Timeline: Genomic Imprinting: the Emergence of an Epigenetic Paradigm

PERSPECTIVES

Evidence of genomic imprinting TIMELINE Early observations, particularly in insects and plants, indicated that the appearance of Genomic imprinting: the emergence a particular visible trait in offspring could differ depending on whether it was transmit- of an epigenetic paradigm ted from the mother or the father. In some of the early studies, imprinting effects were observed cytogenetically and, as such, were Anne C. Ferguson-Smith seen to affect whole chromosomes. However, Abstract | The emerging awareness of the contribution of epigenetic processes to genetic experiments suggested that parental- origin effects could also act at the level of genome function in health and disease is underpinned by decades of research in the gene. model systems. In particular, many principles of the epigenetic control of genome function have been uncovered by studies of genomic imprinting. The phenomenon Whole chromosome effects. Historically, of genomic imprinting, which results in some genes being expressed in a parental- there have been several examples of -origin-specific manner, is essential for normal mammalian growth and parental-origin-specific ‘marking’ of whole chromosomes documented in the litera- development and exemplifies the regulatory influences of DNA methylation, ture. Indeed, the term ‘imprinting’ was first chromatin structure and non-coding RNA. Setting seminal discoveries in this field coined by the cytogeneticist Helen Crouse1 alongside recent progress and remaining questions shows how the study of in 1960 to describe the programmed imprinting continues to enhance our understanding of the epigenetic control elimination of one or two paternally derived of genome function in other contexts. X chromosomes in sciarid flies. Sciarid zygotes inherit two paternally derived and one maternally derived X chromosome. In Genomic imprinting is a remarkable epige- well-established roles in many other contexts, female embryos, a single paternally inherited netically regulated process that causes genes such as in stem cell programming, cancer epi- X chromosome is eliminated, but in males to be expressed in a parental-origin-specific genetics and cis-acting mechanisms of gene both paternally inherited X chromosomes manner rather than from both chromosome regulation. As such, genomic imprinting was are selectively lost from somatic nuclei. homologues. Thus some imprinted genes one of the first, and remains one of the most Crouse recognized that the imprint iden- are expressed from the paternally inherited informative, paradigms for understanding tifying the X chromosome as maternal or allele, and other genes are expressed from the the consequences of interactions between the paternal in origin was determined by the maternally inherited allele. Parental-origin genome and the epigenome. sex of the germ line through which it was effects in plants, insects and mammals were A historical perspective of some of these inherited1. Parental-origin effects were also noted around 40 years ago. However, it was discoveries is presented here (with key described in the sex determination mecha- the subsequent elegant embryological and events shown in the TIMELINE), illustrating nism of coccid insects2. These insects lack sex genetic manipulations in the mouse that how embryological studies, combined with chromosomes and in males the paternally placed imprinting on the map and led to its classical and molecular genetic studies, have derived chromosome set becomes hetero- recognition as an important paradigm of provided a framework for the more sophis- chromatic, inactive and is not transmitted to epigenetic inheritance. This, coupled with ticated epigenetic and genomic approaches offspring. Epigenetic differences between the detailed genome mapping studies on human that are applied today. This Timeline article maternally and paternally inherited coccid patients with disorders exhibiting parental- includes consideration of the wider implica- chromosome sets have been described but origin effects in their patterns of inheritance, tions of the chromosomal secrets revealed the underlying processes that establish the provided the molecular basis for the iden- through imprinting research and their parent-specific imprint are not understood. tification of the first endogenous imprinted impact on our understanding of epigenetic Sex chromosome dosage compensation genes and the genomic features and epi- inheritance. The extent and functional is a well-established whole chromosome genetic mechanisms responsible for their implications of genomic imprinting are con- model in which parental-origin effects are mono-allelic expression. These early studies sidered, its regulatory mechanisms reviewed studied. In mammals, females with two X of genomic imprinting established essential and its contribution to health and disease chromosomes achieve parity with males in roles for differential DNA methylation, allele- discussed. Finally, some of the unresolved their X-linked gene dosage through epige- specific histone modifications, large non-coding issues and wider questions that are emerging netic inactivation of one X chromosome. RNAs and dynamic developmental changes are suggested, thus providing a glimpse of In marsupials, X-chromosome inactivation in the epigenetic programme. These factors the challenges and exciting prospects facing is imprinted, with the paternally inherited that influence imprinted domains now have the future of this field. X chromosome being inactive in somatic

Timeline | Key events in imprinting research

Pronuclear transfer experiments Johnson6 describes a Preferential paternal prove the functional Identification21,23,24,26 of the The term ‘imprinting’ mouse mutant with X-chromosome inactivation non-equivalence of parental PWS identified as an imprinted first imprinted genes in is coined1 parental-origin effects is described in mammals4 genomes in the mouse13,14 disorder in humans40 mammals: Igf2r, Igf2 and H19

1960 1970 1974 1975 1980 1984 1985 1989 1990 1991

An imprinted gene is first Lubinksy et al.111 identify Recognition of uniparental Specific genomic regions are Publication of the first recognized in plants5 parental-origin effects in BWS disomy in humans38 discovered that function differently imprinting map in the mouse112 depending on their parental origin9

BWS, Beckwith–Wiedemann syndrome; CTCF, CCCTC-binding factor; ICR, imprinting control region; Igf2, insulin-like growth factor 2; Igf2r, IGF2 receptor; piRNA, PIWI-interacting RNA; PWS, Prader–Willi syndrome; RNA-seq, high-throughput RNA sequencing.

cells3. Interestingly, in mice, X-chromosome Genetic studies in the mouse also con- abnormalities in their behaviour, growth inactivation is similarly imprinted, but ducted in the 1970s suggested evidence and/or viability. Although the effects of egg specifically during pre-implantation stages for imprinting on autosomes. In 1974, cytoplasm or the uterine environment were and in extra-embryonic lineages including Johnson6 described a deletion at the Tme not excluded8, these results suggested dif- the placenta4. In mice, random inactiva- locus that showed in utero lethality when ferential expression from the two parental tion initiates in all embryonic components maternally inherited but not when transmit- chromosome homologues. Over subsequent around the time of implantation. The ted through the male germ line. Mapping years, Cattanach and colleagues9,10 used this molecular mechanism that distinguishes the of the phenotype for this and other Tme approach to screen the whole mouse genome paternal from the maternal X chromosome deletions indicated that the parental-origin for defective outcomes that were caused and establishes imprinted X-chromosome effect localized to a 0.8–1.1 Mb region of by altering the dosage of parental chromo- inactivation is currently unknown. In par- chromosome 17. This provided the basis for somes. Those studies identified around 13 ticular, it will be interesting to learn the the identification of the first endogenous subchromosomal regions for which there extent to which the underlying epigenetic imprinted gene (see below). is a requirement for both a maternally and features conferring parental-origin-specific Around the same time, Cattanach, paternally inherited chromosome region identity in phenomena involving whole Beechey and Searle were working with mice for normal development (see also the chromosomes overlap with those regulating harbouring either Robertsonian translocations MouseBook imprinting catalogue). Most the imprinted expression and repression of or reciprocal translocations, and they were able murine imprinted genes identified to date individual genes. to manipulate the parental origin of par- map to these regions. Uniparental disomy ticular chromosome regions. Their research conceptuses and their wild-type littermates Early evidence that specific genes are extended the earlier work of Snell7, who continue to be used to understand imprint- imprinted. In 1970, one of the first convinc- had first observed a genetic outcome now ing phenotypes and for molecular analyses in ing pieces of experimental evidence for the coined ‘non-complementation lethality’ in which expression and epigenetic features on imprinting of specific genes was described translocation intercross mice. Translocation the maternally versus the paternally inher- in plants. Through rigorous experiments on heterozygotes can give rise to unbalanced ited chromosomes need to be distinguished. the inheritance of maize kernel coloration, gametes — eggs and sperm that are dupli- Kermicle5 recognized that R alleles carried cated or deficient for chromosomal regions Embryological investigations of imprinting. by the female gametophyte may be function- that were involved in the translocation. So, Perhaps the defining experiments that ally different from those carried by sperm. when such translocation heterozygotes are proved the functional non-equivalence With remarkable foresight, Kermicle stated intercrossed, the fusion of complementary of mammalian parental genomes were that the effect he observed was a “parage- unbalanced gametes (for example, an egg the elegant pronuclear transplantation netic rather than a conventional genetic with a maternally duplicated region fer- analyses performed by the Solter and Surani phenomenon” and concluded that the effect tilized by a sperm with deficiency for the laboratories11–14 and published in the early was influencing the expression, not the same region) would be expected to result 1980s. Newly fertilized mouse eggs were genetic make-up, of the R allele. The term in fully viable, balanced diploid zygotes manipulated by removing and replacing the ‘epigenetic’ has now superseded Kermicle’s with so-called uniparental disomy of a whole paternal pronucleus with a second mater- original ‘paragenetic’ description. The rec- chromosome, or uniparental duplication/ nal one to generate a diploid, genetically ognition that the ‘imprint’ is not dependent deficiency for a particular chromosomal bimaternal (also known as gynogenetic or on the DNA sequence, but rather the paren- region. However, they noted that in some of parthenogenetic) conceptus. Alternatively, tal germline environment through which these balanced cases, normal complemen- the maternal pronucleus was replaced with the gene passes, now defines the process of tation did not occur and the uniparental a second paternal one to generate a dip- imprinting. duplication or deficiency embryos had loid, genetically bipaternal (androgenetic)

Timeline | Key events in imprinting research The de novo DNA Identification of Discovery81–83,113 that methyltransferase Discovery of a requirement differential DNA methylation-sensitive A large, non- DNMT3A is shown A maternal-zygotic factor is identified for the piRNA pathway to methylation at Identification binding of CTCF regulates coding RNA is to establish male as being essential for maintaining DNA establish germline methylation imprinted of a germline- reciprocal imprinting of shown to regulate and female methylation imprints during at an imprinted domain in domains48–50 derived ICR54 H19 and Igf2 imprinting77 germline imprints63 pre-implantation reprogramming75 the paternal germ line107

1993 1995 2000 2001 2002 2004 2008 2009 2011

Evidence from a Dnmt1 Imprinting is discovered DMNT3L is shown to be Manipulation of An RNA-seq Demethylation of histone H3 knockout mouse that DNA in marsupials required for de novo imprinted loci on data set from lysine 4 is found to be required methylation is essential for the (non-eutherian germline methylation maternal chromosomes reciprocal hybrids for establishing a maternal maintenance of imprinting51 mammals)87,99 imprinting64 allows a bimaternal is used to identify germline methylation mark70 mouse to develop novel imprinted to term19 transcripts30

conceptus. The failure of embryos without It is noteworthy that in humans, partheno distal chromosome 7, compared to normal the two different parental genomes to pro- genetic bimaternal ovarian teratomas and littermates, also indicating repression of Igf2 ceed past mid-gestation suggested that the androgenetic bipaternal conceptuses on the maternally inherited chromosome mammalian genome possessed genes that (complete hydatidiform moles) have related and expression from the paternally inherited were somehow ‘marked’ differently on the phenotypes to those in the mouse. allele24. Located approximately 90 kb down- two parental genomes11–14. This was further stream from the murine Igf2 gene lies the emphasized in studies proving that the fail- Identification of imprinted genes H19 gene, a non-coding RNA of unknown ure of uniparental conceptuses was not due Identification of the first endogenous function that contains a conserved mam- to oocyte cytoplasmic defects15,16. Although imprinted genes. The first three endogenous malian microRNA, miR‑675 (REF. 25). Using not the first use of the term, ‘imprinting’ was imprinted genes in the mouse were identi- hybrid embryos in which expression from specifically applied in this context to refer to fied in 1991. The first to be published was the the two parental alleles could be distin- the phenomenon describing the non-genetic gene responsible for the parental-origin effect guished by strain-specific polymorphisms, difference that distinguishes the two paren- that Johnson had described at the Tme locus Tilghman and colleagues26 found that H19 tal genomes, resulting in their functional on mouse chromosome 17. By assessing the was expressed from the maternally inherited non-equivalence. expression of positionally cloned candidate chromosome. Hence two adjacent genes, Parthenogenetic conceptuses devel- genes falling within the minimal deletion Igf2 and H19, show reciprocal patterns of oped tissues predominantly of embryonic region, Barlow and colleagues21 showed that imprinted expression (FIG. 1Ba). Interestingly, origin, with a failure in the development the gene encoding the insulin-like growth H19 and Igf2 are generally co-expressed, of the extra-embryonic lineages, whereas factor 2 receptor (Igf2r) was expressed solely sharing mesodermal and endodermal androgenetic conceptuses developed from the maternally inherited chromosome; enhancers. Therefore, the study of the organ- predominantly extra-embryonic lineages the paternally inherited copy was repressed. ization and regulation of this imprinted and lacked, or had very underdeveloped, Subsequent studies22 indicated that Igf2r is domain provided an excellent paradigm embryonic components. This suggested one of a cluster of imprinted genes on the for the mechanistic studies to decipher that the two parental genomes provided proximal region of mouse chromosome 17 imprinting control, outlined below. reciprocal functions, at least during the first (FIG. 1Aa). IGF2R functions both as a half of gestation, and that the absence or mannose‑6‑phosphate receptor and also Ongoing identification of imprinted genes. overexpression of imprinted genes exclu- as a receptor for insulin-like growth It is noteworthy that the four studies describ- sively expressed from either the maternal or factor 2 (IGF2). ing the first three endogenous imprinted paternal genome caused the developmental The Igf2 gene was shown in two studies genes in mouse used four different methods failure. Subsequent studies that mixed to be imprinted too. Mice carrying a tar- to determine their imprinting. These four androgenetic or parthenogenetic cells with geted deletion of Igf2 on the distal portion of approaches — targeted deletion, allele- normal cells to form chimaeras provided chromosome 7 showed a growth deficiency specific activity in hybrids, positional clon- evidence of further roles for imprinted phenotype on paternal transmission but not ing and the use of uniparental duplication/ genes in the development of particular on maternal transmission. Expression ana- deficiency conceptuses from translocation embryonic lineages, such as mesodermal lyis showed that Igf2 was transcribed from intercrosses — illustrate the range of dif- derivatives and the brain17,18. More recent the paternally inherited chromosome and ferent methodologies that allow expression experiments, in which the dosage of repressed on the maternally inherited one23 from maternal or paternal chromosomes imprinted genes was genetically manipu- — imprinting that is reciprocal to that of to be distinguished. Subsequently, many lated, have confirmed that perturbed Igf2r. In a second study, Igf2 was found to be more imprinted genes have been identified. imprinting is the only barrier to successful repressed in embryos with maternal unipa- To date approximately 100 imprinted genes parthenogenetic development in mice19,20. rental duplication and paternal deficiency of have been validated as being imprinted in

#/CVGTPCNN[OGVJ[NCVGF C /CVGTPCNN[GZRTGUUGFIGPG /CVGTPCN

+IHT 5NEC 5NEC 5NEC 2CVGTPCNN[GZRTGUUGFIGPG

2CVGTPCN $KCNNGNKECNN[GZRTGUUGFIGPG #KTP

D 4GRTGUUGFIGPG /CVGTPCN

1UDRN 6PHTUH %CTU 0CRN65NEC -EPS 6TRO UUE #UEN /GVJ[NCVGF+%4 6PHTUH 6PHTUH 2JNFC %FMPE %F 6URCP

2CVGTPCN 7POGVJ[NCVGF+%4

-EPSQV /GVJ[NCVGFUGEQPFCT[&/4 E /CVGTPCN 7POGVJ[NCVGFUGEQPFCT[&/4

0GUR )PCU ∗ 2CVGTPCN Figure 1 | Imprinted clusters in mammals. 0GURCU)PCUZN 'ZQP# Schematic representations in mouse of four )PCU imprinted clusters that are regulated by maternally methylated germline imprinting control F regions (ICRs) (Aa–d) and three clusters that are /CVGTPCN regulated by paternally methylated germline ICRs (Ba–c). For all seven clusters, targeted dele- #5+% 295+% 7DGC #VRC tion of the ICR in the mouse has proven their role as elements controlling parental-origin-specific gene expression across the whole imprinted 2CVGTPCN domain. Aa | The insulin-like growth factor 2 (TCV/CIGN 5PWTH5PTRP UPQ40#U 7DGCCU receptor (Igf2r) cluster. Ab | The Kcnq1 cluster. Kcnq1 encodes a tissue-specifically imprinted voltage-gated potassium channel that is not $2CVGTPCNN[OGVJ[NCVGF imprinted in cardiac muscle. Ac | The Gnas clus- C &/4 &/4 &/4 ter is named after the guanine nucleotide bind- /CVGTPCN %6%( ing protein, α-stimulating (Gnas) gene. Note that although the germline differentially methylated *&/& * 'PJCPEGTU region (DMR) encompasses both the neuro endocrine secretory protein antisense (Nespas) 2CVGTPCN and Gnasxl promoters, the ICR itself (indicated by the asterisk) covers the Nespas promoter. +PU +IH Ad | The Snrpn cluster, which in humans is associated with Prader–Willi syndrome (PWS) and D Angelman syndrome. Ba | The Igf2–H19 cluster /CVGTPCN %6%( harbouring the Igf2 gene and the non-coding RNA gene H19, which contains the microRNA 2WVCVKXG miR‑675. DMR0 is placenta-specific and its GPJCPEGTU &/& germline status is not known. Bb | The RAS 2CVGTPCN protein-specific guanine nucleotide releasing factor 1 (Rasgrf1) cluster. The tandem repeats #46CPFGOTGRGCVU CUITH are required for the paternal germline methylation of the ICR. Bc | The Delta-like homologue 1 E OK40#U OK40#U (Dlk1)–Dio3 cluster. Multiple imprinted, non- /CVGTPCN coding RNAs are expressed from the maternally inherited chromosome. For example, AntiRtl1 encodes seven microRNAs (miRNAs). The small +)&/4 )VN #PVK4VN %&5PQ40#U /KTI nucleolar RNA (snoRNA)-containing gene is also 2CVGTPCN known as Rian. The genes and clusters are not drawn to scale. CTCF, CCCTC-binding factor. Figure is modified, with permission, from REF. 52 $GICKP &NM 4VN &KQ © (2007) Elsevier Science.

the mouse (see the Catalogue of Imprinting of inheritance (BOX 1). These disorders are imprint must be erased in the germ line such Effects and the Medical Research Council usually caused by uniparental disomy or that appropriate parental-origin-specific Harwell Genomic Imprinting homepage). deletions. Indeed, uniparental disomy as a identity can be established in the gametes for Many, though not all, of these imprinted concept was described in humans by Engel38 the next generation. genes are located in clusters (FIG. 1). in 1980 through the recognition of meiotic In vivo allele-specific expression analyses errors that gave rise to gametes with the loss What is the mark? DNA methylation is — in normal hybrids, in genetic models of (nullisomy) or addition (disomy) of certain the only epigenetic modification known to defective imprinting or in knockout mice chromosomes. More recently an increased fulfil all four of these properties. In mam- — have proven that some imprinted genes incidence of imprinted disorders has been malian cells, DNA methylation modifies show tissue-specific imprinting or cell-type- associated with assisted reproductive predominantly CpG dinucleotides and is specific absence of imprinting. For example, technologies39. associated with a transcriptionally repressed Igf2 is bi-allelically expressed specifically Of the many notable contributions, state47. Not long after the identification of in the choroid plexus and leptomeninges of the analyses of patients with Beckwith– the first imprinted genes, it was shown that the brain23. Another paternally expressed Wiedemann syndrome (BWS) — an the two parental chromosomes at imprinted imprinted gene, Delta-like homologue 1 overgrowth disorder that is also associ- loci were differentially marked by DNA (Dlk1), shows selective absence of imprint- ated with an increased incidence of child- methylation48–50. Such regions are known as ing in the postnatal neurogenic niche27 (see hood tumours — and the neurological differentially methylated regions (DMRs). below). By contrast, several other imprinted disorders Prader–Willi syndrome (PWS) Not only did this finding of parental-origin- genes are imprinted in one or few tissues, and Angelman syndrome40–45 provided specific DNA methylation provide a starting with the placenta being a predominant site important early advances in identifying and point for determining the developmental of tissue-specific imprinting28,29. understanding the regulation of imprinted dynamics of the epigenetic programme at Most recently, the emergence of next- genes. PWS and Angelman syndrome are these genes, but it also resulted in the evolu- generation sequencing technology and the two distinct syndromes that map to the tion of an epigenetic paradigm for studying genome-wide identification of strain-specific same domain on human chromosome 15, cis-acting mechanisms of gene regulation. polymorphisms have allowed sequencing but that differ in the parental origin of Such mechanisms can act over short or long of the transcriptomes of tissues from the underlying range of genetic and epi- distances and can reveal links between the reciprocal hybrid mice, with the potential genetic defects46. Studies of patients and epigenetic state, the chromatin structure to identify more elusive tissue-specific clinical samples led to improved genomic and genome function. Key experiments imprinted genes. This approach has con- resolution of clusters of human imprinted from Li, Beard and Jaenisch51 in which firmed the previously identified imprinted genes, and analyses of the consequences the maintenance DNA methyltransferase genes and has resulted in the identification of microdeletions enabled the mapping of DNMT1 was deleted in mice, proved the of a few additional genes30–32. However, regional imprinting control regions (ICRs), requirement for DNA methylation in many putative imprinted genes identified by which are specific loci that are required genomic imprinting. allele-specific genome-wide transcriptome for the imprinting of all genes within a approaches are yet to have their imprinting cluster (FIG. 1). Where and when are imprints established? status validated. Hence, although the mouse as a model The identified DMRs fall into two categories: Approaches to predict imprinting organism has been pivotal in determin- those that acquire their DMR status after fer- based on sequence characteristics or ing the epigenetic mechanisms regulating tilization (somatic or secondary DMRs) and epigenetic features remain challenging, and imprinting, the contribution of human those that become differentially methylated to date only a minority of novel predicted genetic studies to our understanding of in the germ line (germline DMRs). Somatic imprinted genes have been validated experi- imprinting control cannot be underesti- DMRs are sometimes tissue-specific and mentally33,34. Interestingly, a particular mated. In addition to the generation of new they depend on the presence of a germline class of retrotransposon-derived imprinted knowledge, these studies have generated DMR52. Mapping microdeletions in patients, genes with X-chromosome homology diagnostic tools for the clinic and clarified and the targeted deletion of the germline has been successfully recognized using the validity of the mouse models for more DMRs in mice, showed that these DMRs this approach35. Nonetheless, sequencing detailed analyses. are the crucial ICRs that are essential for whole transcriptomes and determining the mono-allelic expression within an imprinted parental origin of the transcripts by map- Epigenetic mechanisms of imprinting cluster53–58. Germline DMRs with methyla- ping reads back to sequenced heterozygous In differentially marking the two parental tion that is acquired during oogenesis are genomes (such as those of reciprocal hybrid chromosomes, the process of genomic found at promoters of protein-coding genes mouse strains) currently holds the most imprinting has four key mechanistic prin- or non-coding RNA genes, whereas those promise for investigating the extent and ciples. First, of course, it must be able to with methylation acquired in the paternal cell-type-specificity of imprinting. influence transcription. Second, it must be germ line are found in intergenic regions52 heritable in somatic lineages such that the (compare FIGS 1A and 1B). The evolutionary Insights from human disease. With a few memory of parental origin is faithfully prop- implications of this difference between the exceptions36,37, imprinting is found to be agated into daughter cells during cell divi- positions of the maternally and paternally conserved between mice and humans, sion. Third, it is likely to be initiated on the methylated DMRs have been discussed and valuable insights into the identity and paternally and maternally inherited chro- elsewhere59. organization of imprinted loci have come mosomes at a time when they are not in the In both male and female primordial from the study of human disorders that same nucleus; that is, during gametogenesis germ cells, epigenetic marks start to become show parental-origin effects in their patterns or immediately after fertilization. Finally, the erased once the migrating cells enter the

genital ridges at around day 11.5 of mouse The de novo methyltransferase owing to the inappropriate activation of LTR gestation60. This erasure includes loss of DNMT3A is required for the establishment retrotransposons and non-LTR retrotrans- methylation at the ICRs. Subsequently, the of DMRs in the germ line63. Its regula- posons in spermatogonia and spermato- developing germ cells acquire new epige- tory factor, DNMT3L, is also required for cytes66. This link between the silencing of netic states and this reprogramming includes the establishment of germline imprints64. ‘parasitic’ elements in the germ line and the the establishment of sex-specific DNA meth- DNMT3L interacts with DNMT3A and epigenetic machinery causing imprinting ylation marks at ICRs. In males, these new influences the structure of the enzyme and was considered as early as 1993 (REF. 67) and methylation imprints begin to be acquired its ability to bind and methylate DNA65. may yet hold the elusive answer to arguably during late fetal development61. By contrast, DNMT3L is also essential for the repression the most important outstanding question in females the process starts postnatally dur- of retrotransposons in the male germ line: in the imprinting field: how and why does ing the growing oocyte phase in the early male germ cells lacking this factor undergo the epigenetic machinery recognize and neonatal period62. meiotic catastrophe and pachytene arrest differentially mark particular regions in the male and female germ lines? Is it related to a host defence system? Or does the answer Box 1 | Imprinting and inheritance lie in the properties of the underlying DNA In 1974, Lubinsky and colleagues111 reported parental-origin effects in familial Beckwith– sequence65, or in germline-specific transcrip- Wiedemann Syndrome (BWS). In their Lancet paper they noted, “affected offspring of either sex tional activity across the DMR region68? Does born only to female but not to male carriers” in the pedigree. This turned out to be a classic the answer lie in the presence or absence of example of inheritance of an imprinted disorder. The figure shows a hypothetical pedigree of other types of modification such as histone familial inheritance of an imprinted disorder through five generations, illustrating the parental- methylation69,70, does it depend on other origin-specific mode of inheritance of the disease. Here, the mutation is in a maternally expressed currently unknown mechanisms or perhaps imprinted gene and, therefore, when father transmits the mutation, offspring are unaffected some combination (or all) of the above? because the paternally inherited allele is normally repressed. Half of his offspring will be carriers (as in generation II). The female offspring will transmit the mutation in the active, maternally inherited On fertilization, the egg and sperm allele to 50% of their children, who will be affected, as is clearly evident in generation III. transmit parental-origin-specific differen- One of the explanations for the BWS pedigree that Lubinsky et al. suggested was that the BWS tial methylation to the new conceptus and gene acted “through factors mediated by the ovum but not by the sperm”. We now know that a dramatic epigenetic reprogramming events subset of BWS cases can be caused by maternally inherited mutations in the cyclin-dependent occur that are associated with the acquisi- kinase inhibitor 1C (CDKN1C) gene, a maternally expressed imprinted cell cycle regulator. tion of totipotency and pluripotency71. Imprinting of CDKN1C is regulated by methylation that is established in the female germ line During this time the paternally inherited (FIG. 1Ab), so BWS can be caused either by mutation in CDKN1C itself, or by the absence of genome undergoes active DNA demeth- methylation on the maternally inherited imprinting control region (an epimutation). Other ylation, perhaps through a mechanism mechanisms that cause BWS include paternal uniparental disomy for chromosome 11. involving a hydroxymethylcytosine inter- It is important to emphasize the distinction between parental-origin effects that are due to 72,73 imprinting and sex-linked inheritance. In sex-linked inheritance, the trait maps to sex chromosomes mediate , with a replication-dependent and hence, when transmitted to offspring, males and females differ in their manifestation of the passive demethylation of the maternally trait. This is fundamentally different from parental-origin effects associated with genomic inherited genome. Coincident with these imprinting, in which it is the sex of the transmitting parent that matters. Imprinted traits almost events, chromatin remodelling and dynamic exclusively map to autosomes and importantly, when transmitted to offspring, males and females changes in histone modifications occur74. To are equally affected (see the figure). Furthermore, the manifestation of the imprinted trait will be act as an epigenetic memory that is inherited silenced and will seem to skip a generation when inherited from one carrier parent: for example, from the germ line and is stable throughout the trait is not observed when inherited from the father, but re-emerges in the offspring of his development, methylation imprints must daughters (see generations III–V in the figure). be resistant to such reprogramming events. So how are imprints maintained in an envi- + ronment that is undergoing such extensive epigenetic change? Recent evidence suggests that the resistance to reprogramming is ++ conferred through the targeting of the epigenetic machinery to ICRs. Factors important for this process have been identified, including the KRAB zinc finger protein ZFP57 +++ (REF. 75) and developmental pluripotency- associated protein 3 (DPPA3; also known as PGC7 or Stella)76. +8 The influence of ICRs Maternal methylation. Several imprinted clusters of genes are regulated by maternally 8 methylated ICRs at promoters that repress large non-coding or multi-functional transcripts, the activity of which on the paternal /CNG (GOCNG #ȭGEVGF %CTTKGT chromosome is required for the repression of protein-coding genes in cis on that

chromosome. One of the best characterized Paternal methylation. The best charac- conserved between mammalian species25,86,87. examples of this comes from studies of terized paternally methylated ICR is the Their analysis has taught us several perhaps antisense Igf2r RNA (Airn), a non-coding H19‑DMR. This intergenic DMR is required unexpected but important principles about RNA that regulates imprinting at the Igf2r for normal imprinting of H19 and the recip- the relationship between DNA methylation cluster77 (FIG. 1Aa). On the unmethylated, rocally imprinted linked gene Igf2 (REF. 55). and gene expression more widely. Two of paternally inherited allele, the ICR is a The element functions through interaction these principles are particularly notewor- promoter for the Airn transcript, which is with the zinc-finger protein CCCTC-binding thy. First is a role for DNA methylation in expressed in an antisense direction on the factor (CTCF), which binds DNA in a meth- repressing promoters of non-coding tran- paternally inherited chromosome. On the ylation-sensitive manner and preferentially scripts and the regulatory importance of the maternally inherited chromosome, where targets the unmethylated maternal chromo- non-coding transcripts themselves. When the Airn promoter is germline-methylated some81–83 (FIG. 1Ba). When bound, CTCF unmethylated and expressed, the non-cod- and repressed, Igf2r is expressed. Deletion insulates the Igf2 promoter from downing RNA prevents transcription of adjacent of the unmethylated Airn promoter region, stream enhancers that it shares with H19 and protein-coding genes (FIG. 1A). The genome or premature truncation of the Airn RNA facilitates H19 promoter–enhancer interac- is rich in large, non-coding transcripts, itself, results in bi-allelic expression of Igf2r tions by generating loops that influence generally of unknown function, but with though the failure to repress the paternally chromatin topology84,85. Igf2 is therefore not the potential to contribute to the repression inherited allele54,75,77. It is interesting that activated on the maternal chromosome. By of adjacent protein-coding genes. Second, Airn also represses the other imprinted contrast, CTCF does not bind to the meth- the promoters of some repressed imprinted genes in the cluster, including the tissue- ylated, paternally inherited chromosome, genes are secondary DMRs — regions that and temporal-specific Slc22a3 solute carrier so the enhancers are free to interact with acquire their methylation after fertilization gene located further 5′ of Airn. Evidence the Igf2 promoter, and the H19 promoter is in response to a germline DMR. Secondary suggests that the Airn RNA can recruit and repressed and becomes methylated. Some methylation is often acquired after the allele target repressive histone modifications other paternally methylated DMRs, such has become repressed88, perhaps even as a to the Slc22a3 promoter in a tissue- and as the IG‑DMR on mouse chromosome 12 consequence of transcriptional repression stage-specific manner78. At least three other (FIG. 1Bc), do not bind CTCF and hence may rather than having an initiating role. It is imprinted clusters regulated by maternal influence imprinting of the adjacent genes often assumed that the acquisition of pro- germline methylation seem to be controlled using a different, currently unknown mecha- moter methylation is instructive for gene by large, multifunctional transcripts, the nism86. It therefore seems that, although a repression, but imprinting studies indicate expression of which is required for the consistent theme in imprinting control is the that determining the temporal relationship repression of adjacent protein-coding genes establishment of germline-derived DMRs, between the acquisition of methylation and in cis68,78–80 (FIG. 1Ab–d). However, the extent how they act in cis to confer mono-allelic the repression of transcription is important to which these transcripts act to occlude expression differs from locus to locus. for interpreting whether methylation has protein-coding transcription, or influence indeed caused repression. Other insights the recruitment of inactivating epigenetic Principles of imprinted regulation applied that have emerged from imprinting stud- states to repressed imprinted domains, more widely. Nonetheless, the mechanisms ies that can be applied more widely include remains to be determined. of imprinting at the above loci seem to be contributions to understanding the relative

Glossary

Choroid plexus Histone modifications Pachytene A rich network of blood vessels located in the Reversible post-translational modifications, such A stage of meiosis in which the chromosome homologues brain that is responsible for the production of as methylation and acetylation, that occur on the are closely synapsed. This is the stage when crossing-over cerebrospinal fluid. amino-terminal tails of core histone proteins. between the homologous chromosomes occurs.

Coccid insects Leptomeninges Pronucleus Scale insects of the order Hemiptera. Two of the three layers of membrane that protect The haploid nucleus from a male or female gamete. the brain and the spinal cord; cerebrospinal fluid Complete hydatidiform mole circulates between these two layers. Reciprocal translocation A product of conception that has two paternally The interchange of genetic material between two derived genomes and is devoid of maternally inherited LTR retrotransposons chromosomes that are non-homologous. chromosomes. It develops as a rapidly growing mass, A long terminal repeat (LTR)-containing class is derived from cells that would normally contribute Robertsonian translocation of genetic element that can replicate and to the placenta and lacks fetal tissue. A chromosomal abnormality in which two acrocentric insert into a host genome through an RNA chromosomes become joined by a common centromere. CCCTC-binding factor intermediate. (CTCF). A highly conserved zinc finger protein that Sciarid flies influences chromatin organization and architecture Neurogenic niche Dipteran insects of the genus Sciara, also known and is implicated in diverse regulatory functions A specific microenvironment in which neural stem as fungus gnats. including transcriptional activation, repression cells can respond to endogenous and exogenous and insulation. cues and can undergo self-renewal, proliferation Uniparental disomy and/or differentiation. A cellular or organismal phenomenon in which both Heterochromatic chromosome homologues are derived from one parent A cytogenetic term to describe chromosomes or Ovarian teratomas with none derived from the other parent. It can be the chromosomal regions that remain condensed and A tumour, derived from egg cells, which consists of cells result of fertilization involving a disomic gamete and a heavily stained during interphase. that resemble fetal tissue-derived cells. gamete that is nullisomic for the homologue.

hierarchy of epigenetic changes associated may facilitate the maintenance of regional the maintenance of the neural stem cell with developmental programming, the influ- activity or repression and indeed may be pool and the subsequent generation of ence of epigenetic states on DNA–protein modulated developmentally, as has been new neurons in the adult and throughout interactions and the relationships between shown for other non-imprinted regions of the remaining lifetime of the animal. This DNA and histone modifications. the genome90,91. However, it has been shown selective absence of imprinting correlates that the histone H3 lysine 4 trimethylation with the acquisition of methylation at the Histone modifications. Despite correlations (H3K4me3)-modified chromatin may shield germline DMR on the maternal allele and between the presence of active and repres- DNA from methylation, a finding based suggests that gain or loss of imprinting sive histone modifications at imprinted loci on the lower affinity of the DNMT3A– may be a dynamic, epigenetically regu- and the mono-allelic behaviour of imprinted DNMT3L complex for chromatin har- lated mechanism that controls gene dos- genes, there is no substantial evidence that bouring this mark69. Furthermore, and in age in particular developmental contexts27 these modifications are instructive for support of this, in the female germ line, (FIG. 2). The extent of such modulation, imprinting. Rather, such marks contribute demethylation of H3K4 is required in order and whether this may also apply to other to the hierarchy of epigenetic states that is that de novo DNA methylation can occur at imprinted genes, remains to be elucidated. built on DNA and chromatin in response to some ICRs70. It therefore seems likely that Rather than complicating our understand- the germline DMR89. These modified states histone modifications might be instructive ing of imprinting, these observations per- in the germ line, thus contributing to the haps illustrate the importance of dynamic C%CPQPKECNKORTKPVKPI recruitment of the heritable germline DNA changes in epigenetic states as a mechanism imprint. Further analysis is required to to control gene dosage as the need arises. determine the extent to which modulation Further analyses of these events may con- /CVGTPCN of histone modifications is required for tribute more generally to our understand- germline DNA methylation. ing of the epigenetic control of dosage in +)&/4 )VN normal developmental contexts, in envi- 2CVGTPCN Function and evolution of imprinted genes ronmentally stimulated diseases (for exam- Over the years, the phenotypic characteriza- ple, in cancer and adult-onset metabolic tion of patients with imprinted disorders and disease) and in mechanisms underlying the &NM mice with loss or gain of allelic activity at phenotypic consequences of ageing. imprinted genes has elaborated on the early D0GWTQIGPKEPKEJG embryological and genetic studies indicating Evolutionary forces. Imprinting is believed that imprinted genes function to control pre- to have evolved independently in plants, /CVGTPCN natal growth, placentation and the develop- and is not found in lower vertebrates. ment of lineages such as the musculoskeletal Comparisons of imprinting between a +)&/4 )VN system and the brain92–94. More recently, wide range of eutherian and non-eutherian 2CVGTPCN several studies have indicated that imprinted mammals have shown both conservation genes also play important parts in postnatal and lack of conservation of imprinting. For processes including adaptation to feeding, example, imprinting has not yet been found &NM social behaviour and metabolism95–97, pro- in prototherian mammals (the egg-laying cesses that may be particularly responsive to platypus and the echidna). In metatherian environmental influences. Perhaps genomic mammals (the marsupials) only a subset of /CVGTPCNN[ 4GRTGUUGFIGPG GZRTGUUGFIGPG imprinting, as an epigenetic mechanism to loci imprinted in eutherians show conser- regulate gene dosage, might have evolved in vation of imprinting. Domains such as the 2CVGTPCNN[ GZRTGUUGFIGPG response to extrinsic and/or intrinsic signals Igf2–H19 and Igf2r clusters are imprinted in to allow levels of these genes to be modu- marsupials25,87,99 but others, such as the Dlk1 /GVJ[NCVGF+%4 7POGVJ[NCVGF+%4 lated as conditions required. Consistent with and Snrpn imprinted clusters, are not; their /GVJ[NCVGFQPN[KPVJGPGWTQIGPKEPKEJG this are findings that different imprinted organization and imprinting evolved after the genes show a wide range of tissue- and/or divergence of eutherians and metatherians /GVJ[NCVGF 7POGVJ[NCVGF temporal-specific imprinting during from their common ancestor100,101. UGEQPFCT[&/4 UGEQPFCT[&/4 development94–98. These findings imply that different evolutionary selective pressures, acting at different Figure 2 | Tissue-specific0CVWT imprintingG4GXKGYU^ )GPGVKEUeffects. Dynamic control. Evidence in support of times on different gene clusters, led to the Schematic representation of the Delta-like the idea that the control of gene dosage evolution of dosage control by imprinting. homologue 1 (Dlk1) imprinted locus. a | The may be a modulatable process has come This might reflect the different physiological state of the imprinted Dlk1 and Gtl2 (also from the study of the paternally expressed roles of imprinted genes and their ability to known as Meg3) genes in most tissues (canoni- imprinted gene, Dlk1 in mice (FIG. 2). adapt to new levels of functional complex- cal imprinting). b | In the postnatal neurogenic During embryogenesis Dlk1 is exclusively ity through changes in their gene dosage. niche, there is a selective absence of imprinting at the Dlk1 gene, and this increase in overall expressed from the paternally inherited For example, it is well-established that Dlk1 expression is required for neurogenesis27. chromosome. However, in the neurogenic imprinted genes contribute to the regulation Selective activation of the maternal allele of niche shortly after birth Dlk1 selectively of nutritional resources to the fetus in utero. Dlk1 is associated with increased methylation loses its imprinting, and its dosage dramati- This occurs predominantly through the at the IG differentially methylated region cally increases. This is coincident with a placenta, a recently evolved feto-maternal (IG‑DMR) imprinting control region. requirement for both parental alleles for organ that mediates the supply and demand

of nutrients to the growing fetus. Even mar- associated with the KAP1 chromatin Identifying the function of these marks and supial mammals have a yolk sac and/or pla- co-repressor complex75,105, the specific integrating them into existing epigenetic centa for their short gestation102. Genes such presence of H4K20me3 (REFS 91,106), the frameworks will require the development of as Igf2 and Igf2r, which are important for recruitment of PIWI-interacting RNA novel methods for identifying, sequencing regulating resource acquisition through the (piRNA)-mediated silencing mechanisms107 and manipulating them, and for determining placenta, are imprinted in both marsupials and targeting by DNMT3A–DNMT3L their place in the increasingly colourful epi- and rodents. based on sequence periodicity65,66,108. genetic palette that controls our DNA, paints Evolution of imprinting at the Igf2 and To what extent does the underlying our chromatin and builds our chromosomes. 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M. & Beechey, C. V. in Chromosomes for continuing or even strengthening efforts at one locus acts by influencing gene expres- Today (eds Fredga, K., Kihlman, B. & Bennett, M.) 135–148 (Unwin Hyman, London, 1990). in animal genetics. These principles, which sion at a second locus that is remote from 113. Kanduri, C. et al. Functional association of CTCF with apply equally to other model organisms, the first), and gene–environment interac- the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive. Curr. Biol. include: the wealth of literature accumulated tions. Curiously, although gene loci identi- 10, 853–856 (2000). over 100 or more years for models such as the fied in human GWASs explain only a small Acknowledgements mouse and rat; the genome resources that, as proportion of the heritability of complex The author acknowledges the many scientists whose research for humans, have accelerated the pace of gene traits, such as body mass and blood pressure, has contributed to the genetic, embryological and epigenetic studies on parental-origin effects and genomic imprinting, identification for a wide range of phenotypes; studies in rodents explain several times the and apologizes to those whose work it has not been possible and the opportunities for in vivo phenotyp- total genetic variance for similar traits2,3. to mention or cite in the confines of this Timeline article. The author is grateful to previous and current members of the ing and gene targeting that have catalysed In the rat, integrating linkage analysis Ferguson-Smith team for their contributions to the ideas pre- our understanding of genetic mechanisms. with expression profiling has proved a par- sented here, and thanks the colleagues with whom she has spent many a long hour debating and discussing the past, However, genome-wide association ticularly powerful approach. The application present and future of genomic imprinting and epigenetic studies (GWASs) in humans have recently of this approach using adipose tissue led processes. identified hundreds of genes and gene loci to the identification of Cd36 as an insulin Competing interests statement for common human diseases. Furthermore, resistance gene in rats and humans4. This The author declares no competing financial interests. new sequencing technologies have acceler- was among the first complex trait genes

FURTHER INFORMATION ated the pace of discovery for Mendelian to be positionally cloned in any mammal. Anne C. Ferguson-Smith’s homepage: traits, providing insights into their molecular Building on this integrative strategy led to http://www.pdn.cam.ac.uk/staff/ferguson basis by direct study of the human diseases detection of thousands of rat expression Catalogue of Imprinting Effects: http://igc.otago.ac.nz/home.html rather than models. Surely it is not sufficient quantitative trait loci (eQTLs) in multiple Epigenesys (EU Network of Excellence): to continue studying animal models just tissues5,6 and identification of rat genes for http://www.epigenesys.eu International Human Epigenome Consortium: because it has always been this way? cardiac mass, cardiac failure, glomerulo- http://www.ihec-epigenomes.org One of the arguments in favour of con- nephritis and hypertension1,7, all of which MouseBook imprinting catalogue: http://www.mousebook. org/catalog.php?catalog=imprinting tinuing studies of model organisms is that show conserved function in humans. A high Medical Research Council Harwell Genomic Imprinting the genetic studies of common human dis- proportion of other complex trait genes homepage: http://www.har.mrc.ac.uk/research/ genomic_imprinting eases have significant limitations. The genes identified in the rat have conserved phe- University of Cambridge Centre for Trophoblast Research: and gene loci found by GWASs are mostly notypes in humans, in several cases more http://www.trophoblast.cam.ac.uk of small effect and explain a relatively low strongly so than corresponding mouse mod- ALL LINKS ARE ACTIVE IN THE ONLINE PDF proportion of the overall heritability for a els. Particular examples are: the polycystic