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105 Transgenerational Epigenetics. http://dx.doi.org/10.1016/B978-0-12-405944-3.00010-6 Copyright © 2014 Elsevier Inc. All rights reserved. Heritable Generational Epigenetic Effects through RNA Nicole C. Riddle Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA Chapter 10 Chapter Outline Introduction 105 Transgenerational Inheritance Via the Gametes 106 Gametic RNA Populations 106 Small RNA Pathways 106 RNA-Guided Genome Rearrangements in Ciliates 108 Tetrahymena thermophila 108 Oxytricha trifallax 109 RNA Transport in Plant Systems 109 Paramutation in Maize 110 RNA Transfer Via the Gametes in Arabidopsis Thaliana 111 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 Conclusion 116 Acknowledgements 116 References 116 Abbreviations 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 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 sur- vival; 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 meth- ylation and chromatin modifications come to mind. Epi- genetic 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 ELSEVIER

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  • Heritable Generational Epigenetic Effects through RNANicole C. RiddleDepartment of Biology, University of Alabama at Birmingham, Birmingham, AL, USA

    Chapter 10

    Chapter OutlineIntroduction 105Transgenerational Inheritance Via the Gametes 106Gametic RNA Populations 106Small RNA Pathways 106RNA-Guided Genome Rearrangements in Ciliates 108

    Tetrahymena thermophila 108Oxytricha trifallax 109

    RNA Transport in Plant Systems 109Paramutation in Maize 110RNA Transfer Via the Gametes in Arabidopsis Thaliana 111

    Transgenerational Inheritance of RNAi in Caenorhabditis Elegans 111The piRNA Pathway, Transposon Silencing, and Hybrid Dysgenesis in Drosophila Melanogaster 112Transgenerational Epigenetic Inheritance in Mammals 113

    piRNA Inheritance and Imprinting in Mouse 114miRNA Inheritance and Paramutation in the Mouse 115

    Transgenerational Epigenetic Inheritance in Humans 115Conclusion 116Acknowledgements 116References 116EV

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    Transgenerational Epigenetics. http://dx.doi.org/10.1016/B978-0-12-405944-3.00010-6Copyright 2014 Elsevier Inc. All rights reserved.

    AbbreviationsDMRs Differentially methylated regionsdsRNA Double-stranded RNAENCODE Encyclopedia of DNA ElementsEndo-siRNAs Endogenous siRNAsGFP Green fluorescent proteinH3K9me2 Histone 3 lysine 9 dimethylationIAP Intracistral A particleIES Internal eliminated sequencelncRNAs Long non-coding RNAsmRNAs Messenger RNAsmiRNAs MicroRNAspri-miRNAs Primary miRNA transcriptspiRNAs PIWI-interacting RNAsRdDM RNA-directed DNA methylationRISC RNA-inducing silencing complexRNAi RNA interferencerRNAs Ribosomal RNAsscnRNAs Scan RNAssiRNAs Small interfering RNAsviRNA Virus-derived small interfering RNAsTEs Transposable elementstRNAs Transfer RNAs

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    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 sur-vival; 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 meth-ylation and chromatin modifications come to mind. Epi-genetic 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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    epigenetic inheritance. RNAs carry out a wide array of functions depending on their sequence composition; these include enzymatic functions (ribozymes), regulatory func-tions (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 transgen-erational 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 can-didate 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

    While transgenerational effects can be mediated by many means (communication via the placenta in mammals, anti-bodies 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 com-paring 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 con-tributed (however, see below). Analysis of the oocyte cyto-plasm 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.

    GAMETIC RNA POPULATIONS

    Transcriptome studies from oocytes show that the mater-nally 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). MicroR-NAs (miRNAs), for example, feature prominently among the maternally supplied small RNAs,5,6 as do classes of small

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    interfering RNAs (siRNAs) for example, endogenous siRNAs (endo-siRNAs) in the mouse7,8 and PIWI-inter-acting 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 specifica-tion, 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, sev-eral hundred to several thousand transcripts are detected in sperm (for examples see 13,1517), 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 pres-ence 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.

    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 2030 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 epigen-etic gene silencing, and many others.21,22 Small RNAs are of great interest in studies of transgenerational epigenetic

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    inheritance because they can potentially provide sequence specificity. They can act as guides to specific genomic loca-tions by sequence homology and are known to recruit vari-ous proteins to target sites, including epigenetic modifiers such as DNA methyltransferases.23

    Generally speaking, there are three basic RNAi path-ways, which have been elaborated upon by various spe-cies (for an introduction to the small RNA pathways, see 24). This description is based on the Drosophila mela-nogaster 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 DCR-2, 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

    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). Pro-duction 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 miRNARISC causes translational repres-sion of matching mRNAs and mRNA destabilization, lead-ing 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

    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 homolo-gous 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

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    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 chro-matin 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 inher-ited from one generation to the next can produce epigen-etic effects and thus mediate transgenerational epigenetic inheritance.

    RNA-GUIDED GENOME REARRANGEMENTS IN CILIATES

    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 mac-ronucleus. While the exact number of these nuclei depends on the species, the micronucleus represents the germline and is transcriptionally silent, while the macronucleus cor-responds 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 rear-ranged compared to the micronucleus, DNA is eliminated, and chromosomes are fragmented (reviewed in 26). In Tetra-hymena thermophila, approximately 30% of the micronu-clear 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). Stud-ies of these processes in several ciliate species demonstrate EL

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    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.

    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 elimi-nation,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 pre-cursors are produced specifically during sexual reproduc-tion 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 reten-tion of the IES in the new macronucleus.33,34 As the paren-tal macronucleus does not contribute DNA to the progeny, a model of genome scanning has been proposed.29,32

    The genome-scanning model suggests that bidirec-tional 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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    FIGURE 10.2 Nuclear differentiation in Tetrahymena. Left panel: The zygotic product contains a diploid nucleus (MIC), and the parental macro-nucleus (MAC). Middle panel: The zygotic diploid nucleus divides to produce two new micronuclei (MIC). Right panel: One of the new nuclei differenti-ates into the new macronuleus (MAC), while the other remains, forming the new micronucleus (MIC). The old parental macronucleus degenerates (light grey sickle shape).

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    where they scan the macronucleus by subtractive hybridiza-tion 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 mac-ronucleus. These remaining scnRNAs are shuttled back to the developing macronucleus, where they recruit chromatin proteins to homologous sequences (the IESs) and coordi-nate their elimination from the new macronucleus. DNA elimination by this mechanism takes place in Tetrahymena and Paramecium, leading to the elimination of 1530% of the micronuclear genome.28 Thus, communication through RNA between the developing F1 macronucleus and the parental macronucleus orchestrates the somatic develop-ment in the F1.

    Oxytricha trifallax

    Oxytricha, another microbial eukaryote, employs a different RNA-dependent mechanism to achieve macronuclear devel-opment. In Oxytricha, only 5% of the germline genome is retained in the somatic macronuleus.25,35 Instead of mark-ing 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 mac-ronucleus 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 piR-NAs into the parental macronucleus will lead to retention of homologous sequences in the next generation,36 demon-strating that RNA mediates genome rearrangements in Oxy-tricha. 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 macro-nuclear 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

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    developed several complex systems to orchestrate the genome rearrangement necessary to generate their somatic macro-nucleus. The species examined to date all utilize RNA-based transgenerational communication, indicating that this mode of information transfer might be commonly used.

    RNA TRANSPORT IN PLANT SYSTEMS

    Some of the earliest support for RNA-based transgenera-tional 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 tis-sues. Depending on the plant species, this switch to the repro-ductive phase is terminal (in annual plants such as Arabidopsis thaliana), or can happen repeatedly (in perennials such as trees). Independent of the plants growth habit, the late specifi-cation 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 macro-molecules between cells, as well as systemically through the entire plant. Intercellular transport is mediated by plasmo-desmata openings that span the cell wall and allow com-munication between the cytoplasm of two neighboring cells. The plasmodesmata provide connections between cells and facilitate the exchange of macromolecules, including sev-eral 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 col-leagues 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 mediat-ing 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 vas-cular system that moves nutrients throughout the plant in what is commonly known as sap. In addition to various

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    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 tis-sue, 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 Long-distance 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.

    PARAMUTATION IN MAIZE

    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 4852). Paramutation is best understood for genes affect-ing 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, homozy-gous B plant is crossed to a purple homozygous B-I plant (reviewed in 54). In F1, all plants are heterozygous, exhibit-ing 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 geno-type, 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 paramuta-tion suggest that RNA might be the factor communicat-ing between the two alleles in trans. Mutants defective for either the initiation or maintenance of paramutation often represented RNAi pathway components that had been pre-viously identified in Arabidopsis thaliana. Plant RNAi pathways differ somewhat from the three basic pathways described above. In plants, siRNAs can direct DNA meth-ylation (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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    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 molecu-lar mechanisms as well, overall the study of paramutation supports a role for RNA in mediating its transgenerational epigenetic effects.

    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 par-ent, 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.

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    RNA TRANSFER VIA THE GAMETES IN ARABIDOPSIS THALIANA

    A second line of evidence for RNA-mediated transgen-erational 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 gen-erative nucleus produces the two sperm nuclei needed for double fertilization.

    During gamete formation in many systems including mammals, epigenetic information such as DNA methyla-tion and imprinting is reprogrammed to return to a plu-ripotent state in the zygote.6466 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 vul-garis discovered by Linneaus,67 the SUPERMAN epialleles causing homeotic transformations in Arabidopsis thali-ana,68 and the FWA epialleles causing late flowering also in Arabidopsis.69 Despite the leaky nature of the repro-gramming, 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 gameto-phyte, 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 mainte-nance 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 reactiva-tion of TEs,70 an unusual observation, as maintenance of TE silencing in the germline is paramount to prevent com-promising 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 retrotranspo-son 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

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    early embryo.73 Thus, it appears that siRNAs in pollen can guide TE silencing in the next generation and thus commu-nicate 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 develop-ment, as are SGS3 and RDR6,74 both of which are involved in the non-cell autonomous RNAi pathway.7577 Interest-ingly, 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 intercel-lular 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 recov-ery 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 cen-tral cell, GFP levels are reduced in the egg cell. This result indicates that the miRNA was able to move from the cen-tral 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, trans-generational epigenetic inheritance via siRNAs appears to be responsible for maintaining TE repression to ensure genome integrity.

    RANSGENERATIONAL 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 par-ent 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 silenc-ing and mRNA degradation in the cytoplasm (cytoplasmic RNAi) or through transcriptional silencing by chromatin modification (nuclear RNAi).23 In C. elegans, the inheri-tance of silencing induced by dsRNA to the F1 generation

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    depends on the presence of a functional nuclear RNAi path-way.81 nrde () mutants lacking a functional nuclear RNAi pathway are capable of a silencing response to dsRNA treat-ment, but they are unable to maintain this silencing in the F1 past the embryonic stage.81 In wild-type F1, H3K9me3 (his-tone 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 trans-mitted 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.8285 the piRNA pathway,8688 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 bind-ing protein91) are required for generating the silencing sig-nal, 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 inheri-tance 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 consis-tent 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 silenc-ing 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 technologi-cal advances in sequencing technologies. Rechavi and col-leagues investigated a special case of RNAi, virus-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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    | 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 transcrip-tional 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 can-didate for a transmitted silencing factor, as they are gener-ated in the germline.87 Despite lack of data from C. elegans, based on their localization patterns and data from other sys-tems (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.

    HE piRNA PATHWAY, TRANSPOSON SILENCING, AND HYBRID DYSGENESIS IN DROSOPHILA MELANOGASTER

    Research in Drosophila melanogaster has contributed greatly to our understanding of epigenetic processes and has led to several discoveries relating to RNA-mediated trans-generational 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 9395). 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. Pri-mary piRNAs originate directly from the long piRNA pre-cursor transcripts, while secondary piRNAs are produced by an amplification loop termed ping-pong amplification (reviewed in 93). In the female germline, piRNAs are mater-nally deposited in the oocyte and function in the embryo to silence TEs.96,98101 Their germline function has made piR-NAs of great interest to researchers focused on transgenera-tional 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 steril-ity 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

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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, dys-genesis 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 germ-line 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 mater-nally 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 sup-plied; 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 pater-nally inherited I-element during gametogenesis and steril-ity.99 These findings were confirmed using the P-element dysgenesis system, indicating that piRNAs of several types are maternally deposited and induce transgenerational epi-genetic silencing.

    Several research groups tested this model of TE silenc-ing experimentally. Ronsseray and colleagues used a trans-silencing system to show that insertion of a transgene in this case a P-element within a piRNA cluster at the sub-telomere can induce silencing of a second P-element con-struct sharing sequence similarity at a second location in the genome (105 and earlier references therein). This trans-silencing 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

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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 incor-poration of sequence elements in piRNA loci can lead to silencing of matching sequences in trans, and that piR-NAs 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 match-ing 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 heterochro-matin, is recruited to the ectopic site, as is SU(VAR)3-9, one of the three H3K9 histone-methyltransferases in Dro-sophila.110 Together, these data demonstrate that recruit-ment of piRNAs leads to the formation of a silent chromatin structure. These findings suggest that the piRNAs transmit-ted through the oocyte enable the offspring to efficiently silence matching sequences such as TEs.

    The small RNAs are also responsible for hybrid dysgen-esis 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 match-ing the misregulated TEs in the maternal lineage.111 In the wild-type situation, TE-derived small RNAs are transmit-ted through the germline to the next generation where they induce silencing of TEs matching their sequences. Interest-ingly, 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 mecha-nisms 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.

    RANSGENERATIONAL EPIGENETIC INHERITANCE IN MAMMALS

    In mammalian systems especially, the study of transgen-erational 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 off-spring (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,

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    Parent F1 ospring F2 ospring

    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.

    it is not surprising that unequivocal evidence for transgen-erational epigenetic inheritance is rare in humans and not linked to a particular molecular mechanism.

    There are several natural cases of epigenetic transgener-ational 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)) inser-tion drives ectopic expression of agouti in a DNA methyl-ation-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 meth-ylation 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; how-ever, 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 methyltrans-ferase 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 epi-genetic 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 involve-ment of another mechanism, which may be RNA mediated.

    piRNA Inheritance and Imprinting in Mouse

    The best evidence we have for RNA-mediated transgenera-tional inheritance in mammals is from the mouse. While

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    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 mamma-lian systems as well. The basic pathways have been con-firmed 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, evi-dence for transgenerational epigenetic inheritance exists in mammalian systems as well.

    In addition to their traditional role of transposon silenc-ing 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 methyl-ated regions (DMRs). The DMRs control the expression levels of imprinted loci by regulating access of the tran-scriptional machinery. Methylation at the DMRs is reset every generation during gametogenesis; first the old meth-ylation 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 sug-gests the additional involvement of piRNAs and a non-cod-ing 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 trans-poson-derived sequence in the DMR, the piRNA pathway proteins MILI1, MIWI2, and MitoPLD (the mouse homo-log 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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    mapping to Rasgrf1 were lost.118 In addition to the piR-NAs, 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 imprint-ing behavior in a transgene.119 Thus, this case of imprint-ing requires a small RNA component as well as a longer non-coding RNA, both of which are potentially inherited through the germline.

    miRNA Inheritance and Paramutation in the Mouse

    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-34cs role in zygotic development (more recent knockout stud-ies reveal no developmental defects in mice lacking miR-34c121), 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 abnor-mal RNAs that are incorporated into the sperm. Injection of RNA from a Kittm1Alf/+ heterozygous mouse into wild-type zygotes results in the white tail tip/white feet pheno-type 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

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    replicated the Kittm1Alf/+ phenotype.52 Follow-up work dem-onstrated 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 trans-mitted 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 devel-opment do not involve unusual inheritance patterns, two additional examples of paramutation-like behavior have been described and linked to miRNAs in mouse. miR-124 injec-tions 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 genera-tion.127 Thus, miRNAs appear to be able to mediate trans-generational epigenetic inheritance in mammalian systems, similar to what is observed in some plant systems.

    RANSGENERATIONAL EPIGENETIC INHERITANCE IN HUMANS

    Given the challenges associated with studies of transgen-erational 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 Epigenom-ics Project, we have accumulated large amounts of epig-enomic 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 epig-enomes are in the face of the reprogramming that occurs during human gametogenesis and during early develop-ment, and if they indeed can be inherited.

    Several lines of evidence suggest the occurrence of trans-generational 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). How-ever, these studies are difficult to interpret, as several dif-ferent mechanisms could explain the observed inheritance patterns, including, but not limited to, transgenerational epigenetic inheritance. A few case studies also seem to sup-port the idea that some epigenetic information in the form of DNA methylation can accidentally escape reprogramming. For example, a study of PraderWilli/Angelman syndrome patients with imprinting defects suggests that the imprinting

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    defects are due to incomplete erasure of the grandmothers 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 defi-nite 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

    The experimental evidence reviewed above supports a role for RNA-mediated transgenerational epigenetic inheritance in most model systems. The RNAs involved tend to be non-coding 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 transcrip-tion. 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 candi-date RNA and ectopically initiated by an artificial introduc-tion of the RNA. While such tests are challenging to carry out, they are necessary to unequivocally demonstrate RNA-mediated transgenerational epigenetic inheritance and will allow the field to move forward. These tests are especially important given growing interest in the idea that todays environmental stresses can affect subsequent generations and that this might be mediated by induced expression changes and inheritance of associated RNAs.

    ACKNOWLEDGEMENTSThe author would like to thank the members of her lab and three anon-ymous reviewers for their helpful comments on the manuscript. In ad-dition, she would like to apologize to all her colleagues whose work she was unable to cite due to space limitations.

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