Epigenetic Gene Regulation During Development and Under Adverse

Environmental Conditions

Marisa Bartolomei, PhD

University of Pennsylvania

Perelman School of Medicine

LEARNING OBJECTIVES

At the conclusion of this presentation, participants should be able to:

● Describe epigenetic gene regulation in the mammalian embryo.

● Describe the role and epigenetic regulation of imprinted genes in development.

● Identify potential epigenetic risks associated with ART.

DISCLOSURE

● No Disclosures

Mammals Have Thousands of Cell Types, Which Originate from Single Genome in the Fertilized Egg

1 Genome Genetic information

obtained from gametes

Cell type specific organized information

DNA

Chromatin

> 1000 Epigenomes

Epigenetics

“Epi”= above, over, outside or beside

Molecular definitions: defined chemical changes to the DNA and the proteins that package DNA

Events that are above and beside genetics; beyond DNA sequence

Epigenetics Vs Genetics

There are Many Levels of Epigenetic Information

Two Main Components to the Epigenetic Code

DNA methylation Histone Modifications

Cytosine Guanine

DNA Methyltransferase

(DNMT) DNAHistone

Histone TailsNucleosome

H3

H2B

H2AH4

Histone tails protrude from the nucleosome and can serve as templatesfor epigenetic modifications

Luger et al., Nature 1997; 389:251-260

Structure of the Nucleosome

Bhaumik et al., Nature Struct Mol Biol 2007; 14:1008-1016

Post-translational modifications of amino acids occur primarily on thehistone tails

Histone Modifications

Chromatin and Gene Expression Euchromatin vs Heterochromatin

Two Main Components to the Epigenetic Code

DNA methylation Histone Modifications

Cytosine Guanine

DNA Methyltransferase

(DNMT) DNAHistone

Histone TailsNucleosome

DNA Methylation

• Mostly occurs at cytosines in CpGs, which are sparsely distributed (70-80% are methylated)

• Under-represented in the genome largely because 5mC is mutagenic –deamination of 5mC generates T

• Generally a repressive mark through preventing binding of transcription factors or assembling repressive chromatin structure

• Dynamically regulated during mammalian development

Most of the Mammalian Genome Reprograms DNA Methylation During Development

Sperm genome:packaged with protaminesOnly 5-15% of histones retained in mature spermatozoa (human)

Post fertilization:Removal of protaminesDeposition of new histones…. Histone chaperones (Hira)

Remodelling Sperm Genome After Fertilization

Barton et al, 2001

Zygotic reprogramming : DNA demethylation

5mC

DAPI

Imprinted genesNon-imprinted genes

Genomic Imprinting is Mammalian-Specific and Results in Monoallelic, Parent-of-Origin-Specific Expression

Gene Direction ofImprint

Function of Gene Product Role in Embryo Growth/Behavior

IGF2 Paternal Positive regulator of growth Growth

H19 Maternal Negative regulator of growth Suppression of growth

IGF2R Maternal Negative regulator of growth Suppression of growth

GRB10 Maternal Negative regulator of growth Suppression of growth

GRB10Paternal (neuron-specific)

Signal adaptor Aggression

UBE3AMaternal(neuron-specific)

Ubiquitin ligase & transcriptional co-activator

Memory, learning, motor function

PEG3 PaternalZinc finger protein; control of apoptosis

Sex-specific behavior

NDN PaternalRegulator of neuronal growth anddifferentiation

Spatial learning; socialization

NESP Maternal Secretory pathway Exploratory behavior

GNAS Maternal Signal transduction Cognition & sleep

Gene Direction ofImprint

Function of Gene Product

Role in Placenta

PEG3 PaternalZinc finger protein; control of apoptosis

Growth

PEG1 Paternal Hydrolase Growth

MASH2 MaternalHelix-loop-helix transcription factor

Spongiotrophoblastdevelopment

PHLDA2 MaternalPleckstrin homology domain protein

Spongiotrophoblastrestriction

CDKN1C Maternal Cell cycle regulatorSpongiotrophoblastrestriction

SLC22A3 Maternal Cation transporter Nutrient transfer

Functions of Imprinted Genes

From Kalish et al, 2014

Imprinted Genes Reside in Clusters and are Regulated by ICRs which are DNA Methylated on a Single Parental Allele

Imprinting control region (ICR)

DNA methylated ICR

ICRs are Differentially Methylated in the Germline and Escape DNA Methylation Reprogramming after Fertilization

In Vitro Fertilization

Angelman Syndrome

Imprinting Disorders Associated with IVF Exhibit Loss of ICR Methylation

Macroglossia

Hemihyperplasia

Organomegaly

Omphalocele

Beckwith-Wiedemann Syndrome

Intellectual disabilitymicrocephalyseizuresataxiaeasily provoked laughter

Nicholls and Knepper

From Jenn Kalish

IVF is associated with an higherthan expected incidence of the imprinting disorders

Beckwith-Wiedemann syndrome and Angelman Syndrome

In Vitro Fertilization

WHY???

Techniques Used in Assisted Reproductive Technologies (ART) Occur When the Genome is Epigenetically Reprogrammed

Vrooman and Bartolomei, Reproductive Toxicology, 2017

The Problem

How can we address whether procedures used in ART disruptepigenetic gene regulation?

Ultimately, our goal is to improve the technology.

Strategy—Use a Mouse Model

Investigate morphology and epigenetic gene regulation

in the mouse embryo and placenta

ET x

transfergestation to blastocystmating

S+ET x

transfergestation to blastocystmatingsuperovulation

IVF

transferculture to blastocystIVFsuperovulation

xNatural

gestation to termmating

ART Paradigm-Can We Associate Specific Procedures with Pathology?

Placental Phenotype at Term-Weight

a

b b

c

one-way ANOVA, p<0.0001

weight (ET, SET, IVF)

a

b b

c

one-way ANOVA, p<0.0001

Lisa Vrooman and Eric de Waal: HMG, 2015

1000 μm

Junctional zone

Labyrinth zone

Natural

EmbryoTransfer

(ET)

Superovulation and Embryo Transfer

(S + ET)

IVF

H&E

Placenta Morphology Worsens with Each Additional Manipulation

a

abbc

c

one-way ANOVA, p<0.0001

Lisa Vrooman and Eric de Waal: HMG, 2015

Placental Phenotype at Term

weight (ET, SET, IVF)

junctional zone (SET and IVF)

Imprinting Control Regions Regulate Imprinting and Exhibit Parental-Allele Specific DNA Methylation

ICR

ICR

Imprinted Genes

ICR=Imprinting Control Region

Unmethylated CpG

Methylated CpG

Maternal Allele

Paternal Allele

C GG C

CH3

CH3

Placental Phenotype at Term-ICR Methylation

Lisa Vrooman and Eric de Waal: HMG, 2015

H1

9/Ig

f2 IC

RP

eg

3 IC

R

Sn

at4

IC

R

weight (ET, SET, IVF)

junctional zone (SET, IVF)

loss of ICR methylation (IVF)

p<0.01

p<0.001p<0.001

Variance ratio test

Placental Phenotype at Term-Global DNA Methylation

Lisa Vrooman and Eric de Waal: HMG, 2015

LUMA Assay

weight (ET, SET, IVF)

junctional zone (SET, IVF)

Loss of ICR methylation (IVF)

Loss of Global DNA methylation (IVF)

CONCLUSIONS I

Placentalweight

JunctionalZone Overgrowth

ICR DNA Methylation

Global DNAMethylation

Aberrant ImprintedGene Exp

ET = = = =

S +ET = = =

IVF

In comparison to naturally conceived groups

Lisa Vrooman and Eric de Waal: HMG, 2015

Natural

ET

S+ET

x

transfergestation to blastocystmating

x

transferculture to blastocystIVFsuperovulation

x

gestation to termmating

transfergestation to blastocystmatingsuperovulation

x

mating culture to blastocyst transferS+EC+ET

S+IVF+EC+ET

ART Paradigm-Can We Associate Specific Procedures with Pathology?

superovulation

CONCLUSIONS II

● Embryonic time course reveals that embryo is initially smaller

● Placenta subsequently overgrows

● Embryo culture alone is no worse than IVF procedure

● Epigenetic abnormalities are associated embryo culture

Lisa Vrooman & Eric Rhon-Calderon

Gametogenesis Preimplantation Development

Assisted Reproductive Technologies (ART) Expose the Early Embryo to Suboptimal Conditions

Superovulation Embryo TransferEmbryo Culture

Modified from Smallwood and Kelsey, 2012

Imprinted genes

Center of Excellence in Environmental Toxicology

Joanne ThorvaldsenChristopher KrappLisa VroomanLaren RiescheAimee JuanSuhee ChangBlake CaldwellNicole Matos-RoblesEric Rhon-CalderonRexxie PrasayaNikita ChoudharyMayra RomeroGid MerzAsha DahiyaDuy Nguyen

Erin FischerOlivia ChaoEric de Waal

Jennifer Kalish (CHOP)

Richard Schultz, Paula Stein Monica Mainigi, Christos Coutifaris, Teri Ord

NIGMS, NICHD, NIEHS, NIDA, NIBIB