Genomic Imprinting

POSTGRADUATE SEMINAR ON

GENOMIC IMPRINTING PATEL HIREN M REG NO. 04-0822-2008 ANBT-699 MAJOR ADVISOR

MINOR ADVISOR

Department of animal biotechnology College of Veterinary Science and Animal Husbandry Anand Agricultural University Anand - 388 001.

Topics to be Discussed Introduction Evidences that gene are Imprinted Imprinted genes in Mice How does Imprinting occur Imprinting Mechanism Imprinting Cycle X-Chromosome inactivation Imprinted gene in sheep Imprinting Disorder Conclusion Future prospects 2

INTRODUCTION People inherit two copies of their genes—one from their mother and one from their father Usually both copies of each gene are active, or “turned on,” in cells Sometime only one of the two copies is normally turned on Which copy is active depends on the parent of origin Some genes are normally active only when they are inherited from a person’s father or a person’s mother Genomic imprinting is an epigenetic phenomenon by which the two parental alleles3

Conti… Genomic imprinting in mice was first deduced from nuclear transplantation experiments

(McGrath and Solter, 1983)

The nonequivalence of maternally and paternally contributed genomes was first identified in elegant nuclear- transfer studies

(McGrath and Solter, 1984)

 There is conservation of imprinting between mice and humans, But one exception, the Insulin-like growth factor 2 receptor (Igf-2r) (Barlow et al., 1991)

 Imprinted genes are organized in clusters, and two of the clusters, on mouse chromosomes 7 and 17, contain both maternally and paternally expressed genes (Zemel

et al., 1992)

4

Conti…

DNA, once methylated, will tend to stay methylated, thus providing a mechanism for the stable maintenance of an imprint during cell division and differentiation

(Bestor and Verdine , 1994)

Imprinted genes are often characterized differential DNA methylation regions (DMRs)

by

In somatic cells, DMRs such that for one parental allele CpGs in a region are methylated, while for the other parental allele they are not (Shemer and Razin , 1996)

Methylation is further supported by the demonstration that mice deficient in Dnmt1-gene function show a loss of imprinting at almost all loci tested

(Shemer et al,. 1997)5

Pedigree of imprinted maternally expressed phenotype (Barlow and Stewart, 1991)

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Evidences that gene are Imprinted

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Neuclear Transplantation Studying An early observation pointing to

genomic imprinting was the finding of aberrant development of artificially constructed isoparental embryos in mice

Gynogenetic (two female pronuclei) Androgenetic (two male pronuclei) (McGrath & Solter , 1984)

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Neuclear transplantation

Fertilized diploid embryo (zygote) the maternal and paternal nuclei do not fuse for 12 hours 10

Neuclear Transfer shows both Parental Genomes needed for Mouse Embryonic Development

McGrath & Solter (1984) Cell 37:179, Surani et al., (1984) Nature 308:548

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12

Conti

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Uniparental Disomy Uniparental disomy (UPD) occurs when an individual receives both copies of a chromosome from one parent only Child inherits the genes from one parent only (uniparental) In mice with certain translocations that produced uniparental disomies for particular chromosomes (Cattanach and jones, 1994)

One type of cross generated foetuses containing two copies of a large portion of one types of 14 chromosome

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Cont… In mice chromosome no 7 shows such types of translocation One crossed foetuses containing two copies of a large portion of maternal chromosome no 7 but no copies from paternal side These mice fetuse were developmentally retarded, small placenta and died in utero at midgestation Reciprocal cross, resulting in two paternal and no maternal chromosome 7 homologues, conceptuse died much earlier 16

Haig's Model The evolutionary conservation of imprinting suggests that the phenomenon might provide some selective advantage The challenge is now to determine the function and mechanism of allelic inactivation by imprinting To date the most compelling model has been provided by Haig and his colleagues That is know as ‘‘Conflict Theory’’ (Moore and Haig, 1991) 17

Haig' Model: Conflict Imprinting evolved in mammals because Theory the conflicting interests of maternal paternal genes within a litter

of and

In non monogamous, the mother provides significant maternal resources to the offspring both during intrauterine and suckling after birth Successful passage of paternal genes into the next generation is best ensured by having the embryos consume maternal resources The mother's interests are best served by distributing her resources more equitably among litters 18 (Moore and Haig, 1991)

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cont… Mouse embryos are sensitive to the levels of the paternally expressed growth factor insulin-like growth factor II (IGF-2) (Robertson, 1991)

A complete loss of function of the gene encoding the growth factor, Igf-2, leads to a 40% reduction in birth weight Fetuses that lack Igf-2r are approximately 30% larger than normal, have elevated circulating levels of IGF-2, and die around birth (lau and Barlow , 1994) 20

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Imprinted Genes in Mice In the early l990s, experiments with either gene “knock-out” or naturally occurring strains of mice in which the two parental alleles could be distinguished, showed that:

Insulin-like

growth factor 2 (Igf2) gene was expressed only from the paternal allele Insulin-like growth factor 2 receptor(Igf2 R) gene was expressed only from the maternal allele (DeChaira et al,. 1991)

The maternally expressed H19 gene also functions to lower the concentration of Igf2 by suppressing its transcription on maternal22

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http://www.mgu.har.mrc.ac.uk/impr 25 inting

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DNA Methylation Covalent modification of the DNA is important for gene silencing Most genes have GC rich areas of DNA in their promoter regions, These are referred to as CpG islands Methylation of the C residues within the CpG islands leads to gene silencing DNA methylation is restricted to C in a 5’-CpG -3’ dinucleotide (Ariel et al,. 1994)

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Enzymatic control of DNA Methylation

Three mammalian DNA methylases:

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Cytosine Methylation  5-methylcytosine  Chemically unstable  Prone to deamination  Resulting in thymine  Inefficient recognition of mismatch by DNA repair mechanisms  Cytosine deamination gives Thymine, which is recognized by DNA repair systems 31

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ØMT = DNA methyltransferase ØHDAC = Histone Deacetylase ØMeCP2 = Methyl-CpG-binding protein 33

Morgan et al. (2005) Hum Mol Genet 14, 47-58 34

DNA Methylation Experiment  Improper or absent of DNA methylation

of imprinted gene may lead to abnormal growth

 Mouse embryos with a targeted deletion of the DNA methyltransferase gene showed abnormal expression of at least three imprinted gene (Li et al,. 1993)

 DNA methyltrasferase inhibitor ‘5azacytidine(AzaC)’ can reactivate transcription from this allele

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DNA Methylation Experiment

(Li et al., 1993 Nature 266:362) 36

What happens to mouse embryos that lack DNA methylation?

Colum P. Walsh, J. Richard Chaillet & Timothy H. Bestor. Nature 37 Genetics 20:116 (1998)

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Mechanism of Imprinted Gene Silencing

To dissect the molecular mechanism of imprinting, we have to focused on a region of approximately 300 kb on the distal portion of mouse chromosome 7

An imprinted region on the distal end of mouse chromosome 7. The positions of the imprinted genes Mash-2, Ins-2, Igf-2 and H19 on mouse chromosome 7 (Guillemot et al,. 1995)

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Molecular Mechanism

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Molecular Mechanism

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DNA Methylation at CpG island The clustering of four imprinted genes raised the possibility that, they are controlled by a signal or signals that act over large distances

A search for candidates for the epigenetic mark focused from parent-specific DNA methylation The best candidate for the epigenetic mark at the H19/Igf-2/Ins-2 locus is paternal-specific DNA methylation of the

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DNA Methylation at CpG island Evidence in favor of DNA methylation as the epigenetic mark at the locus comes from a targeted disruption of the DNA methyltransferase gene in mice Embryos that are homozygous for a null mutation in the gene, and therefore have dramatically reduced levels of DNA methylation, express both alleles of H19 and have silenced both Igf-2 genes (Li et al,. 1993) 43

Enhancer Competition Model model rests on the The enhancer competition premise that H19 and Igf-2 utilize the same enhancers Only two enhancers have been identified in the vicinity of the Igf-2 and H19 genes, at + 9 and + 11 kb relative to the start of transcription of the H19 gene H19 promoter is activated by enhancer, Igf-2 gene is not expressed from maternal allele H19 is methylated, enhancer is unable to interact with the H19 and Igf-2 gene is expressed from paternal side

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Enhancer Competition Model IGF2

H19

Insulin-like growth factor receptor 2

non-coding RNA

E

E

Endoderm-specific enhancer

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Enhancer Competition Model OFF

ON

IGF2

H19

E

E

E

E

OFF

ON CHCHCH CH

IGF2

3 3 3

3

H19

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Deleting Enhancer To ask whether enhancer are required for expression of both H19 and Igf-2, a line of mice was generated in which the enhancers were deleted, and the consequence to the expression of H19 and Igf-2 was assessed

(Leighton et al,. 1995)

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Cont…

When the enhancer deletion was inherited from females, decline in H19 RNA in all tissues of endodermal origin The levels of Igf-2 RNA were unaffected by the maternal deletion of the enhancers, Igf-2 is normally silent

(Leighton et al,.

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Cont…

When the enhancer deletion was inherited from males, the levels of Igf-2 RNA declined in exactly the same manner as H19 RNA in the maternal heterozygotes, while the levels of H19 RNA were unaffected

(Leighton et al,.

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Deleting H19 Gene To test the dependence of Igf-2 imprinting on the H19 gene, a strain of mice was generated that carries a deletion of the paternal-specific methylation domain, including the structural H19 gene itself and 10 kb of 5' flanking sequence

(Leighton et al,.

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C ont … H19 deletion

Paternal inheritance of the would have no phenotypic consequence This proved, both the maternal H19 allele and the paternal allele of Igf-2 were unaffected When the H19 deletion was inherited from a female, the neonatal progeny expressed both paternal and maternal Igf-2 alleles in all tissues examined

(Leighton et al,.

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Differentially Methylated Regions (DMRs) Imprinted genes are often characterized by differential DNA methylation There are two types of DMRs: Primary DMRs- In oocytes and sperm Secondary DMRs -after fertilization For most imprinted genes examined, DMRs are closely associated with CpG islands and direct repeats (Jeffrey et al,. 2001) 53

Differentially Methylated Regions (DMRs) DMRs acquire their allelic methylation after fertilization

The local spacing and co operativitybetween DMRs and core DMRs may be important for the propagation of the imprinting signal Also important for stabilization of specific epigenotypes in imprinting clusters The disruption of any of these elements could result in failure to maintain or establish a parental epigenotype (Reik and Walter. 1998)

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Role of Tandem Repeats in Allele Specific Methylation

 (+) methylation and (-)de-methylation signals  (R) may act as methylation centers  Gray/Red disks- The extent of methylation may be limited by counteracting demethylation signals  Trefoilstructures-Trans-acting factors interfere with methylation or its spreading  A gradient of methylation is represented by differently shaded circles: (Miguel et al,. 55 (white circles) lack of methylation 1998)

Role of Tandem Repeats in Allele Specific Methylation

 (+) methylation and (-)de-methylation signals  (R) may act as methylation centers  Gray/Red disks- The extent of methylation may be limited by counteracting demethylation signals  Trefoilstructures-Trans-acting factors interfere with methylation or its spreading  Trans-acting factors (black ovals) may act alternatively in the demethylation pathway resulting in a dominant demethylation (Miguel et al,. 56 signal, spreading over the entire region (allele 1998) 2)

Imprinting Control Regions How does an ICR (ICRs) act to regulate

monoallelic expression in somatic cells? (A)  ICRs appear to be gene regulatory elements that can significantly affect the expression of genes in cis  The Igf2/H19 ICR is a chromatin insulator that blocks the interaction of enhancers with the Igf2 promoter  When H19 gene is methylated, CTCF cannot bind to the insulator, and the activator is able to activate the transcription of the Igf2 (Jeffrey gene et al,.

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Imprinting Control Regions How does an ICR (ICRs) act to regulate

monoallelic expression in somatic cells? (B)  These ICR regulatory elements can be turned “off” on one parental allele and “on” on the other parental allele by the presence and absence of DNA methylation  This brings about allelic activity and inactivity in trans, or monoallelic (Jeffrey et al,. expression 2001)

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Imprinting Control Regions (ICRs)

(Jeffrey et al,.

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Role of RNA in IMPRINTING Imprinted gene clusters contain

multiple imprinted mRNA genes and at least one imprinted noncoding RNA (ncRNA)

Two imprinted ncRNAs have now been shown to act as cis-acting domain silencers This indicate that RNA-mediated silencing may be a central feature of genomic imprinting 60

Most imprinted clusters

genes

are

Cont…

found

in

Each imprinted cluster is regulated by one imprint control element or ICE In an imprinted cluster are imprinted protein-coding mRNA genes; however, at least one is always an imprinted ncRNA Imprinted ncRNAs is that they show reciprocal parental-specific expression The reciprocal expression of imprinted mRNAs and ncRNAs has long been thought to indicate that ncRNAs play a role in and Denise, silencing the mRNA genes in(Florian an imprinted 2006)

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ncRNA- Mediated Silencing Transcription of ncRNAs could induce silencing of genes laying several hundred kilobase pairs upstream and downstream Two possibilities could be considered [A] ncRNA transcription could activate a domain repressor contained within the ncRNA transcription unit

(Florian and Denise, 2006)

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ncRNA- Mediated Silencing Transcription of ncRNAs could induce silencing of genes laying several hundred kilobase pairs upstream and downstream Two possibilities could be considered [B] ncRNA transcription could repress a domain activator contained within the ncRNA transcription unit

(Florian and Denise, 2006)

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Antisense RNA The Igf2r and Kcnq1 ICRs contain the promoter of an antisense RNA that may cause repression of the sense transcript in (Florian and Denise, cis 2006)

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Chromatin Accessibility Model Imprinted domains must contain particular

DNA sequences that distinguish them from the rest of the genome

According to this model open chromatin would allow modification of multiple changes Methylation of CpG or binding of repressive chromatin proteins or both , lead to compacted chromatin In compacted chromatin modification is prevented

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Hypercondensation over Chromatin Domains causes Transcriptional Silencing

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Chromatin Accessibility Model

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(Jeffrey et al,. 2001)

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Imprinting Cycle SOMATIC TISSUE

p m READIN G

p m

GERMLI NE

ERASURE ERASURE

GERMLI NE ESTABLISHMENT

MAINTENANCE 70

Imprinting Cycle PGCs, like somatic cells, presumably also inherit one maternally and paternally imprinted genome They develop and differentiate at 11.5 dpc, both genomes must become maternalized or paternalizedaccording to germ cell sex (new imprints) For the female germ line, both alleles become fully methylated or maternalized in fetuses at 16.5 days post-coitum (dpc) The male germ line, both alleles become fully (Jeffrey methylated or paternalized by 18.5 dpcet al,. 2001)

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Cont…

This establishment of ICR methylation in male germ cells corresponds to the time of genome-wide de novo methylation This is occurring in germ cells of both sexes from approximately 15.5 dpc to 18.5 dpc At that time germ line is not dividing

(Jeffrey et al,. 2001)

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Imprinting Cycle

(Jeffrey et al,. 2001)

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Cont…

(Jeffrey et al,. 2001)

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DNMTs are active in germ During development cellsof Spermatid and oocyst more amount of DNMTs are find in their nuclei

(Miguel et al,. 1998)

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Reik W. et al, Science 293, p1089

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Reik W. et al, Science 293, p1089

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X-Chromosome Inactivation molecular features are

Many between genomic inactivation

imprinting

shared and X

Both are cis-acting epigenetic silencing mechanisms Both show a positive correlation between expression of a ncRNA and silencing (Reik and Lewis, 2005)

It has been suggested that X inactivation was a driving force in the evolution of genomic imprinting 79

X- Inactivation Center (XIC) XIST gene, encoded at site of X inactivation center

It is untranslated RNA in regional silencing in cis (Panning et al,. Accumulation of Xist transcripts along the 1997) length of the inactive chromosome

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Figure 7 -78 Molecular Biology of the Cell, 4th Edition

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Mature gametes Primordi al germ cell

X-Chromosome Inactivation Zygot Establishme e nt of M X

P

X a X a

M P

X

Germlin e imprinti

M P

Trophectod erm

Erasur e Erasur e

M P M P

Blastoc yst X a Xi

M P

Inner cell Mass

Xi X a X a Xi

imprinting & X-

M P

X a Xi

Placent a M X P Xi a

Embr yo

Random XInactivation

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Imprinted Gene in Sheep

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Imprinted Gene in Sheep In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus While H19, IGF2R, GRB10 and p57KIP were maternally expressed GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21 (Alexandra et al,. 2008) 84

Cont… Differential methylation of the H19 CTCF III upstream region methylated IGF2R DMR2- Maternaly methylated

-

paternaly

In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically The majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage (Alexandra et al,. 2008) 85

Imprinti ng Disorde r 86

Imprinting Disorder Any type of condition which leads to loss of imprinting may lead to various disease condition Where the chromosome involved in the UPD is imprinted there may be complication for that individual In maternal UPD, A condition called Prader Willi Syndrome In paternal UPD, A condition called angelman syndrome

87

Cont…

The interest in genomic imprinting and epigenetics in animal husbandry :

occurrence of a fetal overgrowth syndrome during assisted reproduction techniques (ART) in ruminants This overgrowth is known offspring syndrome" or LOS It is characterized by

as

"large

A significant increase in birth weight (8% – 50%) Increase in gestational length Breathing problems at birth 88 An increased frequency of perinatal death

Imprinting Disorder in sheep There is a link between imprinted genes and muscle development in lamb Influence of the imprinted IGF2 gene on muscle growth and fat deposition

(van Laere et al,. 2003)

An even more prominent effect is seen of the so called callipyge mutation on the muscle development of the hind legs of sheep Callipyge lambs are born normal and the first signs of muscular hypertrophy are first detectable in the loin and hindquarters at 4–6 weeks of age

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Imprinting Disorder in sheep

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Conclusion Major reprogramming of the imprints occurs in the germ lines of developing embryos Regions of differential methylation have been identified at all imprinted genes Genome-wide alterations in methylation occur in the pre implantation and early post implantation embryo Multiple cis-acting sequences are probably required for the creation and maintenance of the methylation state

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Cont… DMRs are CpG-rich and associated with direct repeats Imprinted gene clusters should contain at least one ICE ICE will carry an epigenetic mark on one parental chromosome Imprinted DNA sequence modified only in one gamete 92

Cont… Regulation mechanisms such as antisense transcription and enhancer competition play an important role in the imprinting process Mostly imprinted gene are conserved in different species Imprinting and mutation both prevent gene expression , but in mutation DNA sequence is change while in in printing no change in DNA The imprinting process is reversible

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Future prospects In animal husbandry our interest is to produce more producible animal For achievement of more production we are going for cross breeding Genomic imprinting may affect the production of traits which is regulated by imprinted gene Thus the emerging area of epigenetics holds promises of being of interest to both the farmer, the veterinarian and for the area of animal models for the years to

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