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Environmental Effects on GenomicImprinting in Development and Disease

Rakesh Pathak and Robert Feil

AbstractGenomic imprinting mediates the parent-of-origin-specific, mono-allelic expres-sion of many protein-coding genes and noncoding RNAs. This paradigm forepigenetic gene regulation plays diverse roles in mammalian development,growth and behavior. Mechanistically, it involves parentally inherited DNAmethylation marks that control clusters of imprinted genes. Perturbation ofthese epigenetic imprints affects embryonic and postnatal development andleads to complex diseases in humans, including different types of diabetes. Thischapter discusses imprinted genes, with emphasis on those that control metabo-lism and cellular proliferation, several of which encode proteins of the insulin-likegrowth factor/insulin signaling pathway. Nutrition, chemical pollutants, and otherenvironmental cues can readily perturb DNA methylation imprints, not onlyduring development, but sometimes even in adults. Such epigenetic alterations(“epimutations”) may affect imprinted gene expression and, hence, can havedeleterious effects on phenotype. In the future, clinical and environmentalimprinting studies will gain from taking a broader approach that considers notonly the imprinted gene loci themselves, but also similarly controlled loci locatedelsewhere in the genome.

KeywordsEpigenetics • Environment • Genomic imprinting • DNA methylation • Growth •Metabolism • IGF/Insulin pathway • Endocrine disruptor

R. Pathak • R. Feil (*)Institute of Molecular Genetics (IGMM), Centre National de Recherche Scientifique (CNRS),UMR-5535, University of Montpellier, Montpellier, Francee-mail: [email protected]; [email protected]

# Springer International Publishing AG 2017V.R. Preedy, V.B. Patel (eds.), Handbook of Nutrition, Diet, and Epigenetics,DOI 10.1007/978-3-319-31143-2_92-1

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List of AbbreviationsART Assisted Reproductive TechnologyBPA Bisphenol ABWS Beckwith-Wiedemann SyndromeICR Imprinting control regionIGF Insulin-like growth factorINS InsulinIUGR Intra-uterine growth restrictionncRNA Noncoding RNASRS Silver Russell SyndromeTNDM Transient neonatal diabetes mellitus

ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Roles of Genomic Imprinting in Development, Growth and Metabolism . . . . . . . . . . . . . . . . . . . . . . 4Imprinting (De)regulation, the INS/IGF Signaling Pathway and Diabetes . . . . . . . . . . . . . . . . . . . . . 9Imprinting Disorders and Effects of Assisted Reproductive Technology . . . . . . . . . . . . . . . . . . . . . . . 11Nutrition and Toxic Components Influence Genomic Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13A Broadening Outlook on Imprinting Deregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Dictionary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Introduction

Epigenetic mechanisms contribute to the establishment and maintenance of stablepatterns of gene expression during development and throughout postnatal life. DNAmethylation at cytosine residues is the best studied epigenetic modifications inmammals. It plays essential roles in cells and tissues. These include stable repressionof endogenous retroviruses and the tissue-specific silencing of developmental genes,particularly germ line genes, which become silenced in the embryo. DNA methyl-ation also contributes to X-chromosome inactivation. This is a gene dosage mech-anism in female embryos that leads to the repression of most genes on one of the twoX chromosomes.

The current chapter focuses on another gene dosage mechanism for whichcytosine methylation is essential, namely genomic imprinting (Bartolomei andFerguson-Smith 2011). It introduces the roles of imprinted gene expression inmammalian development and its perturbation in disease and makes the link withthe insulin/IGF signaling pathway, metabolism and growth control. The chapter alsodiscusses how diet and environmental cues may perturb the epigenetic regulation ofimprinted genes and that this can have long-lasting phenotypic consequences (Feiland Fraga 2012).

Mammalian genomic imprinting evolved coincident with the emergence of vivi-parity and the growing importance of placentation and the evolution of defense

2 R. Pathak and R. Feil

mechanisms against transposable elements. The oldest imprinted genes arose about170 million years ago, during the Jurassic period. Today, more than 100 protein-coding genes and hundreds of noncoding RNAs (ncRNAs) are known to beimprinted in mice and humans (Peters 2014). To understand what the mono-allelicexpression of imprinted genes entails, it is important to remember that imprintingwas discovered only 30 years ago, when two different laboratories found that boththe maternal and the paternal genome are strictly required for embryonic develop-ment (McGrath and Solter 1984; Surani et al. 1984). Particularly, parthenogenetic(with two maternal genomes and lack of the paternal genome) and androgeneticembryos (with two paternal genomes) were found to not develop to term and hadgross developmental abnormalities. About half of all the known imprinted genes areexpressed from the maternal genome only, whereas the other half are expressed fromthe paternally inherited genome only. In mono-parental embryos, consequently,individual imprinted genes are either not expressed at all or are overexpressed, andthis explains the gross developmental effects observed in the mono-parentalembryos. Also maternal or paternal uniparental disomy of individual chromosomesis associated with developmental phenotypes (Cattanach and Kirk 1985).

Imprinted genes are organized in evolutionarily conserved clusters. At each“imprinting cluster”, there is an essential regulatory region that acquires allelic DNAmethylation in one of the two parental germlines (Peters 2014; Sanli and Feil 2015).The mono-allelic methylation marks at these “imprinting control regions” (ICRs) aremaintained throughout development and mediate the mono-allelic imprinted expres-sion of genes at the respective domains (Fig. 1). Most ICRs acquire their allelic DNAmethylation in the oocyte; at only some it is acquired during spermatogenesis.

What makes ICRs unique is that, exceptionally, they maintain their differentialmethylation throughout pre- and postimplantation development (Kelsey and Feil2013). This contrasts with the bulk of the genome, which undergoes global waves ofdemethylation and de novo methylation during preimplantation development andgastrulation. How precisely this remarkable epigenetic stability is controlled is notwell understood. DNA methylation imprints at ICRs are consistently associatedwith repressive histone modifications, however, including histone H3 lysine-9 tri-methylation (H3K9me3) and H4 lysine-20 trimethylation (H4K20me3), and showallelic binding of heterochromatin protein 1 (HP1g) as well (Kelsey and Feil 2013).Furthermore, the histones H3 associated with DNA methylation imprints are notcanonical histones, but a variant histone called H3.3 (Voon et al. 2015). This varianthistone and regulatory protein complexes are recruited onto the chromatin in anallele-specific manner and collectively contribute to the stability of the allelicmethylation state of ICRs in the embryo.

The epigenetic maintenance of ICRs is essential to ensure faithful mono-allelicexpression of imprinted genes. In case this maintenance process is perturbed, thiscauses pathological phenotypes. In humans, losses or gains of DNA methylation atICRs are causally involved in a growing number of congenital “imprinting disor-ders” (Eggermann et al. 2015, Hirasawa and Feil 2010). Given their essential roles indevelopment, growth metabolism and behavior there has been a tremendous interestin imprinted gene domains. Of particular interest have been the well-conserved

Environmental Effects on Genomic Imprinting in Development and Disease 3

imprinting clusters that control fetal- and postnatal growth, and the imprinted genesthat are part of the insulin-like growth factor/Insulin (IGF/INS) signaling pathway(Delaval et al. 2006; Moore et al. 2015; Peters 2014).

Though ICRs are epigenetically stable, even slight changes in DNA methylationcan be readily detected in molecular assays. Once aberrant methylation changes haveoccurred, they persist in the embryo and this perturbs imprinted gene expression.These unique characteristics and the causal link with diseases including diabetes andintrauterine growth disorders have made genomic imprinting an excellent paradigmto explore the epigenetic effects of the environment and nutrition (Feil and Fraga2012). This constitutes the overall theme of the chapter, which focuses on imprintedgene loci that control growth and metabolism, their involvement in specific humandiseases and the effects of nutrition and toxic components (Tables 1 and 2).

Roles of Genomic Imprinting in Development, Growth andMetabolism

Ever since the discovery in mice of the first imprinted genes – Igf2r, Igf2, and H19(Barlow et al. 1991; Bartolomei et al. 1991; DeChiara et al. 1991) – a main focus ofresearch has been on epigenetic regulation and how at individual domains, the ICR

PGC (male)

SpermOocyte

Maintenance

AdultZygote

Embryonic Development

Postnatal Development

Maintenance

Paternal genome

Maternal genome

DNA methylation Imprint

Embryo

PGC (female)

Fig. 1 Germline establishment and somatic maintenance of parental imprints. Shown is theestablishment of paternal DNA methylation imprints and maternal DNA methylation imprints duringspermatogenesis and oogenesis, respectively. After fertilization of the oocyte, these parental DNAmethylation imprints are maintained in all somatic cells, during embryonic development and afterbirth. During early embryonic development the imprints are relatively susceptible to environmentaland stochastic methylation changes, which may affect imprinted gene expression and phenotype.


brings about mono-allelic expression in cis (Abramowitz and Bartolomei 2012).A lot is known also about the biological functions of imprinted gene expression,particularly in development, growth, and metabolism (Peters 2014). For instance, theinsulin-like growth factor-2 (Igf2) gene on mouse chromosome 7/human chromo-some 11p15 (DeChiara et al. 1991) is a major regulator of fetal growth. It isexpressed from the paternal genome in mesodermal and endodermal tissues. Igf2is flanked by the insulin gene (Ins2), which is also imprinted and expressed from the

Table 1 Human imprinting disorders

Imprinting disorder (ID)

Chromosome/ imprinted domain

Epigenetic/geneticalteration

Clinical features

Beckwith-WiedemannSyndrome

(BWS)

Chr. 11p15:IGF2-H19: ICR

KCNQ1: ICR

Hypermethylation

Hypomethylation

Pre- and postnatal overgrowth, organomegaly, macroglossia, omphalocele, neonatal hypoglycemia, hemihypertrophy,increased tumor risk

Pseudohypo parathyroidism

Type-1B(PHP1B)

Chr. 20q13:

GNAS

Paternal UPD(20)

Aberrant methylation

Resistance to PTH and other hormones; Albright’s hereditary osteodystrophy, subcutaneous ossifications, feeding behavior anomalies, abnormal growth

Silver-Russell Syndrome

(SRS)

Chr. 11p15:IGF2-H19: ICRIGF2-H19: IGF2

KCNQ1: CDKN1C

HypomethylationPoint mutations

Point mutations

IUGR, postnatal reduced growth, macrocephaly at birth, hemihypotrophy, prominentforehead, triangular face, feedingdifficulties

Transientneonataldiabetes(TNDM)

Chr. 6q24:

PLAGL1: alt-TSS

Paternal UPD(6)paternal duplication

Methylation defect

IUGR, transient neonatal diabetes,hyperglycemia withoutketoacidosis, macroglossia,omphalocele

Kagami-Ogata Syndrome (KOS14)

Chr. 14q32:

DLK1-DIO3: ICR

DLK1-DIO3: MEG3

Paternal UPD(14)

Maternal deletion,hypermethylationMaternal deletion

IUGR, polyhydramnios, abdominaland thoracic wall defects, bell-shaped thorax, coat-hanger ribs

Temple Syndrome

(TS14)

Chr. 14q32:

DLK1-DIO3: ICRDLK1-DIO3: MEG3

Maternal UPD(14) Paternal deletion

Aberrant methylation

IUGR, reduced postnatal growth, neonatal hypotonia, feeding difficulties in infancy, truncal obesity, scoliosis, precocious puberty, small feet and hands

Angelman Syndrome

(AS)

Chr. 15q11-q13:

SNRPN: UBE3A

Paternal UPD(15)

Maternal deletionPoint mutations

Feeding problems, developmental delays, pronounced speech impairment, hyperactivity, severe movement and balance disorders, bouts of laughter

Prader-Willi Syndrome

(PWS)

Chr. 15q11-q13:

SNRPN: ICRSNRPN: NECDIN

Maternal UPD(15)Paternal deletionsLoss of methylationLoss of methylation

Obesity, short stature, decreased muscle tone, hypogonadism,decreased mental capacity


Table 2 Nutritional and toxicological effects on imprinted genes

SpeciesDevelopmentalwindow

Epigeneticalteration/expressionchange

Tissues/cell typesaffected

Phenotypicobservations References

Gestationalstarvation

Human Peri-conception(first stages ofdevelopment)

Alteredmethylation inadult offspring(IGF2, INS,MEG3, GNAS)

Blood(adult)

Correlates withmetabolic andmental phenotypesin adults

Tobi et al.(2014)

High-fat diet Rat,mouse

Early gestation Alteredimprinted geneexpression

Varioustissues

Reduced fetalgrowth,Type-2 diabetes inoffspring

Morita et al.(2014),Sferruzzi-Perri et al.(2013)

Increased“methyldonor”(folate-richdiet)

Human Adult life Increased DNAmethylation,including atIGF2-H19

Blood Folate treatmentgiven to hyper-homocysteinaemiapatients

Ingrosso etal. (2003)

Alcoholconsumptionin pregnantfemales, inadult males

Human,mouse

Embryonicdevelopment,malegametogenesis

Reduced DNAmethylation atIGF2-H19locus and ICRs

Malegermcells,blood,frontalcortex

Fetal alcoholsyndrome in offspring, reducedfertility in alcoholicmales

Masemola etal. (2015),Ouko et al.(2009),Stouder et al.(2011)

Lead/cadmiumPollutionexposureduringpregnancy

Human Embryonicdevelopment

Alteredmethylation atICRs inoffspring

Embryo,placenta

Correlates witheduced fetal growthand childhoodobesity

Park et al.(2017)

Bisphenol-AExperimentalexposure ofpregnantfemales

Mouse Peri-conceptual,embryonic dev.

Expressionchanges andloss ofmethylation(Snrpn,Kcnq1ot1,Cdkn1c)

Embryo,placenta

Expression andmethylationchanges can beinherited to nextgeneration

Susiarjo etal. (2013,2015)

VinclozolinExposure ofpregnantfemales

MouseRat

Embryonicdevelopment

Reducedmethylation atIgf2-H19,increasedmethylation atmaternal ICRs

Germcells ofmaleoffspring

Expression andmethylationchanges can beinherited to nextgeneration

Stouder andPaoloni-Giacobino(2010)

MethoxychlorExposure ofpregnantfemales

Mouse Embryonicdevelopment

Reducedmethylation atIgf2-H19, gainof methylationat maternalICRs

Germcells ofmaleoffspring

– Stouder andPaoloni-Giacobino(2011)

Assistedreproductiontechnologies

Mouse,farmanimals,human

Peri-conception,embryonicdevelopment

Gains andlosses of DNAmethylation atICRs

Tissues,blood

In animals, diversephenotypicabnormalities. Inhumans, smallincrease inoccurrence of IDs

Dean et al.(1998), Diasand Maher(2013), Feiland Fraga(2012)


paternal allele only in the yolk sac (Deltour et al. 1995; Duvillie et al. 1998; Moore etal. 2001). In the embryo proper and in adult pancreas, Ins2 is expressed from bothalleles. Igf2 and Ins2 are part of the IGF/INS signaling pathway (Fig. 2) and are partof a small imprinted domain. This “Igf2-H19 domain” also comprises the long non-coding RNA (lncRNA) gene H19, which is expressed from the maternal allele andhas a negative effect on fetal growth (Gabory et al. 2010). The Igf2-H19 domain isstructurally conserved in humans, where its perturbation causes two different diseasesyndromes. Its intergenic ICR is methylated on the paternal copy (Fig. 3). On thematernal chromosome, this ICR acquires a specialized chromatin structure throughbinding of a structural protein called CTCF and that of cohesin complexes. Thisdifferential chromatin structuration at the ICR mediates the allelic expression of Igf2,Ins2, and H19 (Abramowitz and Bartolomei 2012).

Another locus that controls fetal growth is the Kcnq1 domain, located next to theIgf2-H19 domain. This 1-Megabase domain comprises several genes that areimprinted in the embryo, placenta, and after birth. These include Cdkn1c (also calledp57Kip2), a negative regulator of the cell cycle, expressed from the maternalchromosome only. Loss of Cdkn1c expression enhances fetal growth, and its over-expression leads to growth restriction. The domain comprises several placenta-specific imprinted genes as well. These include the transcription factor gene Ascl2,which controls spongiotrophoblast development (Guillemot et al. 1995), a processwhich is influenced by the imprinted Cdkn1c gene as well (Zhang et al. 1998). Thedomain’s ICR is intragenic and it expresses a long ncRNA, called Kcnq1ot1, from itsunmethylated paternal copy (Fig. 3). In the preimplantation embryo, the allelicexpression of the long ncRNA induces gene repression on the paternal chromosome,through a process that involves repressive histone methylation (Umlauf et al. 2004).

Grb10 on mouse chromosome 11/human chromosome 7 is another imprintedgrowth regulator. In the mouse, Grb10 is expressed from the maternal chromosomein placenta and in mesodermal and endodermal tissues. It encodes a growth-factorbinding protein (GRB10) that antagonizes the insulin/IGF pathway, by binding tothe cytoplasmic phase of the insulin (IR) and IGF1-receptor (IGF1R) (Fig. 2). Grb10knockout mice show severe overgrowth, whereas a double dose of expression due todeletion of the domain’s ICR reduces fetal growth (Charalambous et al. 2003; Shiuraet al. 2009). The maternal Grb10 expression in the placenta is conserved in humans,which emphasizes the evolutionary importance of Grb10 imprinting in extra-embry-onic lineages (Monk et al. 2009).

Grb10 expression in the brain influences social behavior. In brain, an alternativetranscript is expressed from the paternal chromosome only, both in mice and inhumans (Monk et al. 2009; Sanz et al. 2008). The comparative studies on Grb10emphasize that imprinted genes can have different functions depending on whereand from which promoter they are expressed and that their mono-allelic expression isnot always from the same parental chromosome in different tissues.

Igf2r (also called the mannose-6-phosphate receptor (Barlow et al. 1991)) onmouse chromosome 17 encodes a nonfunctional, antagonistic receptor of IGF2.Consequently, its expression has a negative effect on IGF/INS signaling (Fig. 2).This imprinted gene is expressed from the maternal chromosome only and mutations


lead to severe overgrowth and embryonic lethality. This phenotype can be “rescued”by either loss of Igf2 expression or loss of the nonimprinted IGF1-receptor (Ludwiget al. 1996; Wutz et al. 2001). The extensive research on the imprinted Igf2, Ins2,Igf2r, and Grb10 genes has established that sets of imprinted genes control commonpathways of growth and metabolism.

Targeting studies in the mouse have revealed other roles for imprinted genes aswell. Observed phenotypic effects have often been tissue-specific and imprinting isparticularly important in placental development and function and in neurogenesisand behavior (Peters 2014).

Worth mentioning is Zac1 (also called Plagl1), a transcription factor gene whichis paternally expressed in humans and mice. Its genetic deletion was shown to lead toreduced fetal growth and perinatal death. Interestingly, ZAC1 controls several otherimprinted genes of a network that comprises nonimprinted genes as well (Al Adhamiet al. 2015; Arima et al. 2005; Varrault et al. 2006). ZAC’s imprinted gene targetsinclude Igf2 and H19, Cdkn1c, and also the lncRNA Kcnq1-ot1 expressed by theICR of the Kcnq1 imprinted domain. Recent studies have unraveled the details of

IRIGF1R

IRS1/2

GrowthMetabolismCell Survival

AKT

IGF1Insulin

IGF2

Insulin

IGF1

GRB10 GRB2

Fig. 2 The IGF/INS signalling pathway is controlled by genomic imprinting. The IGF/INSpathway activates intracellular signalling cascades that control the proliferation and survival ofcells, metabolism and growth. Several proteins that are encoded by imprinted genes (blue shapes)play key roles in enhancing, or repressing, the IGF/INS trans-membrane signaling. Whereas IGF2and Insulin have positive effects on the pathway, and hence on metabolism and cellular prolifera-tion, GRB10 and IGF2R have negative effects. This is a simplified figure which shows only some ofthe key proteins of the downstream intracellular cascades.


this trans-regulation. For instance, ZAC1 binds to an enhancer that is shared by Igf2and H19 (Iglesias-Platas et al. 2014).

Other links between growth-related imprinted genes are known as well. Loss ofH19 expression at the Igf2-H19 domain leads to upregulation of six other imprintedgenes. Transgenic H19 overexpression corrects this phenotype, which indicates thatthe H19 ncRNA itself is involved. Possibly, the trans regulation by H19 ncRNAoccurs through interaction with a chromatin repressor protein called MBD1(Monnier et al. 2013). These and other examples show that imprinted genes evolvedcommon biological functions and influence each-other within intricate networks ofimprinted genes.

Imprinting (De)regulation, the INS/IGF Signaling Pathway andDiabetes

Four imprinted genes directly control the IGF/INS pathway (Fig. 2). Multiple othersaffect cellular proliferation and glucose metabolism in other ways (Peters 2014). Inthe mouse, Igf2 is expressed from the paternal chromosome, both in the embryo andplacenta, whereas Igf2r is expressed from the maternal genome only in the sametissues. Ins2 is imprinted during uterine development and shows, like Igf2, paternalexpression in the yolk sac. The receptor binding protein Grb10 is expressed from thematernal genome only. In humans, the situation is similar to that in rodents, but forIGF2R. In humans, this gene is imprinted in a polymorphic manner: a minority ofpeople shows maternal IGF2R expression, the majority express both parental alleles.INS, however, is consistently imprinted in the yolk sac, as it is in mice (Monk et al.2006; Moore et al. 2001).

Glucose metabolism is influenced by other imprinted genes as well. Studies ontransient neonatal diabetes mellitus (TNDM) revealed a key role of ZAC1. TNDM isa transitory form of diabetes in newborns, with hyperglycemia and low insulin levelsduring the first year of life. Unlike in type-I diabetes, there is no evidence for auto-immunity against the pancreatic Β cells. In more than half of the patients, there isloss of the methylation imprint at the ICR of the ZAC1 locus. This leads to biallelicZAC1 expression (Fig. 3). The increased gene dosage impairs glucose-stimulatedinsulin secretion in B cells at fetal and postnatal stages, when ZAC1 is expressedmost highly. Particularly, ZAC1 is thought to induce a pituitary adenylate cyclase-activating polypeptide, which is an activator of glucose-stimulated insulin secretion.In adult pancreatic Β cells, ZAC1 expression is much lower and functionally lessimportant.

In the mouse, loss of Zac1 expression gives rise to altered expression of multipleother imprinted genes, including that of Igf2 and H19 (Al Adhami et al. 2015). In theplacenta, interestingly, the H19 long ncRNA produces a micro-RNA (miR-675) thatreduces the expression of Igf1r. These different findings functionally link theessential ZAC1 transcription factor to Igf2, H19, and Igf1r expression and to glucosemetabolism and cellular proliferation.


A “variable number of tandem repeat” polymorphism upstream of the INS gene inhumans is genetically linked to the clinical progression of type-1 diabetes, whichinvolves the insulin-producing pancreatic B cells (Zhang et al. 2015). Anotherexample is the Krüppel family transcription factor KLF14. This imprinted gene ismaternally expressed in adipose tissues (Parker-Katiraee et al. 2007) and is geneti-cally linked to an increased risk of type-1 diabetes and metabolic phenotypes (Smallet al. 2011; Voight et al. 2010).

Another developmentally essential imprinted locus, the DLK1-DIO3 domain onhuman chromosome 14q32.2, is genetically linked to type-1 diabetes as well (Wal-lace et al. 2010).

The maternally expressed KCNQ1 gene, which encodes a potassium channel, isimportant for B cells as well. In humans, single nucleotide polymorphisms (SNPs) at

ICR

ICR

IGF2 H19

ICR

CDKN1C KCNQ1ICR

M

P

ICR ICR

KCNQ1 domain IGF2-H19 domain

ICR ICR

KCNQ1OT1 ncRNA

CDKN1C KCNQ1 IGF2 H19

M

P

WT

BWS

BWS

ICR

ICRIGF2 H19

M

P

SRS

a

b

ZAC1/PLAGL1M

P

WT

ZAC1/PLAGL1M

P

TNDM

c

d

e

M

P

ZAC1/PLAGL1 domain

Fig. 3 Epigenetic alterations cause imprinting disorders (IDs). (a) The KCNQ1 and IGF2-H19imprinted gene domains both control cellular proliferation, metabolism and growth. The KCNQ1domain has an intragenic ICR that is marked by maternally-inherited DNA methylation (lollipops).On the paternal chromosome, this ICR produces a long ncRNA that mediates gene repression in cis.The flanking IGF2-H19 domain has an intergenic ICR that is marked by paternal (P) DNAmethylation. B, Embryonic loss of this maternal (M ) imprint at the ICR of the KCNQ1 domain inthe embryo induces BWS. Loss of methylation at the ICR of the IGF2-H19 domain leads to SRS.(c) Conversely, aberrant gain of biallelic methylation at this ICR gives BWS. (d) The ZAC1/PLAGL1 imprinted locus is controlled by maternal DNA methylation, which induces paternalallele-specific expression of ZAC1. (e) Loss of the maternal methylation imprint leads to biallelicZAC1 expression, and an increased dosage of this transcription factor, which causes transientneonatal diabetes mellitus (TNDM).


KCNQ1 are linked to type-2 diabetes following maternal inheritance only (Voight etal. 2010).

Loss of expression of the imprinted Grb10 gene in the mouse is associated withincreased glucose tolerance and increased inulin sensitivity. IncreasedGrb10 expres-sion, on the other hand, gives rise to insulin resistance and impaired glucosetolerance. At the guanine nucleotide binding protein a-stimulating (Gnas) imprintedlocus on mouse chromosome-2, mutations that affect its maternal expression bringabout insulin resistance, hyperinsulinemia, and hyper-glycaemia associated withobesity (Peters 2014). Combined, the different studies show that many imprintedgenes are involved in different types of diabetes, but often the underlying mecha-nisms remain to be discovered.

Imprinting Disorders and Effects of Assisted ReproductiveTechnology

A growing number of diseases are known to be caused by imprinted genes. Several“imprinting disorders” (IDs) are characterized by intra-uterine and postnatal growthdefects and are linked directly or indirectly to the INS/IGF signaling pathway (Fig.3). Already introduced above, TNDM (OMIM 601410) is a rare form of diabetes(incidence <1 in 100,000). More than half of the cases show loss of methylation atthe promoter of ZAC (on the maternal chromosome). This leads to biallelic (andincreased) expression of this transcription factor gene. The genetic and environmen-tal factors that underlie the observed “loss of imprinting” in TNDM remain largelyunknown. In genetic studies on rare TNDM pedigrees, however, different loss-of-function mutations were detected at ZFP57 (Mackay et al. 2008). This repressivezinc finger protein binds to the methylated allele of many ICRs and is essential forthe somatic maintenance of the differential DNA methylation at ZAC1 and at otherimprinted gene loci in humans and mice.

Another mostly sporadic ID (Tobi et al. 2014) that shows clinical overlap withTNDM is the Silver Russell Syndrome (SRS, OMIM 180860). This ID is charac-terized by intra-uterine growth restriction (IUGR), postnatal growth retardation,facial dysmorphism, body asymmetry, and feeding difficulties. In a majority ofcases, there is loss of DNA methylation at the ICR of the IGF2-H19 domain onhuman chromosome 11p15 (Eggermann et al. 2016). This leads to loss of IGF2expression, and hence, the IUGR and postnatal growth retardation characteristic ofthe disease (Fig. 3). Other imprinted regions are likely involved in SRS as well,including the portion of human chromosome 7 where the GRB10 resides. DifferentSRS cases were reported that had aberrant methylation at multiple imprinted loci,including the ICR of GRB10. So far, however, no proof has been obtained for acausal involvement of GRB10. CDKN1C has been an attractive candidate gene aswell, because of its role in reducing cellular proliferation and fetal growth, but so farno cases have been causally linked to this gene.

The Beckwith-Wiedemann Syndrome (BWS, OMIM 130650) is characterized byfetal overgrowth and gives rise to large babies at birth. Clinical features include


postnatal overgrowth and an increased incidence of early childhood cancers. Lessfrequent signs of this rare disease (incidence ~1 in 15,000) are macroglossia, earlobecreases, midline abdominal wall defects, and hypoglycemia. BWS is geneticallylinked to human chromosome 11p15. Genetic mutations at the IGF2-H19 andKCNQ1 imprinted domains explain a minority of cases. Epigenetic alterations(“epimutations”) are responsible for the large majority of BWS cases (Fig. 3). Inabout 10% of BWS patients, there is aberrant biallelic DNAmethylation at the IGF2-H19 domain’s ICR, which gives rise to IGF2 expression now from both parentalchromosomes (and hence, increased growth) (Eggermann et al. 2016). This is theexact opposite epimutation as the one that causes most frequently the SRS syn-drome. Thus, there can be aberrant loss or gain of DNAmethylation at the IGF2-H19ICR during early embryogenesis, with completely opposite effects on IGF2 expres-sion and growth.

The large majority of BWS cases are caused by loss methylation at the ICR of theKCNQ1 domain. This induces gene repression on both the parental chromosomes,including at CDKN1C (Fig. 3). CDKN1C is the main culprit, because on their own,genetic mutation of CDKN1C can give rise to BWS as well (Eggermann et al. 2016).

Why is DNA methylation at ICRs altered in IDs? Mouse studies have pinpointedspecific transcription factors and chromatin regulatory complexes that are recruitedto ICRs and that contribute to the maintenance of their differential DNA methyla-tion. In humans, there is the recent example of the pluripotency-associated transcrip-tion factor OCT4 (POU5F). In rare cases of BWS, there are genetic mutations at anOCT4 sequence binding motif at the IGF2-H19 ICR. These mutations give methyl-ation at the ICR and increased (presumably biallelic) expression of IGF2 (Demars etal. 2010; Poole et al. 2012).

BWS, SRS, and TNDM are mostly sporadic diseases and it is thought that themethylation changes at ICRs occur through stochastic events, or because of envi-ronmental influences. In animal studies, in case germ cells and early embryos areremoved from their natural environment for in vitro culturing and manipulation, thisoften perturbs DNA methylation at ICRs. Similarly, DNA methylation changes mayoccur when embryonic stem (ES) cells are cultured for prolonged periods, particu-larly in media supplemented with fetal calf serum (Dean et al. 1998; Market Velker etal. 2012). Somatic cell nuclear transfer (“cloning”) technologies have been reportedto be associated with losses and gains of DNA methylation at imprinted loci as well,and also here, the embryo culturing step is particularly critical.

Assisted reproductive technologies (ART), such as in vitro fertilization andintracytoplasmic sperm injection, involve manipulation of germ cells and in vitroembryo culture. This raised the question whether IDs could be more frequent amongART-conceived babies. Studies in different countries reported a several-fold higherincidence of BWS, SRS, and TNDM among children conceived by ART (Dias andMaher 2013). The absolute occurrence of IDS, however, remained extremely rare inART babies. It remains unclear whether the increased frequency of IDs would be dueto the in vitro manipulation and culture, or whether it is somehow reflects thereduced fertility and advanced age of the couples that go to the fertility clinic. Intheory, the reduced fertility could be linked to aberrant imprints in the germ cells,


independently from the ART technologies. An example of the latter possibility hascome from oligozoospermia, a fertility condition in men characterized by reducedsperm counts. Sperm cells of oligozoospermic men display minor losses and gains ofDNA methylation at ICRs. These defects were detected in purified spermatozoa andelongated spermatids as well, which confirms that reduced male fertility can beassociated with abnormal germline imprints (Marques et al. 2004, 2017). It shouldbe important to explore the consequences of sperm methylation changes inoligozoospermia for the next generation (Filipponi and Feil 2009).

Are the methylation changes observed in ART babies the same or different fromtho alterations observed in classical (not ART-linked) cases of IDs. Ongoing studiessuggest that there could be differences, and there is more often perturbation inconcert at multiple loci in the ART babies compared to naturally fertilized babies(Hiura et al. 2012). Further studies on larger cohorts are now required to reachstatistically relevant numbers. Several adult-onset diseases are known to be statisti-cally more common in ART-conceived individuals than in naturally conceivedcontrols. The same has been observed in mouse studies which were designed tonot have any genetic or age confounders. These studies indicate that assistedreproductive technologies are causally involved at least in the late onset clinicalphenotypes (Vrooman and Bartolomei 2016).

Nutrition and Toxic Components Influence Genomic Imprinting

Gestational starvation can have deleterious effects on DNA methylation during theearliest stages of development. Peri-conceptual exposure to famine in the Nether-lands, during the last winter of the Second World War (1944–1945), correlated withmetabolic and mental phenotypes in individuals of the next generation. Theseintergenerational clinical effects were linked to changes in DNA methylation atIGF2, INS,MEG3 (Maternally expressed gene-3), and GNAS. Several nonimprintedgene loci involved in growth and metabolism, including insulin-receptor (INSR),showed altered methylation levels as well. Exposure to war famine at later stages ofgestation did not give detectable DNA methylation changes in the born individuals(Tobi et al. 2009, 2014).

Imprinted genes do not vary much in their DNA methylation levels betweenindividuals. Certain other genes show much higher interindividual variations and aremore susceptible to dietary influences. In a study performed in villages of ruralGambia, it was found that children conceived during the rainy season had higherDNA methylation levels at several of these so-called metastable genes than childrenconceived during the dry season (Dominguez-Salas et al. 2014; Waterland et al.2010). Food intake is different between these two main seasons, and this studytherefore highlights the importance of nutrition during the early stages ofdevelopment.

In rats, early gestational exposure to a high-fat suboptimal nutrition givesincreased type-II diabetes in the offspring (Sandovici et al. 2011). This phenotypewas linked to aberrant repression of a transcription factor gene involved in Β-cell


differentiation and homeostasis. In mice that were fed a high-fat diet, the inducedobesity correlated with decreased Igf2 expression (Morita et al. 2014).

Specific nutriments can have effects on their own as well. Folates are important forthe maintenance of DNA methylation through their effects on the methionine cycle.Hyper-homocysteinaemia (OMIM 603174) is a disorder in which there is increased S-adenosylhomocysteine. Consequently, there is a lower availability of S-adenosyl methi-onine, the universal methyl-donor for DNA methylation (Feil and Fraga 2012). Somepatients show reduced DNA methylation at the IGF2-H19 locus, and this methylationdefect can be restored by providing a folate-rich diet (Ingrosso et al. 2003). Concor-dantly, in mice fed after weaning with a methyl-donor deficient diet (which lackedfolate, vitamin B12 and choline), there was altered Igf2 methylation and expression.

Female mice that had been fed ethanol during late gestation gave birth to maleoffspring that showed reduced methylation at the Igf2-H19 ICR in their sperm (Stouderet al. 2011). Alcoholism in men has been linked to reduced DNA methylation in theirsperm as well, at the IGF2-H19 ICR and at another paternal ICR (Ouko et al. 2009). Itis unclear whether ethanol affects DNA methylation directly, or whether it perturbsmale germ cell development, which then gives rise to aberrant methylation imprints(Fig. 4). However, alteredH19methylation has been observed in frontal cortex tissue ofalcoholics as well, which seems to suggest that the ethanol exposure itself mediatesmethylation changes (Manzardo et al. 2012). Children with a condition called fetalalcohol syndrome, due to maternal ethanol exposure in utero, showed reduced DNAmethylation at several maternal ICRs in peripheral blood (Masemola et al. 2015).

Endocrine disrupting chemicals have been explored by many laboratories. Someendocrine disruptors have marked effects in mouse models that monitor geneexpression influenced by retrotransposon methylation (Feil and Fraga 2012; Xin etal. 2015). Bisphenol-A (BPA) is used for the production of diverse consumerproducts including polycarbonate plastics. This environmental pollutant mimicsthe action of estrogens and effects metabolism and reproduction. A recent mousestudy (Susiarjo et al. 2013) explored peri-conceptual exposure to BPA at twodifferent doses, which comparable to concentrations to which humans are exposed.In the developing embryos, this led to abnormal expression of several imprintedgenes (Snrpn, Kcnq1ot1, and Cdkn1c), with partial re-activation of the normallysilent alleles. Concordantly, losses of ICR DNA methylation were detected as well.In the placenta, in addition, there was a globally reduced DNA methylation level.Exposure to BPA at later stages of development had no adverse effects. A recentstudy explored whether there could be trans-generational effects of the gestationalBPA exposure and showed expression and methylation changes at Igf2 in both the F1and the F2 generation (Susiarjo et al. 2015).

Similar effects were observed for other endocrine disruptors (Fig. 4). Vinclozolinis a fungicide widely used in agriculture that has antiandrogenic effects. In malesborn from exposed pregnant female mice, there was reduced DNAmethylation in thesperm at paternal ICRs, including the Igf2-H19 ICR. Maternal ICRs, which arenormally not methylated at all in sperm, showed about 10% of DNA methylation inspermatozoa (Stouder and Paoloni-Giacobino 2010). Intriguingly, male animals ofthe F2 generation showed slightly altered DNA methylation levels at ICRs as well.


Similar, small, effects on DNA methylation in the gametes of male offspring wereobserved following gestational exposure to the endocrine disruptor methoxychlor(Stouder and Paoloni-Giacobino 2011).

Other kinds of polluting chemicals are suspected to perturb DNA methylation aswell (Feil and Fraga 2012), but their possible effects on imprinted genes have notbeen explored.

A Broadening Outlook on Imprinting Deregulation

Recent research has given important mechanistic insights into imprinted geneexpression and how its perturbation causes specific diseases in humans. There aremore and more examples of how diet and chemicals can perturb the epigeneticregulation of imprinting and how this can have long term pathological consequences.Most environmental studies so far have focused on a handful of imprinted genes,those that were already known to control development, growth and metabolism.

ICR methylation in sperm

Maternal alcoholconsumption / endocrine disruptor(s) during early gestation

Adult alcoholconsumption

Normal nutrition and environment

a

bICR methylation in sperm

Fig. 4 Endocrine disruptors and alcohol affect paternal DNA methylation imprints. (a)normal diet during gestation gives rise to male offspring that have canonical DNA methylationlevels at imprinting control regions (ICRs) in their sperm. Shown are four spermatozoa, which eachhave full DNA methylation ( filled circles) at the example “paternal ICR” (rectangles to the right).Conversely, “maternal ICRs” (left rectangles) are fully unmethylated, as shown for the examplematernal ICR. (b) ICRs show abnormal methylation levels in sperm of males that are born fromfemales that consumed alcohol during gestation. The same is observed in male offspring of femalesthat were exposed to endocrine disruptors during gestation. Alcohol consumption in adult males(mouse/human) can also lead to loss of methylation at paternal ICRs and aberrant gain ofmethylation at maternal ICRs in sperm.


These “classical” loci include the imprinted genes of the INS/IGF signaling pathwayand other regulators of metabolism and growth.

It should be relevant to consider also the hundreds of imprinted ncRNAs (micro-RNAs, snoRNAs, and long ncRNAs). Though not discussed in this chapter,imprinted ncRNAs are thought to play important roles in the expression of pro-tein-coding genes, and their environmental perturbation may thus have major phe-notypic effects.

In the methylation studies so far emphasis has been on ICRs, but many moredifferentially methylated regions (DMRs) could be studied. Hundreds of novelimprinted DMRs with oocyte-derived DNA methylation imprints were discovered inhumans recently. Most of these are not conserved in mice and could therefore playspecific roles in humans/primates (Sanchez-Delgado et al. 2016). Several DMRs arepolymorphic, which could imply that at these regions there is a less-stable establishmentand/or maintenance of methylation than at the “classical” ICRs. Besides pinpointingadditional imprinted loci that are susceptible to environmental cues, broader approachesmay confirm whether imprinted genes are often perturbed in concert or not. Such afinding could lead to the identification of new imprint maintenance factors.

Another outstanding question is whether besides the DMRs at imprinted loci,there could be epigenetic perturbation of endogenous retroviruses as well. Thegenome’s endogenous retroviruses are normally fully methylated, and since theirrepressive methylation is regulated similarly as at ICRs, it should be interesting tosee whether these sequences are affected concomitantly in pathological situations.Studies in the mouse show that methylation patterns at one type of retrotransposons,intracisternal A particles (IAP elements), are readily perturbed by endocrinedisruptors as well, similarly at for imprinted loci (Feil and Fraga 2012).

To what extent nutriments and environmental pollutants could affect histonemethylation at imprinted loci is not known. This may be relevant because of theimportant role of histone lysine methylation in the allelic repression of imprintedgenes during development.

Another relevant question is whether there is genetic predisposition to specificdietary and environmental effects. It has been found that nutritional effects on DNAmethylation are different between male and female individuals (El-Maarri et al.2007; Waxman and O’Connor 2006). At specific gene loci, SNPs and other geneticvariations can have marked effects on local DNA methylation as well, leading todifferences between the two alleles or between individuals (Hellman and Chess2010; Kerkel et al. 2008). Could genetic diversity between the genomes of individ-uals influence the probability of methylation changes occurring in physiologicalconditions and upon external cues? Imprinting disorders may provide a nice systemto explore this further. In a study on the genetics of BWS, it was found thatnucleotide polymorphisms at IGF2 correlated with loss of methylation at the ICRof the close-by KCNQ1 domain. This finding indicated that the genotype of theregion influences the occurrence of the aberrant methylation and, hence, the BWSsyndrome (Murrell et al. 2004).

At the biological level, it is often unclear whether observed deleterious methyl-ation and gene expression changes are a direct consequence of the environmental


cue, or whether they reflect a developmental change. This issue seems particularlypertinent for methylation changes in gonadal development and gametogenesis.Clearly, there is still a lot to be discovered about the mechanisms and the biologicalconsequences of epigenetic changes in (imprinted) gene expression.

Dictionary of Terms

• Imprinted gene – A gene that shows parent-of-origin-specific mono-allelicexpression that is controlled by epigenetic marks (i.e., DNA methylation) thatoriginate from the germline.

• Imprinted domain – A chromosomal region comprising one or multipleimprinted genes. Imprinted domains comprise between 30 and several megabasesof DNA.

• Imprinting control region (ICR) – A regulatory sequence element thatundergoes germline-derived, parent-of-origin-dependent, epigenetic marking. Itcontrols imprinted gene expression of close-by genes.

• Imprinted gene network – Many imprinted genes show co-regulated tissue-specific patterns of expression that are also influenced by cellular proliferationand environmental cues. In addition, several imprinted genes control the expres-sion of other imprinted genes.

• de novo DNA methylation – Addition of methyl groups onto a DNA sequencewhich is not yet methylation (“new” DNA methylation).

• Epimutation – An alteration in the epigenetic marking of a regulatory sequenceor gene, leading to altered gene expression.

• Endocrine disruptor – A chemical component that has an antagonistic effect onthe action of a hormone (e.g., an estrogen) because of structural resemblance.Different pesticides and chemicals used for plastic production have endocrinedisruptor effects.

• Assisted Reproductive Technology (ART) – The combination of approachesthat are applied in the fertility clinic and which include in vitro fertilization (IVF)and intracytoplasmic sperm injection (ICSI)

Key Facts

• Genomic imprinting is an epigenetic phenomenon in mammals that gives rise toparental allele-specific expression of more than 100 autosomal genes.

• The parent-of-origin specific, mono-allelic, expression of imprinted genes playsessential roles in development, homeostasis, and behavior.

• Several imprinted genes encode proteins of the Insulin-like growth factor/Insulinsignaling pathway. Several others influence this pathway indirectly.

• Imprinted gene expression is controlled by germline-acquired DNA methylationimprints at “imprinting control regions” These are maintained somatically afterfertilization.


• Different congenital diseases of aberrant growth and metabolism are caused byDNA methylation changes at imprinting control regions. These “epimutations”are thought to occur early in development.

Summary Points

• Many imprinted genes are linked to the IGF/INS signaling pathway and/orgrowth and metabolism.

• Different imprinted loci show genetic polymorphisms that are linked to type-1 ortype-2 diabetes mellitus.

• Epigenetic alterations (“epimutations”) at imprinted loci are causally involved inimprinting disorders and in certain forms of type-II diabetes.

• Mechanisms involved in the maintenance of differential DNA methylation aresimilar between imprinted gene loci and common maintenance factors have beenidentified.

• Nutriments and environmental cues can perturb DNA methylation imprints inembryos, during early gestation, or in the adult, and this leads to aberrantimprinted gene expression and phenotype.

• Clinical and environmental imprinting studies will gain from taking a broaderapproach that considers all imprinted loci and also the similarly repressed endog-enous retroviruses.

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