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Chapter 10
Heritable Generational Epigenetic Effects
through RNA
Nicole C. Riddle
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Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA
Chapter Outline
Transgenerational Inheritance of RNAi in Caenorhabditis
Elegans
111
The piRNA Pathway, Transposon Silencing, and Hybrid
Dysgenesis in Drosophila Melanogaster
112
Transgenerational Epigenetic Inheritance in Mammals
113
piRNA Inheritance and Imprinting in Mouse
114
miRNA Inheritance and Paramutation in the Mouse
115
Transgenerational Epigenetic Inheritance in Humans
115
Conclusion116
Acknowledgements116
References116
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Abbreviations
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Introduction105
Transgenerational Inheritance Via the Gametes
106
Gametic RNA Populations
106
Small RNA Pathways
106
RNA-Guided Genome Rearrangements in Ciliates
108
Tetrahymena thermophila108
Oxytricha trifallax109
RNA Transport in Plant Systems
109
Paramutation in Maize
110
RNA Transfer Via the Gametes
in Arabidopsis Thaliana
111
DMRs Differentially methylated regions
dsRNA Double-stranded RNA
ENCODE Encyclopedia of DNA Elements
Endo-siRNAs Endogenous siRNAs
GFP Green fluorescent protein
H3K9me2 Histone 3 lysine 9 dimethylation
IAP Intracistral A particle
IES Internal eliminated sequence
lncRNAs Long non-coding RNAs
mRNAs Messenger RNAs
miRNAs MicroRNAs
pri-miRNAs Primary miRNA transcripts
piRNAs PIWI-interacting RNAs
RdDM RNA-directed DNA methylation
RISC RNA-inducing silencing complex
RNAi RNA interference
rRNAs Ribosomal RNAs
scnRNAs Scan RNAs
siRNAs Small interfering RNAs
viRNA Virus-derived small interfering RNAs
TEs Transposable elements
tRNAs Transfer RNAs
Transgenerational Epigenetics. http://dx.doi.org/10.1016/B978-0-12-405944-3.00010-6
Copyright © 2014 Elsevier Inc. All rights reserved.
INTRODUCTION
Traditionally, studies concerning the transfer of information
between generations have focused on DNA. As the carrier
of the genetic information, DNA provides the blueprint for
the next generation. However, in addition to DNA, parents
transmit information to their offspring through a variety of
other mechanisms. For example, parents often supply their
offspring with resources to enhance their chances of survival; these resources can include nutrition, parental care,
and other assistance. The quality and quantity of these
parentally supplied resources can transmit information to
the next generation. Because this information is inherited
independently of changes in the primary DNA sequence, its
effects are observed as transgenerational epigenetic effects
on phenotypes.
When considering epigenetic inheritance, DNA methylation and chromatin modifications come to mind. Epigenetic studies often focus on these molecular marks,
and the mechanisms for their transmission from parent to
offspring are well understood.1,2 Recently, there has been
increased interest in RNA as a mediator of transgenerational
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SECTION | IV Basic Mechanisms/Processes of Epigenetic Inheritance
epigenetic inheritance. RNAs carry out a wide array of
functions depending on their sequence composition; these
include enzymatic functions (ribozymes), regulatory functions (various small RNAs and long non-coding RNAs
(lncRNAs)), and metabolic functions (ribosomal RNAs
(rRNAs) and transfer RNAs (tRNAs)). The various small
RNA species, several of which participate in epigenetic
pathways, are of particular interest in regard to transgenerational epigenetic inheritance. As an information carrier,
the type as well as the amount of RNA transmitted to an
offspring is relevant. RNA is perceived as a promising candidate molecule for mediating epigenetic effects, due to the
large amount of information it can convey by providing
sequence complementarity to specific genomic regions.
TRANSGENERATIONAL INHERITANCE VIA
THE GAMETES
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While transgenerational effects can be mediated by many
means (communication via the placenta in mammals, antibodies provided in milk, etc.), when considering a potential
role for RNA, transgenerational epigenetic inheritance via
the gametes is the most likely route.3 The gametes link the
parent and offspring generations and contain a wide array
of biomolecules in addition to the DNA. The presence of
this additional material is particularly striking when comparing the female gamete (oocyte) to the male (sperm).
Oocytes tend to be much larger than sperm, because the
maternal contribution to the zygote consists of DNA and
numerous nutrients, while paternally, mainly DNA is contributed (however, see below). Analysis of the oocyte cytoplasm shows maternal loading of specific proteins, RNAs,
and many other molecules. These maternally provided
resources are vital for zygotic development, as they have
to maintain the developing embryo prior to the onset of
zygotic transcription, often through several rounds of cell
division. For example, the primary activation of the zygotic
genome occurs at the two-cell stage embryo in mouse, after
eight division cycles in Xenopus laevis, after approximately
14 division cycles in Drosophila melanogaster, and at the
globular embryo stage in Arabidopsis thaliana (reviewed
in 4). Thus, the gametes provide a means for the inheritance
of RNA in addition to transmitting the parental genomes to
the next generation.
interfering RNAs (siRNAs) – for example, endogenous
siRNAs (endo-siRNAs) in the mouse7,8 – and PIWI-interacting RNAs (piRNAs).7,8 The importance of maternally
supplied RNA can be illustrated by studies of anterior–
posterior axis formation in Drosophila melanogaster
embryos. Their body axis is specified by the localization
of oskar and bicoid mRNAs, which is established during
oogenesis.9,10 Mislocalization of either oskar or bicoid
mRNA in the oocyte leads to defects in segment specification, such as defective heads in the embryo.9,10 These and
other studies demonstrate that maternally supplied RNAs
are required for proper development and can be inherited
by the zygote.
Studies of sperm composition have shown that despite
common opinion, the sperm transmits some information in
addition to providing one-half of the DNA to the offspring
– RNAs, protein, and chromatin marks all can be passed on
through the sperm as well. Expression analysis of sperm
cells is technically challenging due to the small amounts of
RNA there, and the risk of contamination from other cells.
Next-generation sequencing techniques as well as advances
in cell sorting have been instrumental in the analysis of
sperm RNA populations. Studies in several species have
confirmed the presence, not only of mRNAs, but also of
a variety of non-coding RNAs, including long non-coding
RNAs (lncRNAs), small RNA precursors (pri-miRNAs),
and small RNAs (reviewed in 11; for recent studies on
human:12,13; and mouse:14). Depending on the species, several hundred to several thousand transcripts are detected in
sperm (for examples see 13,15–17), and there is some evidence
of conserved function among them.16 In addition, for several
sperm transcripts, transfer of sperm mRNA to the oocyte
has been confirmed (hamster/human:18; ­
Drosophila:16).
Thus, transcriptome studies have demonstrated the presence of complex RNA populations in the gametes and their
possible transfer to the zygote through both the maternal
and paternal lineages. These RNAs have the potential to
mediate transgenerational inheritance, which could explain
the epigenetic, i.e., non-genetic, inheritance of information
seen in many species.
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GAMETIC RNA POPULATIONS
Transcriptome studies from oocytes show that the maternally supplied RNA pool is very complex. The RNAs
include ribosomal RNAs (rRNAs), transfer RNAs (tRNAs),
messenger RNAs (mRNAs), as well as several species of
small RNA (see below for a detailed discussion). MicroRNAs (miRNAs), for example, feature prominently among
the maternally supplied small RNAs,5,6 as do classes of small
SMALL RNA PATHWAYS
A unifying principle linking many epigenetic phenomena is
the involvement of small RNA pathways. RNA interference
(RNAi), discovered in Caenorhabditis elegans, describes
a gene silencing mechanism induced by double-stranded
RNA (dsRNA) and characterized by the production of
small, approximately 20–30 nucleotide RNAs19 (reviewed
in 20). Often, these types of small RNAs, and sometimes
longer non-coding RNAs, are associated with epigenetic
processes, including genomic imprinting, heritable epigenetic gene silencing, and many others.21,22 Small RNAs are
of great interest in studies of transgenerational epigenetic
Chapter | 10 Heritable Epigenetic Effects Through RNA
the RNA-inducing silencing complex (RISC). Using the
siRNA as a guide, RISC targets homologous mRNAs for
cleavage and degradation, resulting in post-transcriptional
gene silencing. The miRNA pathway processes RNAs that
form hairpin structures, which are expressed from miRNA
loci within the genome (Figure 10.1, middle panel). Production of the mature miRNA involves several cleavage
steps (requiring DROSHA and a Dicer, DCR-1), leading to
the incorporation of the mature miRNA into a second type
of RISC. This miRNA–RISC causes translational repression of matching mRNAs and mRNA destabilization, leading to protein loss. The piRNA pathway processes long
single-stranded transcripts produced from so-called piRNA
clusters, which include many transposable elements (TEs)
and various other repetitive sequences (Figure 10.1, right
panel). piRNAs are produced without a Dicer, and instead
utilize the PIWI class Argonaute proteins ARGONAUTE
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inheritance because they can potentially provide sequence
specificity. They can act as guides to specific genomic locations by sequence homology and are known to recruit various proteins to target sites, including epigenetic modifiers
such as DNA methyltransferases.23
Generally speaking, there are three basic RNAi pathways, which have been elaborated upon by various species (for an introduction to the small RNA pathways,
see 24). This description is based on the D
­ rosophila melanogaster pathways, but the same principles apply to the
majority of species (Figure 10.1). The siRNA pathway
processes dsRNA, both from endogenous and exogenous
sources (Figure 10.1, left panel). A Dicer protein – DCR2, a dsRNA-specific endoribonuclease in the RNase III
family – cuts the dsRNA into smaller fragments. The
resulting siRNA duplexes are processed, and the guide
siRNA binds to an Argonaute protein (AGO2) forming
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FIGURE 10.1 The three basic small RNA pathways. In Drosophila melanogaster, there are three basic small RNA pathways, the small interfering
RNA (siRNA) pathway (left), the microRNA (miRNA) pathway (middle), and the PIWI-interacting RNA (piRNA) pathway (right). While the siRNA and
miRNA pathways process double-stranded RNAs (dsRNAs) – hairpin structures in the case of the miRNA pathway – the piRNA pathway processes long
precursor transcripts derived from so-called piRNA clusters. These various RNAs are processed into small RNAs that are capable of degrading homologous transcripts (siRNA and piRNA pathways), repress translation (miRNA pathway), and guide chromatin modifications (mainly piRNA pathways,
endo-siRNA pathway). For details, see text. Adapted from 24
SECTION | IV Basic Mechanisms/Processes of Epigenetic Inheritance
3, AUBERGINE, and PIWI. Mature piRNAs are thought
to guide mRNA cleavage and/or chromatin modification of
transposon sequences, thus ensuring silencing of TEs and
genome stability (reviewed in 24).
RNAs are one of several classes of biomolecules with
the potential to mediate transgenerational epigenetic
inheritance. There is strong evidence for the contribution
of both DNA modifications (5-methylcytosine) and chromatin structure in the form of histone-tail modifications,
to transgenerational epigenetic inheritance (reviewed in
Chapter 9). In the following review, the author will survey
evidence from various systems that RNA species inherited from one generation to the next can produce epigenetic effects and thus mediate transgenerational epigenetic
inheritance.
RNA-GUIDED GENOME REARRANGEMENTS
IN CILIATES
Tetrahymena thermophila
Initial evidence of RNA involvement in ciliate genome
rearrangements came from genetic experiments probing
the mechanisms controlling macronuclear differentiation.
Mutant screens revealed a role for chromatin proteins as
well as for proteins typically found in the RNAi pathways
(reviewed in 27 for Tetrahymena). For example, TWI1, a
Tetrahymena PIWI homolog, is required for DNA elimination,29 as is DCL1, a Tetrahymena Dicer protein.30 In
addition, small RNAs are prevalent in the relevant stages
of macronuclear development. Small RNAs and their precursors are produced specifically during sexual reproduction and are most abundant when the macronucleus and
micronucleus begin their differentiation.31,32 These data
suggest a role for the RNAi pathway in DNA elimination in
­Tetrahymena macronuclar development.
Follow-up experiments provided direct evidence for the
role of RNA in the genome rearrangements. In Tetrahymena,
injection of dsRNA or DNA prevents elimination of specific
sequences (termed internal eliminated sequences (IESs))
from the developing macronucleus. Simply having DNA
homologous to an IES present in the parental macronuleus
(DNA) or a homologous dsRNA present in the cytoplasm
(dsRNA) prevents sequence elimination and causes the retention of the IES in the new macronucleus.33,34 As the parental macronucleus does not contribute DNA to the progeny, a
model of genome scanning has been proposed.29,32
The genome-scanning model suggests that bidirectional transcripts are generated in the germline nucleus.
These transcripts are processed by the RNAi pathway into
small dsRNAs termed scan RNAs (scnRNAs). TWI1 binds
scnRNAs and shuttles them to the parental macronucleus,
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Ciliates are of great interest for research into epigenetic
effects due to the elaborate genome rearrangements that
occur during their sexual reproduction. Generally, ciliates
have two types of nucleus, the micronucleus and the macronucleus. While the exact number of these nuclei depends
on the species, the micronucleus represents the germline
and is transcriptionally silent, while the macronucleus corresponds to the soma and is transcriptionally active (for a
review see 25). Compared to the micronucleus, the genome of
the macronucleus is highly modified; its sequences are rearranged compared to the micronucleus, DNA is eliminated,
and chromosomes are fragmented (reviewed in 26). In Tetrahymena thermophila, approximately 30% of the micronuclear genome is eliminated from the macronucleus; in other
ciliates, the eliminated fraction of the genome can exceed
90%.27 During sexual reproduction, a new macronucleus is
generated from the zygotic nucleus, and the various genome
rearrangements have to be coordinated (Figure 10.2). Studies of these processes in several ciliate species demonstrate
a role for RNA in orchestrating the genome rearrangements
during macronuclear development (for a recent review
see 28), an example of transgenerational epigenetic effects
mediated by RNA.
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FIGURE 10.2 Nuclear differentiation in Tetrahymena. Left panel: The zygotic product contains a diploid nucleus (MIC), and the parental macronucleus (MAC). Middle panel: The zygotic diploid nucleus divides to produce two new micronuclei (MIC). Right panel: One of the new nuclei differentiates into the new macronuleus (MAC), while the other remains, forming the new micronucleus (MIC). The old parental macronucleus degenerates (light
grey sickle shape).
Chapter | 10 Heritable Epigenetic Effects Through RNA
Oxytricha trifallax
RNA TRANSPORT IN PLANT SYSTEMS
Some of the earliest support for RNA-based transgenerational epigenetic inheritance comes from research in several
plant systems. In contrast to animals, plants do not sequester
the germline early in development; rather, at a specified time
during its life, the plant switches from vegetative growth to
its reproductive phase. This switch leads to the production of
flowers and the development of the germline from the same
cell population that used to give rise to all the vegetative tissues. Depending on the plant species, this switch to the reproductive phase is terminal (in annual plants such as Arabidopsis
thaliana), or can happen repeatedly (in perennials such as
trees). Independent of the plant’s growth habit, the late specification of the germline makes it possible for events that impact
the soma to also influence the germline. Thus, in plant systems
there is an increased possibility for disease or environmental
exposures to lead to transgenerational epigenetic memory (for
a recent review see 38).
In addition to their late segregation of the germline,
plants have other physiological characteristics that make
transgenerational epigenetic inheritance via RNA more
likely. Plants are able to transport RNA and other macromolecules between cells, as well as systemically through the
entire plant. Intercellular transport is mediated by plasmodesmata – openings that span the cell wall and allow communication between the cytoplasm of two neighboring cells.
The plasmodesmata provide connections between cells and
facilitate the exchange of macromolecules, including several classes of RNA, from cell to cell (reviewed in 39). The
RNAs exchanged include mRNAs, miRNAs, viral RNAs,
and siRNAs.39 The transport of siRNAs is responsible for
the non-cell autonomous nature of RNAi in plants. Using
a transgene expressing a long dsRNA, Dunoyer and colleagues demonstrated that 21nt small RNAs are the signal
required for spreading of gene silencing by RNAi.40 Molnar
and colleagues came to the same conclusion using grafting
experiments – sequencing of small RNAs from graft and
recipient demonstrated the movement of siRNAs across the
grafting boundary.41 Thus, mobile small RNAs can spread
gene silencing locally, suggesting that other RNAs mediating epigenetic phenomena might do so as well.
Aside from the local transport of RNAs, plants also
have the ability to move macromolecules including RNAs
through their vasculature, in particular through the phloem
(reviewed in 39,42). The phloem is the portion of the vascular system that moves nutrients throughout the plant in
what is commonly known as sap. In addition to various
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Oxytricha, another microbial eukaryote, employs a different
RNA-dependent mechanism to achieve macronuclear development. In Oxytricha, only 5% of the germline genome is
retained in the somatic macronuleus.25,35 Instead of marking specific sequences for elimination in the macronucleus,
sequences are marked for retention. In Oxytricha, 27nt
RNAs of the piRNA class are generated in the somatic macronucleus from long piRNA precursor transcripts.36 These
piRNAs are bound by OTIWI1, then shuttled through the
cytoplasm to the developing macronucleus, where they mark
specific sequences for retention.36 Simple injection of piRNAs into the parental macronucleus will lead to retention
of homologous sequences in the next generation,36 demonstrating that RNA mediates genome rearrangements in Oxytricha. Thus, piRNAs relay information from the parental
macronucleus to the developing F1 macronucleus, a case of
transgenerational epigenetic inheritance mediated by RNA.
In addition, there is evidence that long RNA templates
can be shuttled from the parental macronucleus to the
developing macronucleus in Oxytricha as well. Many of
the Oxytricha open reading frames are “scrambled” in the
micronucleus, and individual sequence fragments have to
be placed in the correct order in the macronucleus to be
able to carry out their functions. Nowacki and colleagues
found that this “unscrambling” appears to be guided by
long RNA templates.37 Injecting long RNA templates with
switched fragment order into the macronucleus resulted
in the incorporation of the new sequence into the macronuclear genome.37 The ability of injected RNA templates
to guide the “unscrambling” of the macronuclear genome
is unique to long RNAs, as attempts to do the same with
piRNAs were unsuccessful.36 This finding indicates that
in Oxytricha there are at least two genome rearrangement
pathways that involved RNA-mediated transgenerational
epigenetic inheritance.
These studies in ciliates demonstrate that RNAs can play
a vital role in mediating epigenetic effects. Ciliates have
developed several complex systems to orchestrate the genome
rearrangement necessary to generate their somatic macronucleus. The species examined to date all utilize RNA-based
transgenerational communication, indicating that this mode
of information transfer might be commonly used.
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where they scan the macronucleus by subtractive hybridization – any scnRNA matching the macronuclear genome will
be eliminated, resulting in a pool of TWI1-bound scnRNAs
homologous to sequences not present in the parental macronucleus. These remaining scnRNAs are shuttled back to
the developing macronucleus, where they recruit chromatin
proteins to homologous sequences (the IESs) and coordinate their elimination from the new macronucleus. DNA
elimination by this mechanism takes place in Tetrahymena
and Paramecium, leading to the elimination of ∼ 15–30% of
the micronuclear genome.28 Thus, communication through
RNA between the developing F1 macronucleus and the
parental macronucleus orchestrates the somatic development in the F1.
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SECTION | IV Basic Mechanisms/Processes of Epigenetic Inheritance
sugars and plant hormones, several studies have shown
that RNAs are moved long distances through the phloem
as well.39 Thus, mRNAs can be moved from a source tissue, where a signal was received, to a sink tissue, where
the physiological response will be carried out. An example
of such behavior is the mRNA from the Flowering locus T
(FT), which is produced mainly in the leaves, but needs to
communicate with the apex to induce flowering.43 Longdistance movement of RNA through the phloem has been
observed for mRNAs, viral RNAs, as well as small RNAs
involved in various epigenetic processes. The transport of
siRNAs through the phloem is thought to be responsible
for the systemic spread of RNAi-based gene silencing
in plants.44,45 The unique properties of plants – the late
specification of the germline and the capacity to transport
RNAs throughout the entire organism – makes them of
particular interest for investigations of transgenerational
inheritance via RNA.
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PARAMUTATION IN MAIZE
subunits of RNA polymerase IV and RNA pol V (RMR6/
RPD1,56 MOP257); they also include an RNA-dependent
RNA polymerase similar to Arabidopsis RDR2 (MOP158)
and a SNF2-like ATPase (RMR159). Based on the discovery
that RdDM pathway proteins affect paramutation, it was
suggested that small RNAs of the RdDM pathway mediate
paramutation.46,60
Direct tests of this model have been difficult, due to
inaccessibility of the tissues in the female gametophyte in
which paramutation is first established. One experiment
looking directly at small RNAs has been carried out for B′
paramutation. B′ paramutation requires seven repeat units
of the 853bp b1TR sequence.61,62 These b1TR repeats are
located approximately 100 kb upstream of the b1 coding
sequence.61,62 The repeats are transcribed bidirectionally,58
and produce siRNAs in B′.57,63 Expression of transgenic
b1TR siRNAs in the absence of the endogenous b1TR repeat
locus was able to recapitulate paramutation.63 While some
data suggest that paramutation might employ other molecular mechanisms as well, overall the study of paramutation
supports a role for RNA in mediating its transgenerational
epigenetic effects.
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Paramutation is an epigenetic phenomenon that refers to an
interaction between two alleles in trans that can permanently
alter their gene expression state (for a recent review see 46).
Since Brink reported paramutation for the maize r1 locus
in 1956,47 cases in various species have been described,
including several cases of paramutation of transgenes
(see 48–52). Paramutation is best understood for genes affecting plant color in maize. For example, the b1 locus encodes
a transcription factor in the anthocyanin pathway leading
to purple plant color;53 null alleles lead to green plant color
(Figure 10.3). Paramutation is observed if a green, homozygous B′ plant is crossed to a purple homozygous B-I plant
(reviewed in 54). In F1, all plants are heterozygous, exhibiting green plant color. The allelic interaction between B-I
and B′ becomes evident in a backcross to the B-I parent,
where all offspring plants are green, irrespective of genotype, indicating that the B-I allele has been altered through
the exposure to B′. These altered B-I alleles, now termed
B′*, are themselves able to convert B-I alleles to the silent
state. Thus, paramutation represents a transgenerational
epigenetic effect.
Explorations of the mechanisms controlling paramutation suggest that RNA might be the factor communicating between the two alleles in trans. Mutants defective for
either the initiation or maintenance of paramutation often
represented RNAi pathway components that had been previously identified in Arabidopsis thaliana. Plant RNAi
pathways differ somewhat from the three basic pathways
described above. In plants, siRNAs can direct DNA methylation (RNA-directed DNA methylation (RdDM)), and
plants have two unique RNA polymerases, RNA pol IV and
RNA pol V, that are part of the RdDM pathway.23,55 The
mutations found to affect paramutation in maize include
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FIGURE 10.3 Paramutation at the b1 locus. Paramutation is illustrated
by the interaction of the B-I and B′ alleles. If homozygous B-I plants with
a purple plant phenotype are crossed to homozygous B′ plants with a green
plant phenotype (parental P generation), all the offspring (F1) show a green
plant phenotype. If F1 plants are backcrossed to the homozygous B-I parent, all offspring are green, irrespective of the genotype, demonstrating
how the B-I allele in the F1 has been changed to a B′* allele, now leading
to green plant color.
Chapter | 10 Heritable Epigenetic Effects Through RNA
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A second line of evidence for RNA-mediated transgenerational epigenetic effects comes from the study of plant
reproductive tissues, in particular, the male gametophyte.
Plants alternate in their development between the diploid
sporophyte and the haploid gametophyte. In angiosperms,
the gametophyte has been reduced to eight cells within the
ovule containing the egg cell (in the female germline) and
three cells in the pollen grain (in the male germline). In
the pollen grain, the haploid nucleus derived from meiosis
divides twice. The first division forms the vegetative nucleus
and the generative nucleus; in the second division, the generative nucleus produces the two sperm nuclei needed for
double fertilization.
During gamete formation in many systems including
mammals, epigenetic information such as DNA methylation and imprinting is reprogrammed to return to a pluripotent state in the zygote.64–66 In plants, resetting can
be incomplete, and the inheritance of epialleles has been
reported. These include, among others, the peloria epiallele
which causes changes in floral morphology in Linaria vulgaris discovered by Linneaus,67 the SUPERMAN epialleles
causing homeotic transformations in Arabidopsis thaliana,68 and the FWA epialleles causing late flowering also
in Arabidopsis.69 Despite the “leaky” nature of the reprogramming, epigenetic resetting is nevertheless an essential
part of gamete formation in plants, and several epigenetic
pathways are involved.
Due to the more accessible nature of the male gametophyte, reprogramming in plants is better understood in the
male germline. In Arabidopsis, the vegetative nucleus of the
pollen grain – which does not contribute DNA to the next
generation – lacks DDM1, a protein required for the maintenance of DNA methylation and heterochromatin structure.70
It expresses DME, a 5-methylcytosine DNA glycosylase
that removes DNA methylation at imprinted loci.71 Thus,
heterochromatin is decondensed, and DNA methylation is
lost.70,72 The loss of DNA methylation leads to the reactivation of TEs,70 an unusual observation, as maintenance of
TE silencing in the germline is paramount to prevent compromising the genome passed on to the next generation.
This apparent paradox was explained when the TEs were
observed to not only reactivate in the vegetative nucleus,
but to produce large amounts of siRNAs.70 Sequencing of
small RNA libraries from sperm cells demonstrated the
presence of siRNAs homologous to Athila – a retrotransposon – thought to originate in the vegetative nucleus.70 These
data led to a model where the TE-derived siRNAs from
the vegetative cells are transported into the sperm nuclei
where they can ensure silencing of the TEs via the RNAi
pathways. Additional support for this model is provided by
studies of RNA-directed DNA methylation in pollen and the
early embryo.73 Thus, it appears that siRNAs in pollen can
guide TE silencing in the next generation and thus communicate epigenetic information transgenerationally.
Support for a similar system in the female gametophyte
is emerging as well. In Arabidopsis, the Argonaute protein
AGO9 is required for normal female gametophyte development, as are SGS3 and RDR6,74 both of which are involved
in the non-cell autonomous RNAi pathway.75–77 Interestingly, AGO9 expression occurs in the maternal companion
cells, not the ovule, and AGO9 protein co-precipitates with
TE-derived small RNAs. These small RNAs target the egg
cell and synergids within the ovule,74 arguing for intercellular movement of the TE-derived small RNAs between
maternal tissues and the gametes as well as between cells
within the ovule. This model is supported by the recovery of maternal siRNAs from endosperm.78 In addition,
experiments using an artificial miRNA targeting green
fluorescent protein (GFP) demonstrate movement of small
RNAs between the central cell – which will give rise to the
endosperm – and the egg cell.79 GFP is expressed in the
egg cell, and the miRNA targeting GFP in the central cell.
In ovules expressing the GFP-targeted miRNA in the central cell, GFP levels are reduced in the egg cell. This result
indicates that the miRNA was able to move from the central cell source to the egg cell and inhibit GFP synthesis
there.79 Thus, data from the male and female gametophytes
indicate that companion cells produce various silencing
RNAs that are moved into the gametes and thus passed on
to the next generation, where they can induce silencing in
the zygote.43,70,79,80 While paramutation might represent
a “malfunction” of the same mechanism, in plants, transgenerational epigenetic inheritance via siRNAs appears
to be responsible for maintaining TE repression to ensure
genome integrity.
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TRANSGENERATIONAL INHERITANCE OF
RNAi IN CAENORHABDITIS ELEGANS
Studies in C. elegans provide several lines of evidence for
heritable transgenerational epigenetic effects that might be
mediated through RNA. This mode of inheritance is best
supported for several classes of small RNAs, and evidence
comes from studies of RNAi – gene silencing induced by
treatment with antisense or dsRNA.19 Interestingly, the
gene silencing induced by treatment with dsRNA in the parent is heritable to the F1 generation.19 This finding implies
that some signal is transmitted through the gametes, capable
of establishing the silent state in the offspring generation.
Generally speaking, gene silencing by RNAi can occur
by two different means, through post-transcriptional silencing and mRNA degradation in the cytoplasm (cytoplasmic
RNAi) or through transcriptional silencing by chromatin
modification (nuclear RNAi).23 In C. elegans, the inheritance of silencing induced by dsRNA to the F1 generation
SECTION | IV Basic Mechanisms/Processes of Epigenetic Inheritance
generation, which are themselves unable to generate the
small RNAs.89 These data suggest that the viRNAs were
inherited from the parent generation and that, despite their
low copy number, they are capable of inducing silencing up
to the F5 generation.
It is likely that viRNAs are not the only small RNA
class that can be transgenerationally inherited. siRNAs are
well known for their ability to target chromatin changes
(H3K9me2/me3) to initiate chromatin-mediated transcriptional silencing. siRNA sequencing studies have shown
that siRNAs are inherited through the gametes and can be
detected until the F3.84 In addition, piRNAs are a good candidate for a transmitted silencing factor, as they are generated in the germline.87 Despite lack of data from C. elegans,
based on their localization patterns and data from other systems (see below for Drosophila), they are likely to be passed
on to the next generation as well. Thus, studies in C. elegans
provide support for the transmission of several classes of
small RNAs to subsequent generations and indicate their
importance for epigenetic inheritance of silencing.
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depends on the presence of a functional nuclear RNAi pathway.81 nrde (−) mutants lacking a functional nuclear RNAi
pathway are capable of a silencing response to dsRNA treatment, but they are unable to maintain this silencing in the F1
past the embryonic stage.81 In wild-type F1, H3K9me3 (histone 3 lysine 9 trimethylation) – a silent chromatin mark –
accumulates at the silenced locus, and homologous siRNAs
are observed as well.81 Because siRNAs appear prior to the
chromatin marks, these data suggest that siRNAs are transmitted through the gametes and serve as silencing signals.81
Interestingly, further studies have demonstrated that
some gene silencing in C. elegans can be inherited past
the F1. This silencing can occur through several of the C.
elegans RNAi pathways, including the siRNA pathway.82–85
the piRNA pathway,86–88 and the viRNA pathway.84,89 For
this to occur, the gene targeted by RNAi has to be expressed
in the germline, and several RNAi pathway components
are required as well.83 Mutant studies indicate that there
is a two-step process involved; some genes such as rde-1
(a PAZ/PIWI domain protein90) and rde-4 (a dsRNA binding protein91) are required for generating the silencing signal, while others, including rde-2 and mut-7, are required
at a later step.83 The silencing signal can be transmitted
through the sperm and does not require the presence of
the target gene.83 Vastenhouw and colleagues confirmed
that the inheritance of persistent silencing does not require
the canonical RNAi pathway genes rde-1 and rde-4, and
instead seems to rely on several chromatin proteins (hda-4,
a HDAC; K03D10.3, a HAT; isw-1, a chromatin remodeler;
and mrg-1, a chromo-domain protein),85 indicating that
while the initiation of silencing occurs through the RNAi
pathway, the long-term inheritance observed up to the F20
generation is chromatin-mediated.
While these earlier studies clearly demonstrate inheritance of silencing, the nature of the inherited factor remains
elusive. Mainly indirect evidence supported a model in which
germline-transmitted RNA serves as the silencing signal in
transgenerational RNAi. Transmission of heritable silencing
induced by dsRNA through the male and female are consistent with this model.82 The silencing factor is diffusible – thus
is not a chromatin mark – and can be inherited in the absence
of the dsRNA target sequence.83 Inheritance of the silencing factor from rde-1- and rde-4-mutant animals incapable
of mounting an RNAi response argues for the transport of
long dsRNAs and against the transport of siRNAs alone.90
WAGO9/Hrde1, which binds siRNAs in the germline, is
required for the inheritance of RNAi induced silencing,92
supporting a model with siRNAs as the inherited factor.
Direct evidence demonstrating the inheritance of
siRNA has only recently been obtained due to technological advances in sequencing technologies. Rechavi and colleagues investigated a special case of RNAi, v­ irus-induced
RNAi (viRNAs), in C. elegans. They could detect ­viRNAs
and observe silencing in worms of the F2, F3, and F4
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112
THE piRNA PATHWAY, TRANSPOSON
SILENCING, AND HYBRID DYSGENESIS
IN DROSOPHILA MELANOGASTER
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Research in Drosophila melanogaster has contributed
greatly to our understanding of epigenetic processes and has
led to several discoveries relating to RNA-mediated transgenerational inheritance. One of these areas of research
focuses on the piRNA pathway. piRNAs and PIWI-class
proteins control transposon silencing and are of vital
importance in the germline (reviewed recently in 93–95).
In Drosophila, piRNAs are derived from piRNA clusters/
loci – sequence regions in the pericentric or subtelomeric
regions of the genome that are TE-dense and to which most
piRNAs map.96,97 These clusters are transcribed, producing
long precursor transcripts that are then processed through
the piRNA pathway to produce functional piRNAs. Primary piRNAs originate directly from the long piRNA precursor transcripts, while secondary piRNAs are produced
by an amplification loop termed “ping-pong” amplification
(reviewed in 93). In the female germline, piRNAs are maternally deposited in the oocyte and function in the embryo to
silence TEs.96,98–101 Their germline function has made piRNAs of great interest to researchers focused on transgenerational epigenetic inheritance.
Hybrid dysgenesis is an epigenetic phenomenon that
has been linked to piRNAs in Drosophila melanogaster. It
occurs when two fly lines are crossed that differ in the TE
families present in their genome. Depending on the direction
of the cross, the resulting offspring are sterile.102 The sterility is caused by the presence of a specific transposon family
(e.g., P-element, and I-element) in one of the lines and not
the other. It was originally observed when wild-caught male
Chapter | 10 Heritable Epigenetic Effects Through RNA
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piRNAs in the aged female – was transmitted to the next
generation, where increased levels of piRNAs matching the
I-element were now produced in young females.109
The above-mentioned experiments show that the incorporation of sequence elements in piRNA loci can lead to
silencing of matching sequences in trans, and that piRNAs can be inherited through the maternal germline. The
final piece of evidence that piRNAs are sufficient to induce
silencing at loci with sequence homology was provided by
Huang and colleagues.110 By introducing sequences matching known piRNAs at an ectopic site, they were able to
recruit PIWI protein to this new site. In addition to PIWI,
HP1a, a chromosomal protein associated with heterochromatin, is recruited to the ectopic site, as is SU(VAR)3-9,
one of the three H3K9 histone-methyltransferases in Drosophila.110 Together, these data demonstrate that recruitment of piRNAs leads to the formation of a silent chromatin
structure. These findings suggest that the piRNAs transmitted through the oocyte enable the offspring to efficiently
silence matching sequences such as TEs.
The small RNAs are also responsible for hybrid dysgenesis in Drosophila virilis.111 This case of dysgenesis differs
from the systems in D. melanogaster, in that several TE
types are misregulated in the dysgenic cross. However, like
in the experiments described above for D. melanogaster, D.
virilis dysgenesis is due to the lack of small RNAs matching the misregulated TEs in the maternal lineage.111 In the
wild-type situation, TE-derived small RNAs are transmitted through the germline to the next generation where they
induce silencing of TEs matching their sequences. Interestingly, it has recently been suggested that misregulation of
TEs due to lack of silencing small RNAs, akin to what is
observed in hybrid dysgenesis, might contribute to hybrid
barriers in plants as well,112 indicating that these mechanisms might have much larger evolutionary implications.
These data from various systems confirm that small RNAs,
and in particular piRNAs, are an excellent example of an
RNA-based transgenerational epigenetic inheritance system.
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flies were crossed to female flies from standard laboratory
strains, because the P-element invaded wild strains after the
initial collection of the laboratory strains. Interestingly, dysgenesis is only observed in one direction of the cross, and this
difference in reciprocal crosses suggested the presence of a
fertility factor in the female parent. Current models ascribe
the sterility to the mobilization of the transposon in the germline of hybrids and a failure to silence the novel TE family.
The discovery that TEs in the Drosophila germline are
regulated by piRNAs provided a candidate mechanism for
hybrid dysgenesis. Brennecke and colleagues showed that in
addition to the maternal loading of piRNA pathway proteins
(e.g., PIWI and AUG103,104), small RNAs were also maternally deposited.99 This maternal loading includes piRNAs
associated with all three Drosophila Argonaute proteins –
PIWI, AGO3, and AUB.99 In dysgenic crosses of males with
an active I-element to females without active I-elements,
very few piRNAs matching I-element were maternally supplied; the few piRNAs identified as matching the I-element
were derived from ancestral inactive copies. The lack of
maternal I-fragment piRNAs led to lower piRNA levels in
the offspring and ultimately the reactivation of the paternally inherited I-element during gametogenesis and sterility.99 These findings were confirmed using the P-element
dysgenesis system, indicating that piRNAs of several types
are maternally deposited and induce transgenerational epigenetic silencing.
Several research groups tested this model of TE silencing experimentally. Ronsseray and colleagues used a transsilencing system to show that insertion of a transgene – in
this case a P-element – within a piRNA cluster at the subtelomere can induce silencing of a second P-element construct sharing sequence similarity at a second location in
the genome (105 and earlier references therein). This transsilencing was dependent on the piRNA pathway components
(squash and zucchini, see also 106), and small RNAs – likely
piRNAs based on their size – matching the P-element could
be detected.105 Khurana and colleagues found in a detailed
study of rare offspring from dysgenic crosses that novel
TE insertions into the piRNA cluster locus 42AB would
result in the production of piRNAs matching the insertion
and reduced transposition of homologous TEs in the next
generation.107 Thus, production of piRNAs correlates with
silencing of matching sequences in the next generation.
Actual transgenerational inheritance of piRNAs was
demonstrated in another study of I-element dysgenesis. The
degree of sterility observed in dysgenic females depends on
their age; increased age led to increased fertility (for example
see 108). piRNA pools from the ovaries of these older females
contained 2.9-fold more sequences matching the I-element
than ovaries from younger females.109 In addition, these
I-element piRNAs were deposited in the oocyte, and their
inheritance could be detected in the embryo prior to zygotic
transcription.109 Thus, an acquired trait – the production of
113
TRANSGENERATIONAL EPIGENETIC
INHERITANCE IN MAMMALS
In mammalian systems especially, the study of transgenerational epigenetic inheritance is complicated by the basic
biology of the organism (Figure 10.4). Due to the long
pregnancy, intimate contact between mother and offspring
through the placenta, and early segregation of the germline,
any treatment the mother experiences also affects her offspring (F1), and the germline of her offspring (and thus the
F2). Therefore, transgenerational epigenetic effects need to
be observed in the F3 generation or beyond. In addition, to
rule out maternal effects or effects that are not mediated
through the gametes, cross-fostering and similar controls are
often necessary. Given the difficulty of such experiments,
SECTION | IV Basic Mechanisms/Processes of Epigenetic Inheritance
114
Parent
F1 offspring
F2 offspring
FIGURE 10.4 Transgenerational effects in mammalian systems. Any effects observed by the mother (left), also affect the developing F1 offspring
(in light grey shown in utero; middle). Due to the early segregation of the germline (*in the embryo on the left), the germline cells can also be affected by
anything affecting the parent animal. These germline cells will develop into the F2 offspring (in dark grey shown in utero of the F1; right). Thus, to ensure
any transgenerational effects seen in mammals are truly epigenetic, they have to be observed in the F3 or beyond.
R
the mechanistic details of the various small RNA pathways
described earlier have been worked out in non-mammalian
model systems, many basic findings hold true in mammalian systems as well. The basic pathways have been confirmed in the mouse, rat, and by studies in human cell lines.
Similar to the previously mentioned model systems, diverse
RNA populations are passed through the gametes to the next
generation in humans as well as other mammalian systems.
The transmitted RNAs include contributions from both the
maternal and the paternal lineage to the zygote. For several
of these RNA types, including a variety of small RNAs, evidence for transgenerational epigenetic inheritance exists in
mammalian systems as well.
In addition to their traditional role of transposon silencing in the germline, piRNAs have been implicated in the
control of imprinting in mice. Alleles at imprinted loci differ
in their expression status depending on their parental origin:
either the maternal allele is expressed and the paternal allele
is silenced; or alternatively the paternal allele is expressed
while the maternal allele is silenced. These allele-specific
differences in expression are accompanied in most cases by
differential methylation at so-called differentially methylated regions (DMRs). The DMRs control the expression
levels of imprinted loci by regulating access of the transcriptional machinery. Methylation at the DMRs is reset
every generation during gametogenesis; first the old methylation pattern is removed, then the new pattern that will be
passed on to the zygote is established (reviewed in 65). Thus,
the current model postulates that imprinting is coordinated
by the inheritance of DNA methylation patterns.
While the above model of imprinting works well for
most loci, recent work on the mouse Rasgrf1 locus suggests the additional involvement of piRNAs and a non-coding RNA. The Rasgrf1 DMR is paternally methylated and
contains a transposon-derived sequence that is required for
proper DNA methylation.117 Due to the presence of a transposon-derived sequence in the DMR, the piRNA pathway
proteins MILI1, MIWI2, and MitoPLD (the mouse homolog of D. melonagaster Zucchini) were investigated and
found to be necessary for imprinting at Rasgrf1. Mutations
in each of the three proteins led to DNA methylation loss
at the Rasgrf1 DMR, and in the MitoPLD mutant, piRNAs
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it is not surprising that unequivocal evidence for transgenerational epigenetic inheritance is rare in humans and not
linked to a particular molecular mechanism.
There are several natural cases of epigenetic transgenerational inheritance in mammals including the agouti viable
yellow (Avy)113 and axin-fused114,115 alleles in mice. The
Avy allele of agouti is probably the best-studied case; here,
a retrotransposon (an intracisternal A-particle (IAP)) insertion drives ectopic expression of agouti in a DNA methylation-dependent manner. The coat color of Avy mice ranges
from full agouti (pseudo-agouti), to variegated, to yellow.
High levels of agouti expression, low levels of DNA methylation at IAP, and yellow coat color correlate. If the Avy
allele is inherited from an Avy/+ heterozygous female with
yellow coat color, the offspring show a trend to more yellow
coat color compared to offspring from Avy/+ heterozygous
females with a darker coat color. This skew in offspring coat
color is interpreted as failure to completely erase epigenetic
marks (e.g., DNA methylation or histone modifications) at
the agouti IAP in the female germline (for a review see 3).
The transgenerational effect on coat color is seen only
for inheritance of Avy through the female germline; however, paternal-effect mutations affecting Avy have been
described. These paternal-effect mutations are mutations
at loci other than agouti, which include Snf2h and Dnmt1.
Snf2h encodes a chromatin remodeler of the ISWI family,
while Dnmt1 encodes the main cytosine DNA methyltransferase controlling maintenance methylation.116 Wild-type
offspring from crosses between Avy/+ mothers and Snf2h or
Dnmt1 heterozygous males showed an unexpected tendency
towards yellow coat color compared to control crosses,
demonstrating a paternal effect.116 While most of the epigenetic effects observed for the Avy allele can be explained
by a tendency to inherit DNA methylation patterns through
the female germline, the trans effect of the Avy allele, as
well as the paternal effects observed, suggest the involvement of another mechanism, which may be RNA mediated.
piRNA Inheritance and Imprinting in Mouse
The best evidence we have for RNA-mediated transgenerational inheritance in mammals is from the mouse. While
Chapter | 10 Heritable Epigenetic Effects Through RNA
miRNA Inheritance and Paramutation
in the Mouse
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miRNAs feature prominently among the maternally and
paternally supplied small RNAs in mouse, and their
absence can lead to developmental defects or lethality,5,6
for example, miR-34c, one of several miRNAs found in the
sperm and zygote, but not the oocyte.120 This observation
indicates that miR-34c is transmitted by the sperm to the
zygote. Using anti-miRNA injections, miR-34c appeared
to be critical for Bcl-2 regulation zygotic development.120
While there is some controversy regarding miR-34c’s
role in zygotic development (more recent knockout studies reveal no developmental defects in mice lacking miR34c121), these data demonstrate that in mouse, as in other
systems, miRNAs can be inherited from one generation to
the next via the gametes.
An inherited long non-coding RNA was suggested to
mediate paramutation at the Kit locus in mouse. Kit encodes
a tyrosine kinase receptor, and a null mutation was generated
by inserting a lacZ-neo cassette (Kittm1Alf).122 The Kittm1Alf
allele is homozygous lethal, and heterozygous mice have a
white tail tip and white feet. Crosses between heterozygotes
produced an excess of mice with the Kittm1Alf/+ phenotype
with many Kit +/+ mice exhibiting white tail tips and feet.52
Additional crosses revealed paramutation-like behavior of
Kittm1Alf in the heterozygote, with the wild-type allele being
altered in the presence of Kittm1Alf to produce the white tail
tip/white feet phenotype typical of Kittm1Alf heterozygotes52
(but see123,124).
Molecular analysis of this phenomenon points to its
dependence on RNA. The Kittm1Alf allele produces abnormal RNAs that are incorporated into the sperm. Injection
of RNA from a Kittm1Alf/+ heterozygous mouse into wildtype zygotes results in the white tail tip/white feet phenotype normally seen in Kittm1Alf/+ heterozygotes. This finding
indicates that the abnormal RNAs produced by the Kittm1Alf
allele can explain its paramutation-like behavior.52 The
degraded nature of the abnormal Kit RNAs suggested the
involvement of one of the small RNA pathways. Thus, two
miRNAs, miR-221 and miR-222, predicted to regulate Kit
were injected into wild-type zygotes; this treatment also
replicated the Kittm1Alf/+ phenotype.52 Follow-up work demonstrated that the paramutation-like behavior of Kittm1Alf
depends on the RNA methyltransferase Dnmt2, and that
RNA methylation is essential for this behavior.125 Together,
these experiments indicate that an RNA component transmitted via the gametes mediates the Kittm1Alf paramutation
behavior, an example of transgenerational inheritance via
RNA in mammals.
While most roles of miRNAs in early mammalian development do not involve unusual inheritance patterns, two
additional examples of paramutation-like behavior have been
described and linked to miRNAs in mouse. miR-124 injections into fertilized eggs lead to an overgrowth phenotype by
targeting Sox9.126 This overgrowth phenotype was inherited
transgenerationally if miR-124 was expressed ectopically in
the sperm.126 Similarly, injection of miR-1 targeting Cdk9
into embryos resulted in cardiac hypertrophy, an increase
in Cdk9 expression, and paramutation-like behavior of this
phenotype.127 miR-1 can be detected in mature sperm, and
the cardiac phenotype is inherited to at least the F3 generation.127 Thus, miRNAs appear to be able to mediate transgenerational epigenetic inheritance in mammalian systems,
similar to what is observed in some plant systems.
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mapping to Rasgrf1 were lost.118 In addition to the piRNAs, a non-coding RNA transcript spanning the DMR was
discovered that appears to be targeted by the piRNAs.118
Follow-up work showed that a Rasgrf1 fragment including
only the DMR and repeat region replicates the imprinting
behavior of the Rasgrf1 locus. The non-coding RNA and
piRNA target sequences were sufficient to induce imprinting behavior in a transgene.119 Thus, this case of imprinting requires a small RNA component as well as a longer
non-coding RNA, both of which are potentially inherited
through the germline.
115
TRANSGENERATIONAL EPIGENETIC
INHERITANCE IN HUMANS
EL
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Given the challenges associated with studies of transgenerational epigenetic inheritance in mammalian systems, it
is not surprising that direct evidence from humans is mostly
lacking. Thanks to improved techniques and several large
consortium studies, such as the ENCODE (Encyclopedia
of DNA Elements) project and the Roadmap Epigenomics Project, we have accumulated large amounts of “epigenomic” data from human cell lines and tissues, which
include DNA methylation as well as chromatin data. Despite
these advances, it remains unclear how stable these epigenomes are in the face of the reprogramming that occurs
during human gametogenesis and during early development, and if they indeed can be inherited.
Several lines of evidence suggest the occurrence of transgenerational epigenetic inheritance, recently reviewed by
Morgan and Whitelaw.128 Cohort studies investigating the
effects of parental and grandparental nutritional status on
offspring phenotypes reported evidence of transgenerational
effects (the Dutch “Hunger Winter”,129 but see 130,131). However, these studies are difficult to interpret, as several different mechanisms could explain the observed inheritance
patterns, including, but not limited to, transgenerational
epigenetic inheritance. A few case studies also seem to support the idea that some epigenetic information in the form of
DNA methylation can accidentally escape reprogramming.
For example, a study of Prader–Willi/Angelman syndrome
patients with imprinting defects suggests that the imprinting
SECTION | IV Basic Mechanisms/Processes of Epigenetic Inheritance
defects are due to incomplete erasure of the grandmother’s
imprint in the paternal germline.132 In addition, cases of
hereditary nonpolyposis colorectal cancer have provided
hints of heritable epialleles in humans (for example see 133),
but again, there are confounding factors that preclude a definite decision for or against epigenetic inheritance.
Overall, transgenerational epigenetic inheritance in
humans is not well established, and there is currently no
evidence for RNA-mediated transgenerational epigenetic
inheritance. Crucial experimental data are lacking and will
likely remain lacking due to the limitations of working with
humans.
CONCLUSION
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The experimental evidence reviewed above supports a role
for RNA-mediated transgenerational epigenetic inheritance
in most model systems. The RNAs involved tend to be noncoding and include both long as well as short transcripts,
with the strongest evidence pointing to small RNAs of the
siRNA, miRNA, and piRNA classes. Much of the data
available to date is indirect evidence for RNA-mediated
transgenerational epigenetic inheritance, and more direct
tests of this model are needed. An ideal experiment would
demonstrate the presence of a candidate RNA in the gamete
as well as the zygote prior to the onset of zygotic transcription. Furthermore, it would reveal the mechanism by which
this RNA mediates an epigenetic effect, and it would show
that this effect can be inhibited in the absence of the candidate RNA and ectopically initiated by an artificial introduction of the RNA. While such tests are challenging to carry
out, they are necessary to unequivocally demonstrate RNAmediated transgenerational epigenetic inheritance and will
allow the field to move forward. These tests are especially
important given growing interest in the idea that today’s
environmental stresses can affect subsequent generations
and that this might be mediated by induced expression
changes and inheritance of associated RNAs.
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ACKNOWLEDGEMENTS
The author would like to thank the members of her lab and three anonymous reviewers for their helpful comments on the manuscript. In addition, she would like to apologize to all her colleagues whose work
she was unable to cite due to space limitations.
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