY ................................................................................................................................................................... 71
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2.2 INTRODUCTION ........................................................................................................................................................ 72
2.2.1 The Dnmt1Δ1o model ....................................................................................................................................... 72
2.2.2 Identification of Dnmt1o .............................................................................................................................. 72
2.2.3 Targeted deletion of Dnmt1o ..................................................................................................................... 74
2.2.4 Localization of DNMT1 in preimplantation embryos ...................................................................... 75
2.2.5 Preimplantation function of DNMT1o .................................................................................................... 77
2.2.6 Fetal development of Dnmt1Δ1o maternal effect offspring ............................................................. 78
2.2.7 Placental development of Dnmt1Δ1o maternal effect offspring..................................................... 80
2.2.8 Chapter 2 aims ................................................................................................................................................. 80
2.3 MATERIALS AND METHODS ................................................................................................................................ 81
2.3.1 Animal husbandry .......................................................................................................................................... 81
2.3.2 Genotyping ......................................................................................................................................................... 82
2.3.3 Collection of placentas .................................................................................................................................. 82
2.3.4 Cryo-histology .................................................................................................................................................. 84
2.3.5 In situ hybridization ....................................................................................................................................... 85
2.3.6 Immunohistochemistry ................................................................................................................................ 86
2.3.7 Immunofluorescence ..................................................................................................................................... 86
2.3.8 Stereology and morphometrics ................................................................................................................ 87
2.3.9 Lipid extraction ................................................................................................................................................ 88
2.3.10 Biostatistics .................................................................................................................................................... 89
2.3.11 Microarray analysis ..................................................................................................................................... 89
2.4 RESULTS ...................................................................................................................................................................... 90
2.4.1 Dnmt1Δ1o maternal effect embryonic viability .................................................................................... 90
2.4.2 E9.5 Phenotypes .............................................................................................................................................. 91
2.4.3 E12.5 Phenotypes ........................................................................................................................................... 93
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2.4.4 E15.5 phenotypes ........................................................................................................................................ 102
2.4.5 E17.5 placental phenotypes .................................................................................................................... 109
2.5 DISCUSSION ............................................................................................................................................................. 120
2.5.1 Fetal viability and growth ........................................................................................................................ 120
2.5.2 Placental labyrinth phenotypes ............................................................................................................. 123
2.5.3 Placental junctional zone phenotypes ................................................................................................. 126
2.5.4 Trophoblast giant cell phenotypes ....................................................................................................... 127
2.5.5 Conclusion ....................................................................................................................................................... 128
3.0 LOSS OF GENOMIC IMPRINTING IN DNMT1O-DEFICIENT PLACENTAS IS ASSOCIATED
WITH SPECIFIC PLACENTAL ABNORMALITIES ...................................................................................... 129
3.1 SUMMARY ................................................................................................................................................................ 129
3.2 INTRODUCTION ..................................................................................................................................................... 130
3.2.1 Loss of genomic imprinting in DNMT1o-deficient placentas .................................................... 130
3.2.2 Transcriptional analysis of imprinted genes .................................................................................... 132
3.2.3 Imprinted DMD methylation analysis ................................................................................................. 132
3.2.4 Association of loss of imprinting and placental phenotypes .................................................... 134
3.2.5 Chapter 3 aims .............................................................................................................................................. 135
3.3 MATERIALS AND METHODS ............................................................................................................................. 136
3.3.1 DNA and RNA purification ....................................................................................................................... 136
3.3.2 Imprinted gene quantitative real-time RT-PCR .............................................................................. 136
3.3.3 Bisulfite genomic sequencing ................................................................................................................. 137
3.3.4 Epityper analysis .......................................................................................................................................... 138
3.3.5 Biostatitics and bioinformatics .............................................................................................................. 139
3.4 RESULTS ................................................................................................................................................................... 140
3.4.1 Loss of imprinted gene expression ....................................................................................................... 140
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3.4.2 Bisulfite genomic sequencing ................................................................................................................. 142
3.4.3 EpiTYPER imprinted DMD methylation analysis ........................................................................... 147
3.4.4 Decreased fetal viability is associated with loss of Peg10 DMD methylation ..................... 157
3.4.5 Placental abnormalities are associated with loss of DMD methylation ................................ 158
3.5 DISCUSSION ............................................................................................................................................................. 166
3.5.1 A broad spectrum of loss of imprinting is revealed in DNMT1o-deficient placentas ...... 166
3.5.2 Association between Ascl2 and Mest gene expression with E/P ratio ................................... 169
3.5.3 Peg10 viability and labyrinth phenotypes ......................................................................................... 170
3.5.4 Loss of Kcnq1 DMD methylation and TGC expansion ................................................................... 172
3.5.5 Loss of Nespas and H19 DMD and junctional zone development ............................................. 174
3.5.6 No placental phenotypes associated with Dlk1, Igf2r or Grb10 DMD methylation .......... 175
3.5.7 Zrsr1 is an imprinted DMD in placenta but Nnat and Nap1l5 are not .................................... 176
3.5.8 Conclusion ....................................................................................................................................................... 176
4.0 KLF14 IS AN IMPRINTED GENE REGULATING PLACENTAL GROWTH .................................... 178
4.1 SUMMARY ................................................................................................................................................................ 178
4.2 INTRODUCTION ..................................................................................................................................................... 179
4.2.1 Klf14 is part of the Mest imprinting cluster ....................................................................................... 179
4.2.2 The Mest imprinting cluster is implicated in placenta development ..................................... 180
4.2.3 KLF14 is a transcription factor regulating metabolism ............................................................... 181
4.2.4 Recombineering is a genetic engineering technique .................................................................... 184
4.2.5 Aims of chapter 4 ......................................................................................................................................... 185
4.3 MATERIALS AND METHODS ............................................................................................................................. 185
4.3.1 Recombineering ........................................................................................................................................... 185
4.3.2 Transfection and selection of ESCs ....................................................................................................... 187
4.3.3 Mouse colony establishment and CRE induced deletion ............................................................. 188
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4.3.4 Klf14 genotyping .......................................................................................................................................... 188
4.3.5 RT-PCR ............................................................................................................................................................. 190
4.3.6 Mest DMD methylation analysis ............................................................................................................. 190
4.3.7 Embyronic and placental analysis ........................................................................................................ 191
4.4 RESULTS ................................................................................................................................................................... 193
4.4.1 Confirmation of Klf14 null allele ............................................................................................................ 193
4.4.2 Klf14 is an imprinted gene expressed in the placenta .................................................................. 195
4.4.3 Klf14 null mice are viable and fertile ................................................................................................... 199
4.4.4 Overgrowth in homozygous Klf14 null placentas ........................................................................... 200
4.4.5 Placenta layer structure in Klf14 null placentas.............................................................................. 202
4.4.6 Placental lipid content increased by high fat diet in Klf14 null placentas ............................ 203
4.5 DISCUSSION ............................................................................................................................................................. 204
4.5.1 Opposing effects of Klf14 and Mest on placental growth ............................................................. 204
4.5.2 Placental and maternal influence on metabolism .......................................................................... 206
4.5.3 Comparison with other targeted Klf14 models ............................................................................... 206
5.0 OVERALL DISCUSSION ............................................................................................................................. 209
5.1 SUMMARY AND SIGNIFICANCE ....................................................................................................................... 209
5.2 FUTURE DIRECTIONS .......................................................................................................................................... 212
5.2.1 Coevolution of genomic imprinting and placentation .................................................................. 212
5.2.2 Imprint-like sequences .............................................................................................................................. 220
5.2.3 Role of placenta in imprinting disorders ........................................................................................... 221
5.2.4 Reproductive technologies ...................................................................................................................... 225
5.2.5 Mechanisms of imprinting ....................................................................................................................... 229
5.2.6 Role of the Kcnq1 cluster in TGC development................................................................................ 231
5.2. Role of the Peg10 cluster in labyrinth development and fetal viability ................................ 233
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