Epigenetic mechanisms in cellular reprogramming

Epigenetics and Human Health

Alexander Meissner
Jörn Walter Editors

Epigenetic
Mechanisms
in Cellular
Reprogramming

Tai Lieu Chat Luong


Epigenetics and Human Health

Series Editors
Prof. Dr. Robert Feil
Institute of Molecular Genetics (IGMM)
Genomic Imprinting & Development laboratory
Montpellier
France
Prof. Dr. Joărn Walter
Universitaăt des Saarlandes FR8.4 Biowissenschaften
Dept of Genetics & Epigenetics
Saarbruăcken
Germany
Priv. Doz. Dr. Mario Noyer Weidner
Schwaăbische Str. 3
Berlin
Germany

More information about this series at
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Alexander Meissner ã Joărn Walter
Editors

Epigenetic Mechanisms in
Cellular Reprogramming


Editors
Alexander Meissner
Dpt. of Stem Cell and Regenerative Biol
Harvard University Broad Institute
Bauer Laboratory
Cambridge
Massachusetts
USA

Joărn Walter
Universitaăt des Saarlandes
FR84. Biosciences
Dept. of Genetics & Epigenetics
Saarbruăcken
Germany

ISSN 2191-2262
ISSN 2191-2270 (electronic)
ISBN 978-3-642-31973-0

ISBN 978-3-642-31974-7 (eBook)
DOI 10.1007/978-3-642-31974-7
Springer Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014958446
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Preface

During development the genome of the fertilised egg is utilised to create a whole

organism with a rich diversity of cell types. While the underlying sequence remains
unchanged, each cell type and developmental stage is reflected in a unique
epigenome. This coordinated process of developmental epigenetic programming
begins in the germ line (early primordial germs cells, PGCs) and cumulates in the
creation of the specialised gametes. Post-fertilisation massive epigenetic
reprogramming establishes the totipotent zygote and pluripotent cells of the early
embryo (inner cell mass, ICM). The latter can be explanted into cell culture and
give rise to pluripotent embryonic stem cells (ESCs) that can be maintained over
long periods.
Over the last decade epigenetic reprogramming processes have been widely
studied in the zygote, in PGCs and in ESCs. The research focused on various
aspects of this topic, most of them being reflected in the selected articles of this
volume including (1) understanding reprogramming events at the level of DNA and
histone modifications, (2) the physiological parameters and enzymes that control
the initiation, the entry and exit from pluripotency, and (3) the differences/similarities of epigenetic reprogramming mechanisms in various pluripotent and totipotent
cells.
The detailed knowledge of the underlying reprogramming mechanisms is of
great importance for many research areas in human health and disease ranging from
stem cell biology to cancer. Examples are a controlled understanding of the cell
intrinsic reprogramming mechanisms activated during the in vitro generation of
induced pluripotent stem cells (iPSCs) from somatic cells and the erroneous
reprogramming mechanisms in somatic (stem) cells leading to massive epigenetic
changes in cancer.
This volume compiles a series of articles featuring the current knowledge of
molecular events accompanying processes of epigenetic reprogramming. The articles focus on mechanisms operating during early embryonic development, the
events that are defining the entry into and exit from pluripotency in ESCs and the
implications of such mechanisms for aberrant reprogramming in the course of
cancer. The reader will obtain a detailed view of the molecular changes occurring
v

vi

Preface

at various epigenetic levels of histone and DNA modifications. All articles feature
references to the important discoveries in the field over the last decade. A glossary
at the end will help the reader to navigate through many of the specific terms used in
epigenetic research.
Cambridge, MA
Saarbruăcken, Germany

Alex Meissner
Joărn Walter


Glossary

Acetylation The introduction, via an enzymatic reaction, of an acetyl group to an
organic compound, for instance to histones or other proteins.
Agouti gene The agouti gene (A) controls fur colour through the deposition of
yellow pigment in developing hairs. Several variants of the gene exist, and for one
of these (Agouti Variable Yellow, Avy) the expression levels can be heritably
modified by DNA methylation.
Alleles Different variants or copies of a gene. For most genes on the chromosomes, there are two copies: one copy inherited from the mother, the other from the
father. The DNA sequence of each of these copies may be different because of
genetic polymorphisms.
Assisted reproduction technologies (ART) The combination of approaches that
are being applied in the fertility clinic, including IVF and ICSI.
5-Azacytidine A cytidine analogue in which the 5 carbon of the cytosine ring has

been replaced with nitrogen. 5-azacytidine is a potent inhibitor of mammalian DNA
methyltransferases.
Bivalent chromatin A chromatin region that is modified by a combination of
histone modifications such that it represses gene transcription, but at the same time
retains the potential of acquiring gene expression.
Bisulphite genomic sequencing A procedure in which bisulphite is used to
deaminate cytosine to uracil in genomic DNA. Conditions are chosen so that
5-methylcytosine is not changed. PCR amplification and subsequent DNA sequencing reveal the exact position of cytosines which are methylated in genomic DNA.
Blastocyst The blastocyst is a structure formed in the early development of
mammals. It is the last stage of preimplantation development in mammals and it
is comprised of outer cell layer—trophoblast, which later develops into placenta,
and of inner cell mass (see ICM), which gives rise to the embryonic tissues. ICM is
vii


viii

Glossary

attached to inner side of the hollow basket-shaped structure, formed by
trophectoderm (trophoblast cell layer).
Bromo domain Protein motif found in a variety of nuclear proteins including
transcription factors and HATs involved in transcriptional activation. Bromo
domains bind to histone tails carrying acetylated lysine residues.
Brno nomenclature Regulation of the nomenclature of specific histone modifications formulated at the Brno meeting of the NoE in 2004. Rules are:
<Histone><amino acid position><modification type><type of modification>.
Example: H3K4me3 ¼ trimethylated lysine-4 on histone H3.
Cell fate The programmed path of differentiation of a cell. Although all cells
have the same DNA, their cell fate can be different. For instance, some cells
develop into brain, whereas others are the precursors of blood. Cell fate is determined in part by the organisation of chromatin—DNA and the histone proteins—in

the nucleus.
Cellular Memory (epigenetic) Specific active and repressive organisations of
chromatin can be maintained from one cell to its daughter cells. This is called
epigenetic inheritance and ensures that specific states of gene expression are
inherited over many cell generations.
ChIP

see chromatin immunoprecipitation.

ChIP on chip After chromatin immunoprecipitation, DNA is purified from the
immunoprecipitated chromatin fraction and used to hybridise arrays of short DNA
fragments representing specific regions of the genome.
ChIP Seq Sequencing of the totality of DNA fragments obtained by ChIP to
determine their frequency and position on the genome. Sequencing is usually
preceded by PCR amplification of ChIP-derived DNA to increase its amount.
Chromatid In each somatic cell generation, the genomic DNA is replicated in
order to make two copies of each individual chromosome. During M phase of the
cell cycle, these copies—called chromatids—are microscopically visible one next
to the other, before they get distributed to the daughter cells.
Chromatin The nucleo-protein complex constituting the chromosomes in
eukaryotic cells. Structural organisation of chromatin is complex and involves
different levels of compaction. The lowest level of compaction is represented by
an extended array of nucleosomes.
Chromatin remodelling Locally, the organisation and compaction of chromatin
can be altered by different enzymatic machineries. This is called chromatin
remodelling. Several chromatin remodelling proteins move nucleosomes along
the DNA and require ATP for their action.


Glossary

ix

Chromo domain (chromatin organisation modifier domain) Protein–protein
interaction motif first identified in Drosophila melanogaster HP1 and polycomb
group proteins. Also found in other nuclear proteins involved in transcriptional
silencing and heterochromatin formation. Chromo domains consist of approx.
50 amino acids and bind to histone tails that are methylated at certain lysine
residues.
Chromosomal domain In higher eukaryotes, it is often observed that in a specific
cell type, chromatin is organised (e.g. by histone methylation) the same way across
hundreds to thousands of kilobases of DNA. These ‘chromosomal domains’ can
comprise multiple genes that are similarly expressed. Some chromosomal domains
are controlled by genomic imprinting.
Chromatin immunoprecipitation (ChIP) Incubation of chromatin fragments
comprising one to several nucleosomes, with an antiserum directed against particular (histone) proteins or covalent modifications on proteins. After ChIP, the
genomic DNA is purified from the chromatin fragments brought down by the
antiserum and analysed.
CpG dinucleotide A cytosine followed by a guanine in the sequence of bases of
the DNA. Cytosine methylation in mammals occurs at CpG dinucleotides.
CpG island A small stretch of DNA, of several hundred up to several kilobases in
size, that is particularly rich in CpG dinucleotides and is also relatively enriched in
cytosines and guanines. Most CpG islands comprise promoter sequences that drive
the expression of genes.
Cytosine methylation In mammals, DNA methylation occurs at cytosines that
are part of CpG dinucleotides. As a consequence of the palindromic nature of the
CpG sequence, methylation is symmetrical, i.e. affects both strands of DNA at a
methylated target site. When present at promoters, it is usually associated with
transcriptional repression.
Deacetylation The removal of acetyl groups from proteins. Deacetylation of

histones is often associated with gene repression and is mediated by histone
deacetylases (HDACs).
DNA demethylation Removal of methyl groups from DNA. This can occur
‘actively’, i.e. by an enzymatically mediated process, or ‘passively’, when methylation is not maintained after DNA replication.
‘de novo’ DNA methylation The addition of methyl groups to a stretch of DNA
which is not yet methylated (acquisition of ‘new’ DNA methylation).
DNA methylation A biochemical modification of DNA resulting from addition
of a methyl group to either adenine or cytosine bases. In mammals, methylation is
essentially confined to cytosines that are in CpG dinucleotides. Methyl groups can
be removed from DNA by DNA demethylation.


x

Glossary

DNA methyltransferase Enzyme which puts new (de novo) methylation onto the
DNA, or which maintains existing patterns of DNA methylation.
Dosage compensation The X chromosome is present in two copies in the one
sex, and in one copy in the other. Dosage compensation ensures that in spite of the
copy number difference, X-linked genes are expressed at the same level in males
and females. In mammals, dosage compensation occurs by inactivation of one of
the X chromosomes in females.
Embryonic stem (ES) cells Cultured cells obtained from the inner cell mass of
the blastocyst, and for human ES cells, possibly also from the epiblast. These cells
are pluripotent; they can be differentiated into all different somatic cell lineages.
ES-like cells can be obtained by dedifferentiation in vitro of somatic cells (see iPS
cells).
Endocrine disruptor A chemical component which can have an antagonistic
effect on the action of a hormone (such as on oestrogen) to which it resembles

structurally. Some pesticides act as endocrine disruptors and have been found in
animal studies to have adverse effects on development, and for some, to induce
altered DNA methylation at specific loci. A well-characterised endocrine disruptor
is Bisphenol-A, a chemical used for the productions of certain plastics.
Enhancer A small, specialised sequence of DNA which, when recognised by
specific regulatory proteins, can enhance the activity of the promoter of a gene
(s) located in close vicinity.
Epi-alleles Copies of a DNA sequence or a gene which differ in their epigenetic
and/or expression states without the occurrence of a genetic mutation.
Epiblast The population of cells in the inner cell mass (see ICM) of a mammalian
blastocyst. It is formed when ICM develops into the embryonic disc, consisting of
two layers: the adjacent to the trophoblast epiblast and the adjacent the blastocoele
(blastocyst cavity) hypoblast.
Epigenesis The development of an organism from fertilisation through a
sequence of steps leading to a gradual increase in complexity through differentiation of cells and formation of organs.
Epigenetics The study of heritable changes in gene function that arise without an
apparent change in the genomic DNA sequence. Epigenetic mechanisms are
involved in the formation and maintenance of cell lineages during development,
and, in mammals, in X-inactivation and genomic imprinting, and are frequently
perturbed in diseases.
Epigenetic code Patterns of DNA methylation and histone modifications can
modify the way genes on the chromosomes are expressed. This has led to the
idea that combinations of epigenetic modifications can constitute a code on top of
the genetic code which modulates gene expression.


Glossary

xi

Epigenetic inheritance The somatic inheritance, or inheritance through the germ
line, of epigenetic information (changes that affect gene function, without the
occurrence of an alteration in the DNA sequence).
Epigenetic marks Regional modifications of DNA and chromatin proteins,
including DNA methylation and histone methylation, that can be maintained from
one cell generation to the next and which may affect the way genes are expressed.
Epigenetic reprogramming The resetting of epigenetic marks on the genome so
that these become like those of another cell type, or of another developmental stage.
Epigenetic reprogramming occurs for instance in primordial germ cells, to bring
them back in a ‘ground state’. Epigenetic reprogramming and dedifferentiation also
occur after somatic cell nuclear transfer.
Epigenome The epigenome is the overall epigenetic state of a particular cell. In
the developing embryo, each cell type has a different epigenome. Epigenome maps
represent the presence of DNA methylation, histone modification and other chromatin modifications along the chromosomes.
Epigenotype The totality of epigenetic marks that are found along the DNA
sequence of the genome in a particular cell lineage or at a particular developmental
stage.
Epimutation A change in the normal epigenetic marking of a gene or a regulatory DNA sequence (e.g. a change in DNA methylation) which affects gene
expression.
Euchromatin A type of chromatin which is lightly staining when observed
through the microscope at interphase. Euchromatic chromosomal domains are
loosely compacted and relatively rich in genes. The opposite type of chromatin
organisation is heterochromatin.
Genomic imprinting An epigenetic phenomenon which affects a small subset of
genes in the genome and results in mono-allelic gene expression in a parent-oforigin dependent way (for a given pair of alleles uniformly either the maternally or
paternally derived copy is active).
Germ line specific stem cells Cells derived from undifferentiated germ cells
which can be maintained without alterations in their characteristics through many
cell divisions.
Heterochromatin A type of chromatin which is darkly staining when observed

through the microscope at interphase. Heterochromatic chromosomal domains,
found in all cell types, are highly compacted, are rich in repeat sequences, and
show little or no gene expression. Extended regions of heterochromatin are found
close to centromeres and at telomeres.
Histone acetylation Post-translational modification of the ε-amino group of
lysine residues in histones catalysed by a family of enzymes called histone
acetyltransferases (HATs). Acetylation contributes to the formation of


xii

Glossary

decondensed, transcriptionally permissive chromatin structures and facilitates
interaction with proteins containing bromo domains.
Histone acetyltransferase (HAT)
amino acids on histone proteins.

An enzyme that acetylates (specific) lysine

Histone code Theory that distinct chromatin states of condensation and function
are marked by specific histone modifications or specific combinatorial codes (see
also epigenetic code).
Histone deacetylase (HDAC) An enzyme that removes acetyl groups from
histone proteins. This increases the positive charge of histones and enhances their
attraction to the negatively charged phosphate groups in DNA.
Histone demethylase (HDM) Proteins catalysing the active enzymatic removal
of methyl groups from either lysine or arginine residues of histones. Prominent
examples are LSD1 and Jumonji proteins.
Histone methylation Post-translational methylation of amino acid residues in

histones catalysed by histone methyltransferases (HMTs). Histone methylation is
found at arginine as mono- or di-methylation and lysine as mono-, di- or
tri-methylation. Modifications are described depending on the position and type
of methylation (mono, di, tri-methylation) according to the Brno nomenclature.
Different types of methylation can be found in either open trancriptionally active or
silent (repressive) chromatin (histone code). Methylated lysine residues are
recognised by proteins containing chromo domains.
Histone methyltransferase (HMT) Enzymes catalysing the transfer of methyl
groups from S-adenosyl-methionine (SAM) to lysine or arginine residues in
histones.
Intracytoplasmic sperm injection (ICSI) Capillary-mediated injection of a
single sperm into the cytoplasm of an oocyte followed by activation to promote
directed fertilisation.
Imprinted genes Genes that show a parent-of-origin specific gene expression
pattern controlled by epigenetic marks that originate from the germ line.
Imprinting

see genomic imprinting.

Imprinted X-inactivation Preferential inactivation of the paternal X chromosome in rodents (presumably also humans) during early embryogenesis and in the
placenta of mammals.
Imprinting control region (ICR) Region that shows germ line derived parentof-origin dependent epigenetic marking which controls the imprinted expression of
neighbouring imprinted genes.
Inner cell mass (ICM) Cells of the inner part of the blastocyst forming the
embryo proper. Inner cell mass cells are the source for ES cells.


Glossary

xiii

Induced pluripotent stem cells (iPS) Cells with an ES cell-like pluripotent
potential derived from differentiated somatic cells by in vitro reprogramming.
Reprogramming is triggered by the activation of pluripotency factor genes and
cultivation in ES cell medium. iPS cells are capable to generate all cell types of an
embryo.
In vitro Fertilisation (IVF) Fertilisation of a surgically retrieved oocyte in the
laboratory, followed by a short period of in vitro cultivation before the embryo is
transferred back into the uterus to allow development to term.
Isoschizomers Restriction enzymes from different bacteria which recognise the
same target sequence in DNA. Often these enzymes respond differently to methylation of bases within their target sequence, which may make them important tools
in DNA methylation analysis. Thus, MspI cuts both CCGG and C5mCGG, whereas
HpaII cuts only the unmethylated sequence.
Locus control region (LCR) Region marked by insulator functions and DNase
hypersensitive sites. LCRs contain binding sites for insulator proteins and enhancer
binding proteins. LCRs control the domain-specific developmentally regulated
expression of genes by long-range interactions with gene promoters.
Maintenance methylation Process that reproduces DNA methylation patterns
between cell generations. Depends in mammals critically (though not exclusively)
on the activity of the ‘maintenance DNA methyltransferase’ Dnmt1. This enzyme
preferentially methylates hemimethylated CpG sites, generated by replication of
symmetrically methylated CpG sequences (see Cytosine methylation), while originally unmethylated sites remain unmethylated upon replication.
Maternal effects Long-term effects on the development of the embryo triggered
by factors in the cytoplasm of the oocyte.
Metastable epiallele Loci, whose epigenetic state is particularly labile, i.e. prone
to be epigenetically modified in a variable and reversible manner. As a consequence
of this lability, various phenotypes may derive from genetically identical cells,
resulting in phenotypic mosaicism between cells (variegation) and also between
individuals (variable expressivity).
Methyl-binding domain (MBD) Protein domain in Methyl-CpG-binding proteins (MBPs) responsible for recognising and binding to methylated cytosine

residues in DNA. Proteins containing MBDs form a specific family of proteins
with various molecular functions.
Methyl-CpG-binding proteins (MBPs) Proteins containing domains (such as
MBD) binding to 5-methyl-cytosine in the context of CpG dinucleotides. MBPs
mostly act as mediators for molecular functions such as transcriptional control or
DNA repair.
Non-coding RNA (ncRNA) RNA transcripts that does not code for a protein.
ncRNA generation frequently involves RNA processing.


xiv

Glossary

Non-Mendelian inheritance Inheritance of genetic traits that do not follow
Mendelian rules and/or cannot be explained in simple mathematically modelled
traits.
Nuclear periphery Region around the nuclear membrane characterised by contacts of the chromosomes with the nuclear lamina.
Nuclear (chromosomal) territory Cell type-specific areas within the nucleus
occupied by specific chromosomes during interphase (G1).
Nucleolus Specific compartments within the nucleus formed by rDNA repeat
domains. Nucleoli are marked by specific heterochromatic structures and active
gene expression.
Nucleosome Fundamental organisational unit of chromatin consisting of
147 base pairs of DNA wound around a histone octamer.
Oogenesis The process by which primary oocyte develops into mature ovum. In
mammals primary oocytes are formed shortly before or shortly after the birth during
the process called oocytogenesis.
Parthenogenesis A form of asexual reproduction in which growth and development of embryos occur without fertilisation, with only oocyte genome (in some
very rare cases—only sperm genomes) contributing to the embryonic genotype.

This form of reproduction occurs naturally in different plant, as well as animal (both
invertebrates and vertebrates) species, but not in mammals. The mammalian egg
can be artificially induced to undergo parthenogenetic development, but the
resulting embryos are not capable of developing to term due to the restrictions
imposed by genomic imprinting (see also: Genomic Imprinting).
Pluripotency Capacity of stem cells to form all cell types of an embryo including
germ cells but not extraembryonic lineages (see Totipotency).
Polycomb group proteins Epigenetic regulator proteins forming multiprotein
complexes (PRCs ¼ polycomb repressive complexes). Polycomb group proteins
possess enzymatic properties to control the maintenance of a suppressed state of
developmentally regulated genes, mainly through histone methylation and
ubiquitination.
Position effect variegation (PEV) A type of clonally heritable variability of gene
expression which relies on epigenetic lability (see also metastable epialleles)
associated with the particular position of a gene within the genome. PEV has first
been observed in the context of gene translocations from euchromatic to heterochromatic environments and is a consequence of variable formation of heterochromatin across the respective locus. PEV may give rise to patches of cells with altered
expression profiles. A classical example is represented by certain mutations in
Drosophila leading to variegated eye pigmentation (‘mottled eyes’).


Glossary

xv

Primordial germ cell Mammalian cells set aside during early embryogenesis
which migrate through the hind gut of the developing mammalian embryo into the
‘Gonadenanlagen’ to form founder cells of the latter germ line.
Pronucleus The haploid nucleus, which is formed from sperm or oocyte genomes
upon the fertilisation and formation of a zygote (see Zygote). The sperm genome is
transformed into paternal pronucleus; the maternal pronucleus originates from the

oocyte genome. Both paternal and maternal pronuclei exist within the same
ooplasm and parental chromosome remains separated until first metaphase stage.
Protamines Small, arginine-rich proteins that replace histones late in the haploid
phase of spermatogenesis (during spermiogenesis). They are thought to be essential
for sperm head condensation and DNA stabilisation. After fertilisation protamines
are removed from paternal chromosomes in the mammalian zygote.
RNA interference (RNAi) Post-transcriptional regulatory effects on mRNAs
(control of translation or stability) triggered by processed ds and ss small RNA
(si-, mi-, piRNAs) molecules. Effects are propagated by enzymatic complexes such
as RISC containing the small RNAs bound by Argonaute proteins.
SAHA Suberoylanilide hydroxamic acid, an inhibitor of certain histone
deactylases, leading to enhanced levels of histone acetylation. See also TSA.
S-adenosylhomocysteine (SAH) Hydrolysed product formed after the methylation reaction catalysed by DNA and histone methyltransferases using SAM as
methyl group donor. SAH is a competitive inhibitor of SAM for most
methyltransferases.
S-adenosyl methionine (SAM) A cofactor for all DNA (DNMTs) and histone
methyltransferases (HMTs) providing the methyl group added to either cytosines
(DNA) or histones (arginine or lysine).
SET domain A domain found in virtually all lysine-specific histone
methyltransferases (HMTs). A protein–protein interaction domain required for
HMT activity and modulation of chromatin structure, frequently associated with
cysteine-rich Pre-SET and Post-SET domains.
Silencer Element in the DNA to which proteins bind that inhibit transcription of a
nearby promoter. Silencer elements are recognised and bound by silencer proteins.
siRNAs small interfering RNAs, RNAs in the size range of 21–24 nucleotides
derived from double-stranded long RNAs cleaved by Dicer. siRNAs are incorporated into the RISC complex to be targeted to complementary RNAs to promote
cleavage of these mRNAs.
Somatic cell nuclear transfer (SCNT) Transfer of the nucleus of a somatic cell
into an enucleated oocyte using a glass capillary to form an SCNT zygote. After
activation of the zygote the genome of the nucleus derived from the somatic cells

becomes reprogrammed to start development.


xvi

Glossary

Spermatogenesis The process by which spermatogonia develop into mature
spermatozoa. Spermatozoa (sperm) are the mature male gametes. Thus, spermatogenesis is the male version of gametogenesis.
Spermiogenesis The final stage of spermatogenesis which sees the maturation of
spermatids into mature, motile spermatozoa (sperm). During this stage, cells no
longer divide and undergo a major morphological transformation. In addition, at
most of the genome, histone proteins are replaced by the more basic protamines.
Stem Cell Non-committed cell which has the capacity to self-renew and divide
many times giving rise to daughter cells which maintain the stem cell function.
Stem cells have the property to differentiate into specialised cells.
Totipotency Capacity of stem cells to produce all cell types required to form a
mammalian embryo, i.e. embryonic and extraembryonic cells (see Pluripotency).
Totipotent cells are formed during the first cleavages of the embryo.
TSA

Trichostatin-A, an inhibitor of certain types of histone deacetylases.

Trithorax group proteins Proteins containing a trithorax like bromo domain:
they are usually involved in recognising histone modifications marking transcriptionally active regions and contribute to maintenance of activity.
Trophoblast

Cells of the blastoderm forming the placental tissues in mammals.

Uniparental Disomy The occurrence in the cell of two copies of a chromosome,

or part of a chromosome, that are identical and of the same parental origin.
X-chromosome inactivation Epigenetically controlled form of dosage compensation in female mammals resulting in transcriptional silencing of genes on surplus
X chromosomes. X-chromosome inactivation is triggered by the non-coding RNA
Xist and manifested by various epigenetic modifications including histone methylation, histone deacetylation and DNA methylation.
Zygote The earliest developmental stage of an embryo. Results from the fusion of
maternal (oocyte) and paternal (sperm) haploid gametes. This stage is often called
‘one-cell embryo’ stage.


Contents

The Oocyte Determinants of Early Reprogramming . . . . . . . . . . . . . . .
Caroline Schwarzer and Michele Boiani

1

Stella and Zygotic Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Toshinobu Nakamura and Toru Nakano

31

Histone Variants and Reprogramming in Early Development . . . . . . . .
Ana Bosˇkovic´ and Maria-Elena Torres-Padilla

43

DNA Methylation Reprogramming in Preimplantation
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Konstantin Lepikhov, Julia Arand, Sarah Fuchs, Jie Lan, Mark Wossidlo,
and Joărn Walter

69

Establishing and Maintaining Pluripotency: An Epigenetic
Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Jing Liao and Alexander Meissner
Epigenetic Regulation of Pluripotency by Polycomb
Group Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Achim Breiling
PRC1-Mediated Gene Silencing in Pluripotent ES Cells: Function and
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Matthias Becker, Nancy Mah, Daniela Zdzieblo, Xiaoli Li, Arvind Mer,
Miguel A. Andrade-Navarro, and Albrecht M. Muăller

xvii


xviii

Contents

The Biology and Genomic Localization of Cytosine
Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Gordon R. McInroy, Neil M. Bell, Gabriella Ficz, Shankar Balasubramanian,
Wolf Reik, and Eun-Ang Raiber
Epigenetic Reprogramming in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Anders M. Lindroth, Yoon Jung Park, and Christoph Plass
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

The Oocyte Determinants of Early
Reprogramming
Caroline Schwarzer and Michele Boiani

Abstract The oocyte is the female germ cell specialized for processing the sperm
genome as well as the only cell in the adult body that can convert, i.e., reprogram,
the genome of a donor somatic cell from a differentiated to a totipotent state. One of
the big open questions in this field pertains to the identity of the natural components
of the oocyte that can achieve nuclear reprogramming. We would like to call them
determinants of reprogramming. In our view the experimental pursuit of these
determinants must be preceded by a review of the oocyte’s properties. These can
be ascribed to qualitative and quantitative traits such as size, nuclear architecture,
cytoplasm-to-nucleus ratio, and molecular makeup of the oocyte. In addition, the
oocyte’s ability to achieve fast and full reprogramming suggests that the nuclear
and/or cytoplasmic molecules in charge of the reprogramming process are abundant. We hypothesize that the reason for such abundance may be simple: these
molecules are normally used to process the sperm genome upon fertilization and are
repurposed for reprogramming. Among these molecules, maternal-effect factors
including transcription factors and chromatin remodeling factors may prime the
reprogramming process and determine its initial speed. Here, we discuss known and
putative factors involved in reprogramming, such as DJ-1, Brg1, Oct4, Glis1, and
Tctp1, that were identified by candidate gene, transcriptomic, and proteomic
approaches. Shedding light on the natural network of reprogramming factors
found in the oocyte will help reveal general principles of cell rejuvenation for the
benefit of aging studies and regenerative medicine.

1 Controversies Surrounding Oocyte Reprogramming
After the discovery of induced pluripotent stem (iPS) cells, which arise after forced
expression of transcription factors such as Oct4, Sox2, Klf4, c-Myc, and Lin28 in
somatic cells (Han et al. 2008; Takahashi and Yamanaka 2006; Yu et al. 2007),
somatic cell nuclear transfer (SCNT) into oocytes has been questioned as being an

obsolete and ethically questionable means of reprogramming. There is little doubt

C. Schwarzer (*) • M. Boiani
Max Planck Institute for Molecular Biomedicine, Mouse Embryology Lab, Muănster, Germany
e-mail: ;
© Springer-Verlag Berlin Heidelberg 2015
A. Meissner, J. Walter (eds.), Epigenetic Mechanisms in Cellular Reprogramming,
Epigenetics and Human Health, DOI 10.1007/978-3-642-31974-7_1

1


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C. Schwarzer and M. Boiani

that medical/clinical progress will be based on iPS cells, since this new technology
circumvents ethical concerns that are associated with the use of human oocytes and
cloned embryos. Mouse iPS cells have recently been differentiated into oocyte-like
cells capable of yielding healthy offspring after fertilization (Hayashi et al. 2012).
SCNT into oocytes is inefficient and flawed compared with sexual reproduction
(in mice, only up to 3 % of cloned embryos implant and develop to healthy
offspring (Wakayama et al. 1998; Wakayama and Yanagimachi 2001), but not
when compared with other reprogramming platforms, including iPS cells. In regard
to the iPS-SCNT comparison, one should not overlook the fact that the coding
transcripts of the iPS cell factors Lin28, Oct4, Sox2, Klf4, and c-Myc are actually
found in oocytes (de Vries et al. 2008; Yan et al. 2010) and that the gold standard of
nuclear reprogramming is to go all the way back to the most undifferentiated state,
i.e., that of totipotency. The achievement of totipotency is, to date, only possible
through the developmental system of an oocyte fertilized by one sperm, or an

ooplasm transplanted with one somatic nucleus, although this might change if the
reports of totipotent embryonic stem (ES) cells receive further substantiation
(Macfarlan et al. 2012). The full reprogramming achieved with oocytes suggests
that the underlying molecular process uses either the same molecular machinery
that supports development after fertilization or a dedicated machinery. Should the
former scenario apply, it would logically follow that reprogramming is not flawed
but simply different because the starting point is also different: differing initial gene
expression signatures and somatic chromatin packed with histones, instead of
sperm DNA packed with protamines. The different starting point may lead to
what many investigators call incomplete reprogramming (Hemberger et al. 2009;
Oswald et al. 2000; Peat and Reik 2012). Should other machinery than those for
sperm processing apply, it would follow that there is some hidden evolutionary
advantage in oocytes preserving the molecular machinery that they seldom use:
SCNT takes place in the laboratory, not in nature.
In this chapter, we elaborate on how oocytes accomplish reprogramming and
which oocytic molecules may be involved in the process. We focus specifically on
mouse oocytes, which have provided precious information on reprogramming in a
mammalian system. We choose to concentrate on early reprogramming from SCNT
to blastocyst, which comprises the time frame of preimplantation development.
This includes the rise of the founder cell lineages (inner cell mass, ICM, and
trophectoderm, TE). The reason behind this focus is that we are interested in
what the oocyte can accomplish in vitro on its own without support from the female
genital tract (reproductive cloning) or from complex in vitro substrates, such as
those used for embryonic stem (ES) cell derivation. It is known that different
somatic nuclei are differently amenable to reprogramming (Fulka et al. 2001;
Gurdon and Wilmut 2011). However, this aspect is superimposed with the heterogeneity of oocytes in regard, for example, to their content in coding transcripts for
the iPS factors (Pfeiffer et al. 2010). It has been shown that nuclear transfer
(NT) with nuclei from more advanced differentiation stages leads to a decrease in
developmental potential of derivative cloned embryos. For example, standard and
serial NT experiments with nuclei of blastomeres of different cleavage stages of

The Oocyte Determinants of Early Reprogramming

3

mouse embryos showed that the differentiation stage of the donor embryos
influences the developmental competence of cloned embryos (Ono and Kono
2006). Specifically, from the 8-cell/morula stage on, a barrier to reprogramming
was apparent from the ability of derivative embryos to yield cloned pups after
embryo transfer in vivo (Ono and Kono 2006).
In this chapter, we approach the molecular mechanisms of reprogramming on
the transcriptional and translational level of gene expression regulation so as to
tackle the elusive reprogramming factors of the oocyte.

2 A Brief History of SCNT
The reason for performing SCNT into oocytes has its roots in the investigation of
the concept of the Weismann barrier (Weismann 1893), that is, the separation of
germ line and soma, and of the role of genome loss in the process of cell differentiation. Almost 60 years ago, when the pioneers of nuclear transfer experiments
paved the way for the modern cloning experiments it was hardly conceivable that
cell differentiation could be reversed, despite the known ability of the oocyte
to process the sperm chromatin and to be totipotent. The first evidence for
reprogramming was provided from the transplantation of nuclei from blastula
cells into enucleated frog (Rana pipiens) oocytes, resulting in normal swimming
tadpoles (Briggs and King 1952). Soon after this achievement, similar studies were
conducted in other species such as Xenopus laevis in the 1950s (Gurdon et al. 1958)
and 1960s (Gurdon 1962a, b). These early nuclear transfer experiments confirmed
that normal development could be supported by the nuclei of differentiated cells;
however, in most cases these cells were not terminally differentiated. Furthermore,
amphibian tissues are capable of natural regeneration, raising the possibility that the

successful cloning of Xenopus laevis could reflect the serendipitous use of native
stem cells as nucleus donors. For this reason, the occurrence of reprogramming in
an experimental setting was disputed until it reached to mammals and was tested
on terminally differentiated cells. The process of experimental dedifferentiation
to totipotency was proven when in 1997 the nucleus from an udder cell was
transplanted into an enucleated sheep oocyte, which resulted in the birth of a
healthy cloned sheep after embryo transfer. Notably, it was also clear that epigenetic change of the genome was reversible (Wilmut et al. 1997). In fact, only
genetic changes such as the loss of chromosomal regions are irreversible. However,
these changes do not necessarily preclude full development after SCNT, as shown
by the cloned monoclonal mouse derived from mature T and B cells (Hochedlinger
and Jaenisch 2002). Now, 17 years after Dolly the cloned sheep (Wilmut et al.
1997) and 16 years after Cumulina the cloned mouse (Wakayama et al. 1998),
SCNT is firmly established also in human oocytes (Tachibana et al. 2013; Chung
et al. 2014; Yamada et al. 2014). Although the ultimate proof of full development is
precluded for ethical reasons, the derivation of diploid ES cell lines from cloned
human embryos is a milestone.


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C. Schwarzer and M. Boiani

3 The Uniqueness of the Oocyte: More than Totipotency
The hypothesis that the molecular machinery in charge of processing the sperm
nucleus after fertilization is repurposed for somatic nuclear reprogramming warrants a concise review of the key properties of mammalian oocytes.
The mammalian oocyte is a female germ cell specialized for reproduction. Its
purpose is to become fertilized by sperm and to start up development, which, upon
implantation, leads to a viable fetus and then a newborn comprised of billions of
cells. Upon fertilization, the embryo undergoes cleavages and forms the founder
cell lineages, the ICM and TE, of which the former develops into the embryonic

germ layers: endoderm, mesoderm, and ectoderm. Pluripotent stem cells can be
derived from culturing the ICM of blastocyst stage embryos under appropriate
conditions. These cells, known as ES cells, retain some of the properties of the
ICM while also encompassing intermediate pluripotent states, which have been
shown to correspond to certain stages of embryonic development, such as naăve and
primed pluripotent states (Hayashi et al. 2008; Nichols and Smith 2009; 2011;
O’Leary et al. 2012). While there are no totipotent cell lines as of yet, individual ES
cells with features of totipotency have been identified within the population of
mouse ES cells (Macfarlan et al. 2012).
Occasionally, the requirement of fertilization as a trigger for development is
bypassed and oocytes become activated without sperm (parthenogenetic activation). Due to lack of paternal contribution, these embryos have karyotypic and gene
expression imbalances, particularly of imprinted genes. As a result, parthenogenetic
embryos can implant but fail to develop to term. ES cells derived from the ICM
of parthenotes can form tumors (teratomata) in vivo (teratoma assay), which are
comprised of all kinds of tissues, albeit with a bias towards certain tissues
(Newman-Smith and Werb 1995). This ability shows that oocytes fulfill the basic
requirements for totipotency even in the absence of the paternal genome. These
properties make the oocyte one of the most unique cells in the body, and also one of
the most valuable: their ovarian stock is limited to finite numbers set aside before
birth, and there is no significant de novo oogenesis taking place thereafter (Tilly
et al. 2009).
When it comes to reprogramming, the uniqueness of the oocyte can be ascribed
to its qualitative and quantitative traits, such as size, chromatin configuration within
the oocyte nucleus (called germinal vesicle, GV), cytoplasm-to-nucleus ratio,
molecular makeup, and perhaps most importantly, endowment in maternal-effect
factors including histones (Li et al. 2010; Wen et al. 2014; Shinagawa et al. 2014).
These factors are qualified as maternal effect because the phenotype of the derivative embryo depends on the genotype of the oocyte and not on the genotype of the
embryo itself. The discovery of these factors was possible in part through advanced
transcriptomic and proteomic studies performed on oocytes. For example, three
protein families were identified that are specific for mouse oocytes but are not

present in ES cells (Wang et al. 2010). These families include, among others,
the ARF family (ADP ribosylation factor family), the Nlrp family (NACHT,


The Oocyte Determinants of Early Reprogramming

5

leucine-rich repeat, and PYD-containing family), which includes Mater (Nlrp5), the
TUDOR family, and the F-BOX family (Wang et al. 2010). Together, these factors
may influence reprogramming and its kinetics from the very start. The relevant
cytoplasmic or nuclear factors are considered to be abundant, because oocytemediated nuclear reprogramming is fast. Reprogramming also requires additional
molecular machinery to govern epigenetic remodeling as well as the regulation of
gene expression during preimplantation development. Epigenetic modifications
include, but are not limited to, DNA methylation and histone acetylation, phosphorylation, and methylation.

4 Qualitative Aspects of Oocyte-Mediated Nuclear
Reprogramming
4.1

Characteristic Traits of the Oocyte Are Species Specific

Oocytes from different species differ in size and morphology, as well as in the
experimental approach to SCNT. The oocyte diameter is approximately 70 μm in
mice, 100 μm in humans, and 125 μm in bovine and can be as large as 1.2 mm in
amphibians (e.g., Xenopus laevis). Therefore, it is not surprising that amphibian
oocytes were so attractive for early manipulation and biochemical studies, and that
first vertebrates to be cloned via SCNT were frogs (Gurdon 1962a, b). The technique of SCNT differs in different species: for example, in amphibians, physical
removal of the maternal chromosomes (enucleation) is not required since these
chromosomes can be inactivated by UV irradiation (functional enucleation). In

mammals, the maternal chromosomes are aspirated from the oocytes using a
micropipette. In domestic animals, such as sheep, cow, and pig, however, the
donor nuclei are electrofused with the ooplasm, whereas in mice the donor nuclei
are typically microinjected into the ooplasm. Moreover, the oocyte’s genotype,
which is very consistent in mice owing to the existence of inbred strains, was shown
to have an effect on cloned embryo development (Gao et al. 2003). For example, in
amphibians, the oocyte is very large in size and therefore several hundred nuclei
can be transferred into the GV of one oocyte in meiotic prophase I (Byrne
et al. 2003). In this setting, reprogramming seems to occur without oocyte DNA
replication and transcription. In almost all mammalian SCNT experiments, metaphase II (MII)-stage oocytes are used as recipients for single nuclei. Mere SCNT
into the oocyte is not sufficient but activation is also required, which is normally
achieved in mice by the addition of strontium chloride (SrCl2) to the calcium-free
culture medium. The activated oocyte then undergoes cleavage and preimplantation
development to the blastocyst stage. Although reprogramming is necessary, it is not
sufficient because the resulting preimplantation embryos require in vitro culture,
which has been shown to influence cloning success (Boiani et al. 2005).


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4.2

C. Schwarzer and M. Boiani

Transcriptional Activity in Germinal Vesicle-Stage
Oocytes

There is increasing evidence that the chromatin configuration of the GV-stage
oocyte is of great importance to the developmental potential of an embryo. The
GV chromatin configuration of fully grown oocytes of many mammalian species

correlates strongly with their transcriptional activity (Christians et al. 1999). In a
subset of GV oocytes, for example, the chromatin is condensed and surrounds the
nucleolus (SN type, transcriptionally inactive), whereas in another subset of
oocytes the nucleolus is not surrounded (NSN type, transcriptionally active) by
heterochromatin. Phenotypically, a main difference between these two types of
oocytes is that the former develops successfully when fertilized while the latter
ceases development at the 2-cell stage (Zuccotti et al. 2002). Thus, there is strong
evidence that the GV chromatin configuration is correlated with oocyte competence, specifically its meiotic and developmental competence (Tan et al. 2009).
During maturation, the oocyte chromatin configuration changes from the NSN type
to an SN type. In mice younger than 15 days, all growing oocytes have NSN type
nuclei; after that some of them become SN type, whereas others retain NSN-type
nuclei even as fully grown oocytes (Zuccotti et al. 1995). It follows that many
cytoplasmic and nuclear properties may differ between SN- and NSN-type oocytes,
which may also explain the observed differences in developmental potential (Inoue
et al. 2008). Dependent on the chromatin configuration of the GV oocyte, gene
expression analyses showed that the level of putative reprogramming factor Oct4 is
different between SN- and NSN-type oocytes, and suggested Oct4 being a key
regulator of developmental potential in MII oocytes. This transcription factor
governs a cascade of Oct4-downstream-regulated genes involved in pathways
such as oxidative phosphorylation, mitochondrial function, and cell death (Zuccotti
et al. 2008). Further, Oct4 is hypothesized to play an important role during
folliculogenesis by regulating the expression of Stella and Foxj2 (Table 1). For
example, when Oct4 is expressed, Stella is upregulated while Foxj2 is
downregulated and the oocyte is in a developmentally competent state (Zuccotti
et al. 2009). In addition to the differences of chromatin configuration in oocytes, it
was shown that an age-related increase in aneuploidy in oocytes is positively
correlated with a SN type of oocyte (Zuccotti et al. 1998). However, a direct
correlation between chromatin state of oocytes (SN or NSN type), their content in
Oct4 protein, and their reprogramming ability has not been established as of yet.

4.3

Different Maturation States and Accompanying
Chromatin Configuration of Recipient Oocytes for SCNT

Almost all mammalian species cloned from somatic nuclei have been generated
using enucleated oocytes in a particular phase of the cell cycle: MII-stage ooplasts.