1.
Genomic Imprinting
•Definition- Differential expression of
two parental alleles
• Only occurs in eutherians (placental,
nonmarsupial) mammals
• Not in other vertebrates
•Of 20-some identified genes, most are
involved in
1. Fetal growth
• Igf2, IgF2r, H19, Grb1
2. Brain development
• Prader-Willi syndrome (PS), Angelman syndromes (AS),
Peg1/Mest

2.
Genomic-imprinting conflict
What is genomic imprinting and why has it evolved?
Expression of a gene depending on whether inherited
from father or mother
Main arenas of imprinting effects on human health:
-Placenta
-Brain
-Carcinogenesis
-Stem cells
-In vitro fertilization
from dad from mom

3.
• In experiments on mice
maternal and
paternal
pronuclei
normal mouse
both
paternal
pronuclei
The placenta is
huge, the embryo
is undeveloped
both
matern
al
pronucl
ei
The embryo is
formed, the placenta
is underdeveloped

4.
A summary of imprinted gene functions during embryogenesis.
Robert N. Plasschaert, and Marisa S. Bartolomei
Development 2014;141:1805-1813
© 2014. Published by The Company of Biologists Ltd

5.
CONFLICT THEORY OF IMPRINTING
-> abundant support from empirical studies of imprinted
genes and growth, in mice and humans
(1) IGF2-IGF2R (Haig & Graham 1991 Cell)
(2) CDKN1C (Andrews et al. 2007 BMC Dev Biol)
(3) GRB10 (Charalambous et al. 2003 PNAS)
Beckwith-
Wiedemann
Syndrome
Silver-
Russell
syndrome
Mighty mouse
Normal sized
human
2 doses IGF2
1 dose IGF2
0 doses IGF2

6.
Genomic imprinting
the unequal expression of the maternal and paternal alleles of a gene
XX
X
Y
Maternal allele
Patternal allele
Patternal
allele
Matternal
allele

7.
• Two major clusters of imprinted genes have been identified in
humans, one on the short (p) arm of chromosome 11 (at
position 11p15) and another on the long (q) arm of chromosome
15 (in the region 15q11 to 15q13).

8.
Mouse models to study genomic imprinting that allow the maternal and paternal chromosome to
be distinguished.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Mouse models to study genomic imprinting that allow the maternal and paternal chromosome to be
distinguished. Mammals are diploid and inherit a complete chromosome set from the maternal and paternal
parent. However, mice can be generated that (1) inherit two copies of a chromosome pair from one parent
and no copy from the other parent (known as UPD), (2) inherit a partial chromosomal deletion from one
parent and a wild-type chromosome from the other parent, and (3) inherit chromosomes carrying single-
nucleotide polymorphisms (known as SNPs) from one parent and a wild-type chromosome from the other
parent. Offspring with UPDs or deletions are likely to display lethal phenotypes, whereas SNPs will allow
the production of viable offspring.

9.
A maternal and paternal genome are needed for mammalian reproduction.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
A maternal and paternal genome are needed for mammalian reproduction. The nuclear transfer technique
used micropipettes and high-powered microscopes to remove the male or female nuclei from a newly
fertilized egg and place them in various combinations into a second “host” fertilized egg that had already
been enucleated, thereby generating anew diploid embryos with two maternal (gynogenetic) or two
paternal (androgenetic) genomes or a biparental genome (wild-type). Gynogenetic and androgenetic
embryos were lethal at early embryonic stages. Only reconstituted embryos that received both a maternal
and paternal nucleus (wild-type) survived to produce living young. These experiments show the necessity
for both the maternal and paternal genome in mammalian reproduction, and indicate the two parental
genomes express different sets of genes needed for complete embryonic development.

10.
Imprint acquisition and erasure in mammalian development.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Imprint acquisition and erasure in mammalian development. Imprints are acquired by the gametes; thus, oocytes and
sperm already carry imprinted chromosomes (first-generation imprints). After fertilization when the embryo is
diploid, the imprint is maintained on the same parental chromosome after each cell division in cells of the embryo,
yolk sac, placenta, and also in the adult. The germ cells are formed in the embryonic gonad and the imprints are
erased only in these cells before sex determination. As the embryo develops into a male, the gonads differentiate to
testes that produce haploid sperm that acquire a paternal imprint on their chromosomes. Similarly, in developing
females, chromosomes in the ovaries acquire maternal imprints (second-generation imprints).

11.
Imprinted genes play a role in mammalian reproduction.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Imprinted genes play a role in mammalian reproduction. Mammals are diploid and reproduction
requires fertilization of a haploid female egg by a haploid male sperm to recreate a diploid embryo.
Only females are anatomically equipped for reproduction, but they cannot use parthenogenesis to
reproduce (the possibility of which is represented by a pink dashed line) because essential
imprinted genes needed for fetal growth are imprinted and silenced on maternal chromosomes.
These genes are expressed only from paternal chromosomes; thus, both parental genomes are
needed for reproduction in mammals. Parthenogenesis is the production of diploid offspring from
two copies of the same maternal genome.

12.
Imprinted genes are expressed from one parental allele and often clustered.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Imprinted genes are expressed from one parental allele and often clustered. Most imprinted
genes (yellow) are found in clusters that include multiple protein-coding mRNAs (IG) and
at least one noncoding RNA (IG-NC). Nonimprinted genes can also be present (NI in
gray). The imprinting mechanism is cis acting and imprinted expression is controlled by an
imprint control element (ICE) that carries an epigenetic imprint inherited from one
parental gamete.

13.
Imprinted expression is regulated by gametic DMRs (G-DMR).
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Imprinted expression is regulated by gametic DMRs (G-DMR). (Left) The
effect of deleting the gametic DMR from the imprinted chromosome (green). (Right) The effect of
deleting the G-DMR from the nonimprinted chromosome (yellow). In many imprinted clusters (e.g.,
Igf2r, Kcnq1, and Dlk1), experimental deletion of the G-DMR only affects the chromosome carrying
the nonimprinted G-DMR. This results in a loss of repression of the imprinted protein-coding mRNA
genes (IG) and a gain of repression of the imprinted lncRNA gene (IG-NC). Note that in some
imprinted clusters (Igf2 and Pws) that are not illustrated here, the methylated G-DMR appears also to
be required for expression of some of the imprinted mRNAs in cis. del, deleted DNA; G-DMR,
gametic differentially DNA-methylated region; NG, nonimprinted gene; arrow, expressed allele;
slashed circle, repressed allele; imprint, epigenetic modification leading to a change in gene expression
in cis.

14.
Two cis-acting silencing mechanisms at imprinted gene clusters.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Two cis-acting silencing mechanisms at imprinted gene clusters. (A) Insulator model for the Igf2 cluster.
The expression pattern for endoderm is shown. On the maternal chromosome, the unmethylated ICE binds
the CTCF protein and forms an insulator that prevents the common endoderm enhancers (E) from
activating Igf2 and Ins2. Instead the enhancers activate the nearby H19 lncRNA promoter. On the paternal
chromosome, the methylated ICE cannot bind CTCF and an insulator does not form; hence the Igf2 and
Ins2 mRNA genes are expressed only on this chromosome. The H19 lncRNA is methylated, most likely
because of spreading from the 2-kb distant methylated ICE, and silenced.

15.
Two cis-acting silencing mechanisms at imprinted gene clusters.
Denise P. Barlow, and Marisa S. Bartolomei Cold Spring
Harb Perspect Biol 2014;6:a018382
©2014 by Cold Spring Harbor Laboratory Press
Two cis-acting silencing mechanisms at imprinted gene clusters. (B) lncRNA model for the Igf2r cluster.
The expression pattern for placenta is shown. On the maternal chromosome, the methylated ICE contains
the Airn lncRNA promoter that is directly silenced by the DNA methylation imprint. The Igf2r, Slc22a2,
and Slc22a3 mRNA genes are expressed only on this chromosome. Mas1 and Slc22a1 are not expressed in
placenta (filled diamond). On the paternal chromosome, the Airn lncRNA promoter lying in the
unmethylated ICE is expressed and silences Igf2r (in part by kicking off RNA polymerase II), Slc22a2, and
Slc22a3 in cis. Note that in both models, the DNA methylation imprint silences the lncRNA and permits
mRNA expression. ICE, imprint control element; gray arrow, expressed allele of an imprinted gene;
slashed circle, repressed allele of an imprinted gene; thick gray arrows, long distance effect in cis.

16.
Paternally expressed imprinted genes that function as growth
promoters (i.e., Igf2, Peg1, Peg3, Rasgrf1, Dlk1) and show
growth retardation in embryos deficient for the gene. There
are also maternally expressed imprinted genes that function
as growth repressors (i.e., Igf2r, Gnas, Cdkn1c, H19, Grb10),
as shown by a growth enhancement in embryos deficient for
the gene.

17.
Categories of imprinted genes
1. Fetal growth genes- Insulin-like growth
factor-like II (IGF2) response pathway
• IGF2
• Igf2r
• Grb10
• H19
• Gnas
• Rasgrf
• Mash2
• Why?- Embryo develops in a parasite-like
relationship with mother.

18.
Categories of imprinted genes
2. Brain development-
• Prader Willi Syndrome- paternal chromosome deletion
• Angelman Syndrome- maternal chromosome deletion

19.
Prader-Willi Syndrome
• 1 in 15,000 live births
• mostly sporatic
• deletion at 15 q11-q13
• diagnosis at 2 years
• compulsive overeaters

20.
A typical Prader-Willi patient
Prader Willi Syndrome- Due to
paternal chromosome deletion

21.
• 1 in 25,000 live births
• mostly sporatic
• 80 % have deletion at 15q11-q13
• Specifically mutation of UBE3A gene
Angelman Syndrome

22.
Angelman Syndrome
•Speech impairment
•None or minimal use of words
•Receptive and non-verbal communication skills higher than
verbal ones
•Movement or balance disorder, usually ataxia of gait
•Behavioral uniqueness: any combination of frequent
laughter/smiling; apparent happy demeanor
•easily excitable personality, often with hand flapping
movements; hypermotoric behavior; short attention span

23.
Angelman Syndrome
Normal
–Angelman Syndrome- maternal chromosome deletion
Or 2 paternal chromosomes
Or an
imprinting
defect

24.
Parent Offspring Conflict Hypothesis (Haig
hypothesis)
• Conflict between male and female over allocation
of maternal resources to offspring
• Dad uses imprinting to direct all resources to
immediate offspring (not future litters)
• Mom uses imprinting to allocate resources to
multiple litters
• Thus, predict paternally expressed genes would
promote growth, maternally expressed genes should
slow it down
• Prediction mostly hold true
• Example- Igf2 (paternally expressed)-if defective=40%
reduction in growth

25.
Parent Offspring Conflict Hypothesis (Haig
hypothesis)
Example – The Igf2 gene and its receptor Igf2r
•Igf2 (paternally expressed)-if defective=40%
reduction in growth
•Igf2r (Igf2 receptor)- if defective=increase growth
•Igf2-/Igf2r- = normal
Another test- Ask if imprinting fails to occur in a monogomous
species
The Beach mouse is entirely monogomous
….but imprinting still occurs, contrary to model

26.
How are imprinted genes silenced?
S. Tilghman, Cell 96:185

27.
How are imprinted genes silenced?
S. Tilghman, Cell 96:185
Dnmt-/- mice-
Many imprinted genes
(e.g. H19) reactivated
..but, Igf2 and Igf2r are
silenced
Mechanism- Methylation interferes with transcription factor binding
Problems with model-
1. Promoters of silent Igf2 and Igf2r alleles are unmethylated
2. One gene, Mash2, is unaffected by loss of methylation

28.
How are imprinted genes silenced?
S. Tilghman, Cell 96:185
Mechanism-
Promoters compete
for a single
enhancer
Problem with model-
Both H19 and Igf2 are expressed if H19 gene replaced
with luciferase
Igf2 H19

29.
How are imprinted genes silenced?
S. Tilghman, Cell 96:185
Epigenetic marker binds to
unmethylated DNA
Mechanism-
Methylation serves two
purposes
1. Inactivate a gene (e.g.
H19)
2. Prevent binding of
epigenetic marker so
that Igf2 is activated
Igf2 H19
Epigenetic insulator prevents enhancer from “talking to” Igf2
Evidence in support: if delete insulator element- both Igf2 and H19 expressed

30.
Evidence for chromatin boundary mechanism
Deletion of ICR- both genes expressed
Identify protein (called CTCF) that binds ICR
CTCF cannot bind methylated DNA
Thorvaldsen and Bartolmei, Science 288:2145, 2000

31.
How are imprinted genes silenced?
S. Tilghman, Cell 96:185
Mechanism- Antisense
transcription of
unmethylated
chromosome blocks
sense strand transcription
Mechanism- Antisense
RNA blocks sense strand
transcription

32.
Prader-Willi Syndrome
Normal
expression
patterns
= ON

33.
Prader-Willi Syndrome
Normal
expression
patterns
= ON
In Prader-Willi deletion,
maternal and paternal
copies are silent
In Angelman, many
genes are activated, but
UBE3A is silenced

34.
Prader-Willi Syndrome
Proposed mechanism of SNPRN imprinting
In Prader Wille, the Switch Initiator Site (SNSIS) is mutated in
paternal chromosome, such that it can’t bind BD RNA
Imprintor gene
RNA
(BD RNA)
SNSIS
SNRPN gene
Female allele
Imprintor gene
RNA
(BD RNA)
SNSIS
SNRPN gene
Male allele
Dnmt
1
2
3