POST-

GRADUATE
SEMINAR
ON

GENOMIC IMPRINTING
PATEL HIREN M
REG NO. 04-0822-2008
ANBT-699
MAJOR MINOR
ADVISOR 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 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)5
Pedigree of imprinted maternally expressed phenotype
(Barlow and Stewart, 1991)
6
7
Evidences that gene are Imprinted

8
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)

9
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 11
12
Conti

13
 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 14
15
 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
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 18
(Moore and Haig, 1991)
19
 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
21
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
23
24
http://www.mgu.har.mrc.ac.uk/impr
inting 25
26
27
 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)

28
29
 Enzymatic control of DNA
Methylation
Three mammalian DNA methylases:

30
 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
32
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 ‘5-

azacytidine(AzaC)’ can reactivate
transcription from this allele 35
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)
38
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) 39
Molecular Mechanism

40
Molecular Mechanism

41
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
42
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
44
expressed from paternal side
Enhancer Competition
Model

IGF2 H19 E E
Insulin-like growth factor receptor 2 non-coding RNA

Endoderm-specific enhancer

45
Enhancer Competition
Model
OFF
ON

IGF2 H19 E E

ON OFF
CHCHCH CH

IGF2
3 3 3 3
H19 E E

46
47
 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,. 48

1995)
 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,. 49
 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,. 50
 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,. 51
Paternal inheritance of the
C ont …
H19 deletion
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,. 52
 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)
54
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,.57
 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
expression (Jeffrey et al,.
2001)
58
Imprinting Control
Regions (ICRs)

(Jeffrey et al,. 59
 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
 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(Florian
an imprinted
and Denise, 61
2006)
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, 62

2006)
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, 63

2006)
 Antisense RNA
The 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)

64
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 65
 Hypercondensation over
Chromatin Domains causes
Transcriptional Silencing

66
Chromatin Accessibility Model

67
68
(Jeffrey et al,.
2001) 69
 Imprinting Cycle
SOMATIC
TISSUE
p
m
READIN
G
p
m

GERMLI
NE
GERMLI
NE

ERASURE ESTABLISHMENT MAINTENANCE

ERASURE 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

methylated or paternalized by 18.5 dpcet al,.
(Jeffrey
2001) 71
 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)

72
Imprinting Cycle

(Jeffrey et al,. 73

2001)
 Cont…

(Jeffrey et al,. 74

2001)
DNMTs are active in germ
cellsof Spermatid and
During development
oocyst more amount of DNMTs are find in
their nuclei
(Miguel et al,. 1998)

75
Reik W. et al, Science
293, p1089 76
Reik W. et al, Science 77
293, p1089
78
 X-Chromosome
Many
Inactivation
molecular features are shared
between genomic imprinting and X
inactivation

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
Accumulation of Xist transcripts along
(Panning the
et al,.
length of the inactive chromosome
1997)

80
Figure 7 -78 Molecular Biology of the 81

Cell, 4th Edition

 X-Chromosome
Mature
Inactivation
Zygot
e
Establishme
nt of
M X X
gametes M imprinting &
P a
X X-
a
Primordi P X
Blastoc
al germ yst
cell M X
Germlin Trophectod a
P Xi
e erm
imprinti M
Inner cell
P
Mass
Erasur
e Placent
a
M X M X
Erasur M Xi P Xi
P a
Xi a
e P X
a Embr
M X yo
P a Random X-
Xi 82
Inactivation
Imprinted Gene
in Sheep

83
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 - paternaly
methylated
IGF2R 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)
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 as "large

offspring syndrome" or LOS
It is characterized by
A significant increase in birth weight (8% –
50%)
Increase in gestational length
Breathing problems at birth
An increased frequency of perinatal death 88
 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 89
Imprinting Disorder in
sheep

90
 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
91
of the methylation state
 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
93
The imprinting process is reversible
 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 94
THANK YOU