POST-GRADUATE
SEMINARON
Department of animal biotechnologyCollege of Veterinary Science and Animal Husbandry
Anand Agricultural UniversityAnand - 388 001.
GENOMIC IMPRINTINGPATEL HIREN M
REG NO. 04-0822-2008
ANBT-699
MAJOR ADVISORDr. C. G. JOSHI
MINOR ADVISORDr. D.N.RANK
2
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
3
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 alleles of a gene are differentially expressed
(Solter, 1988)
4
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)
5
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 by differential DNA methylation regions (DMRs)
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)
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SCHEMA OF IMPRINTING
Pedigree of imprinted maternally expressed phenotype(Barlow and Stewart, 1991)
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Evidences that gene are Imprinted
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Evidences that gene are Imprinted
NEUCLEAR TRANSPLANTATION
UNIPARENTAL DISOMY
HAIG’S MODEL
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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
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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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13
Conti…
Androgenotes
• It have embryonic part small, retarded, or absent
• Extra-embryonic part is OK• After 8 day it dies
Gynogenotes
• Embryonic part OK• Extra-embryonic part is
small, retarded, or absent• Only go to 10 day
Female genome is required for embryonic
developmentMale part is required for extra embryonic
development(Solter, 1988)
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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 chromosome
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Uniparental Disomy
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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
(Cattanach and jones, 1994)
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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)
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Haig' Model: Conflict Theory
Imprinting evolved in mammals because of the conflicting interests of maternal and paternal genes within a litter
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
(Moore and Haig, 1991)
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Haig's Model
•Paternally expressed genes promote embryonic growth•Maternal genes act to restrain the use of maternal resources
Haig's model
predicts that:
•Insulin-like growth factor 2 (Igf-2)•H19•Igf-2r
This model is in remarkably good agreement with
the at least three imprinted genes:
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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)
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Imprinted genes in Mice
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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 maternal chromosome
(Leighton et al,. 1995)
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Imprinted Genes in Mice
Gene Expression Silent allele References
Igf2 Paternal Maternal (DeChaira et al,. 1991)
H19 Maternal Paternal (Brtolomei et al,. 1991)
Insulin-2 Paternal Maternal (Giddings et al,. 1991)
Snrpn Paternal Maternal (Leff et al,. 1992)
Igf-2r Maternal Paternal (Malscheuer et al,. 1993)
Xist Paternal Maternal (Hayashizaki et al,. 1994)
Mash-2 Maternal Paternal (Guillemont et al,. 1995)
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Mouse Chromosome 7
25http://www.mgu.har.mrc.ac.uk/imprinting
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How does Imprinting occur
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How does imprinting occur ?
Many mechanism for gene stamping
Expressed Not Expressed
Site-specific DNA methylation, has received experimental support to date
(Tycko, 1997)
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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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DNA Methylation - Players
DNMTs – DNA Methyltransferases
MBDs – Methyl Binding Domain
proteins
Demethylases
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Enzymatic control of DNA MethylationThree mammalian DNA methylases:
DNMT1
• Generally abundant in all cells• Preference for hemi-methylated
DNA• Copies methylation patterns
after DNA replication
DNMT3A, DNMT3B
• de novo enzymes• Tissue-specific expression• Preference for unmethylated
DNA• Induces novel methylation
marks(Bostick et al., 2007)
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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
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Structure of 5-methylcytosine
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DNA methylation in mammals
MT = DNA methyltransferaseHDAC = Histone DeacetylaseMeCP2 = Methyl-CpG-binding protein
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Deamination of 5-methylcytosine
Morgan et al. (2005) Hum Mol Genet 14, 47-58
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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 ‘5-azacytidine(AzaC)’ can reactivate transcription from this allele
(Tycko, 1997)
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DNA Methylation Experiment
(Li et al., 1993 Nature 266:362)
What happens to mouse embryos that lack DNA methylation?
37
Colum P. Walsh, J. Richard Chaillet & Timothy H. Bestor. Nature Genetics 20:116 (1998)
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Imprinting Mechanism
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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
1 • DNA Methylation at CpG island
2• Enhancer competition model
• Enhancer deletion• H19 gene deletion
3 • Differentially Methylated Regions (DMRs)
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Molecular Mechanism
4• Imprinting Control Regions (ICRs)
• Cis-acting signal• Insulator model
5• Role of RNA in imprinting
• Non coding RNA• Antisense RNA
6 • Chromatin accessibility Model
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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 H19 gene and it’s 5’ flank
(Bartolomei et al,. 1992)
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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)
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Enhancer Competition Model The enhancer competition model rests on the 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
(Yoo- Warren et al,. 1998)
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IGF2 H19 E
Insulin-like growth factor receptor 2 non-coding RNA
Endoderm-specific enhancer
E
Enhancer Competition Model
46
IGF2 H19
IGF2 H19
OFF
OFF
ON
ON
E E
E E
Enhancer Competition Model
CH3CH3CH3CH3
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TESTING THE ROLE OF ENHANCERS IN IMPRINTING
By deleting Enhancer
By deleting H19 gene
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Deleting EnhancerTo 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)
49
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,. 1995)
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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,. 1995)
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Deleting H19 GeneTo 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,. 1995)
52
Cont…Paternal inheritance of the H19 deletion would have
no phenotypic consequenceThis 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,. 1995)
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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 spermSecondary DMRs -after fertilization
For most imprinted genes examined, DMRs are closely associated with CpG islands and direct repeats
(Jeffrey et al,. 2001)
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Differentially Methylated Regions (DMRs)
DMRs acquire their allelic methylation after fertilization
The local spacing and co operativity between 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:
(white circles) lack of methylation (solid circles) complete methylation] (Miguel et al,. 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 Trans-acting factors (black ovals) may act alternatively in the demethylation
pathway resulting in a dominant demethylation signal, spreading over the entire region (allele 2) (Miguel et al,. 1998)
57
Imprinting Control Regions (ICRs)
How does an ICR 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 gene
(Jeffrey et al,. 2001)
58
Imprinting Control Regions (ICRs)
How does an ICR 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 expression
(Jeffrey et al,. 2001)
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Imprinting Control Regions (ICRs)
(Jeffrey et al,. 2001)
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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
(Florian and Denise, 2006)
61
Cont… Most imprinted genes are found in clusters
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 silencing the mRNA genes in an imprinted cluster
(Florian and Denise, 2006)
62
ncRNA- Mediated SilencingTranscription 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)
63
ncRNA- Mediated SilencingTranscription 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)
64
Antisense RNAThe Igf2r and Kcnq1 ICRs contain the promoter of an
antisense RNA that may cause repression of the sense transcript in cis
(Florian and Denise, 2006)
65
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 (Benjamin et al,. 1997)
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Hypercondensation over Chromatin Domains causes Transcriptional Silencing
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Chromatin Accessibility Model
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IMPRINTING CYCLE
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Primary imprinting can be divided into three stages:
• Erasure of the previous imprint • Re-imprinting• Maintenance of the new imprint
(Jeffrey et al,. 2001)
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Imprinting Cycle
pm
pm
GERMLINE
GERMLINE
READING
SOMATIC TISSUE
ERASURE ESTABLISHMENT MAINTENANCE ERASURE
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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 paternalized according 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 methylated or paternalized by 18.5 dpc
(Jeffrey et al,. 2001)
72
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)
73
Imprinting Cycle
(Jeffrey et al,. 2001)
74
Cont…
(Jeffrey et al,. 2001)
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DNMTs are active in germ cells
During development of Spermatid and oocyst more amount of DNMTs are find in their nuclei
(Miguel et al,. 1998)
76
DNA methylation is dynamic & has sex-specific germ cell patterns
Reik W. et al, Science 293, p1089
77
DNA methylation is dynamic & has sex-specific germ cell patterns
Reik W. et al, Science 293, p1089
78
X-Chromosome inactivation
79
X-Chromosome Inactivation
Many molecular features are shared between genomic imprinting and X inactivation
Both are cis-acting epigenetic silencing mechanismsBoth 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
(Lee, 2003)
80
X- Inactivation Center (XIC) XIST gene, encoded at site of X inactivation center
It is untranslated RNA in regional silencing in cis
Accumulation of Xist transcripts along the length of the inactive chromosome
(Panning et al,. 1997)
81
XIST RNA produced from chromosomethat is inactivated
Figure 7 -78 Molecular Biology of the Cell, 4th Edition
82
X-Chromosome Inactivation
P
PPP
P
P
P
P
M
M
MM
M
M
M
M
Zygote
Blastocyst
Embryo
Placenta
Erasure
Erasure
Xa
X
X
Xa
Xa
Xa
Xa
Xi
XiXi Xi
Xi
Xa
Xa
Primordial germ cell
Germline imprinting
Mature gametes
Establishment of imprinting & X-Inactivation
Trophectoderm
Inner cell Mass
Random X-Inactivation
83
Imprinted Gene in Sheep
84
Imprinted Gene in SheepIn 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)
85
Cont…Differential methylation of the
H19 CTCF III upstream region - paternaly methylatedIGF2R DMR2- Maternaly methylated
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)
86
Imprinting Disorder
87
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
(Christopher et al,. 1997)
88
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 as "large offspring syndrome" or LOS
It is characterized by A significant increase in birth weight (8% – 50%)Increase in gestational lengthBreathing problems at birthAn increased frequency of perinatal death
(Sinclair et al,. 2000)
89
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
(Takeda et al,. 2006)
90
Imprinting Disorder in sheep
91
ConclusionMajor 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
92
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
93
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
94
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 come
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