Genomic imprinting is the biological process whereby a gene or
genomic domain exists in a state of epigenetic differentiation that depends
upon its parent of origin. Importantly, the establishment and propagation of
these parent-specific genomic conformations does not alter the primary DNA sequence
comprised of A, C, G, and T nucleotides. Genomic imprints may be covalent (DNA
methylation) or non-covalent (DNA-protein and DNA-RNA interactions, genomic
localization in nuclear space), and the process of imprinting encompasses the
specialized nuclear enzymatic machinery that maintains parental epigenetic markings
throughout the cell cycle. Because of genomic imprinting, the parent of origin
of homologous genetic alleles in diploid individuals can be determined in the
absence of DNA sequence polymorphisms and without recourse to parental DNA samples.
As illustrated in Figure 1, alleles of imprinted genes look and behave differently,
as determined by parent of origin.
Figure 1 : Genomic imprinting results in parent-specific
epigenetic differentiation and monoallelic gene expression. Parental imprints
are established during gametogenesis as homologous DNA passes uniquely
through sperm or egg; subsequently during embryogenesis and into adulthood,
alleles of imprinted genes are maintained in two "conformational"/epigenetic
states: paternal or maternal. The gamete-specific epigenotypes observed
in egg and sperm may go through metamorphosis during embryogenesis into
their somatic forms.
While Gregor Mendel did not provide details of the anatomy of
genes, a fundamental tenet of Mendelian principles of inheritance is that a
gene's parent of origin does not influence its dominance or recessiveness in
phenotype determination. However, in sexually reproductive organisms including
plants, insects, invertebrates, and chordates, the parental origin of genetic
alleles often determines their fates. Mammals have diverged from other sexually
reproductive organisms through the imprinting of a distinct family of genes
involved in embryogenesis. For these imprinted genes, the diploid offspring
distinguishes between maternally-inherited and paternally-inherited alleles,
and selectively expresses only one of them while inactivating the other. Allelic
parental discrimination and silencing at imprinted loci is imperative in the
procreation of wild type mammalian progeny. The life history of these genes-
including when in the past, why, and how they became imprinted- remains a mystery
which fascinates evolutionary and developmental biologists, as well as clinicians
seeking answers and remedies for "non-Mendelian" inherited human genetic
disorders. Most studies of mammalian imprinting have investigated the phenomenon
in mice or humans, but recent studies of a wide variety of mammals, including
monotreme (egg-laying), marsupial (altricial offspring carried in a pouch),
and eutherian ("placental") mammals are helping unravel the origins
and mechanisms of the unique family of imprinted genes. Recent focus on the
physical structure and biochemisty of imprinted chromatin domains also is providing
an image of parental differentiation within the genome. The historical recognition,
evolution, physical chromatin basis, and pathologic consequences of parental
genomic imprinting will be reviewed in this article.
An ancient puzzle for naturalists was the observation that parthenogenetic
reproduction-asexual female reproduction- occurs naturally in many vertebrates
such as birds and fishes but not mammals. However, in 1937 the renowned reproductive
biologist and endocrinologist Gregory Pincus reported that he had successfully
achieved "fatherless rabbits" via parthenogenesis. Such reports of
parthenogenesis discount the need for sperm or male contributions to reproduction.
Partly attributable to Pincus' parthenogenetic rabbit, and the
powerful dual influences of Gregor Mendel's laws of genetic inheritance and
the Watson-Crick model of the DNA double helix, epigenetic memory and inheritance
were not initially widely recognized. That genes could exist in parent-specific
conformations, and that these conformations could be self-templating from one
cell division to the next, simply was not a mainstream viewpoint until recently.
Following the initial report of successful parthenogenesis in
rabbits, early experimental attempts by developmental biologists to produce
parthenogenetic mice consistently failed to develop normally, but the "embryoids"
did show various degrees of development and differentiation along embryonic
lineages. It was therefore believed that successful parthenogenesis was more
a matter of technical optimization of the procedure, and a fundamental need
for sperm-derived nuclear genome is even discounted in some reports. Instead,
the possible explanations included: asynchrony between the parthenogenone developmental
stage and the uterine lining at the time of implantation; alterned nucleus:cytoplasmic
ratio; failure due to absence of a sperm cytoplasmic factor; the expression
of recessive lethal mutations; an incomplete zona reaction; or gene dosage effect
related to X-chromosome imbalance (Graham, 1974).
In parallel with such deliberate experimental manipulations to
improve failing parthenogenesis attempts in mice, human pathologists were serendipitously
approaching an explanation for failed mammalian parthenogenesis from a different
realm of investigation, namely female germ cell tumors. Pathologic analysis
of two peculiar human germ cell tumors provided the conceptual breakthrough
for recognizing the fundamental functional difference between the maternal and
paternal genomes during cell growth and differentiation (Linder et al., 1975;
Kajii and Ohama, 1977; Wake et al., 1978). The histopathologic phenotype of
ovarian teratomas reveals well-differentiated fetal structures of all three
germinative layers (ectoderm, mesoderm, endoderm), while the hydatidiform mole
contains no such elements, only extra-embryonic trophoblast elements. Both of
these tumors arise from ovarian germ cells, and typically have a 46,XX normal
karyotype. However, the teratoma is gynogenetic in origin (Figure 2) while the
hydatidiform mole is androgenetic (Figure 3). Thus, as recognized in the mid
1970's, the developmental potential of ovarian germ cells is determined by the
parental origin of the genome driving its development, indicating a fundamental
distinction between the nuclear genomes of sperm and egg. Further analyses of
pathologic specimenes ruled out a contributory role for parental origin of mitochondrial
DNA or cytoplasmic factors in the differentiation of germ cell tumors.
Figure 2 : Chromosomal studies
of tri-embryo-lineage (endoderm, mesoderm, ectoderm) teratomas reveal
a uniquely gynogenetic constitution.
Figure 3 : Chromosomal studies
of mono-extraembryonic-lineage (trophoblast) hydatidiform moles reveal
a uniquely androgenetic constitution.
As discussed by Wake, Takagi, and Sasaki (J Natl Cancer Inst,
"In contrast to androgenetic ova producing only hydropic villi, parthenogenetic
oocytes in the ovary produce several mature tissues. Remarkable differences
in the end products of both types of conceptuses are of special interest with
regard to the possible physiologic difference between maternally and paternally
derived genome in the egg cytoplasm, influence of implantation site (ovary versus
uterus), and interaction between mother and conceptus in early mammalian embryogenesis.
Parthenogenetic or gynogenetic conceptuses developing in uteri, if existent,
would help resolve these problems."
As pointed out by Wake et al., analysis of human tumors could
not control for potential effects of local environment in guiding developmental
programming, for the teratomas develop with the ovary while hydatidiform moles
develop in utero following passage through the oviduct; furthermore, various
endocrinologic and developmental parameters obviously cannot be controlled for
when studying human pathologic specimens. Nevertheless, pathologic human germ
cell tumor analysis provided an early conceptual framework in the recognition
of the different agendas of paternal versus maternal genomes during development.
Figure 4 : Correlation of
developmental end product with parental chromosome constitution described
by Wake et al., 1978, and discussed above in the text.
Further mouse parthenogenesis and androgenesis experiments of
the early 1980's provided functional evidence of heritable differences between
the maternal and paternal programming of their germ cell genomes while controlling
for many potential confounding factors. The pronuclear transplantation studies
by McGrath and Solter (McGrath and Solter, 1984) and Surani and colleagues (Surani
et al., 1984) provided the requisite parthenogenetic/gynogenetic conceptuses
developing in uteri referred to by Wake et al. The series of pronuclear transplantation
experiments directly confirmed that male and female parent-derived genomes direct
fundamentally different developmental programs in developing embryos. In these
experiments, a mature oocyte is devoided of its pronucleus while leaving the
cytoplasm along with the mitochondria and other organelles intact; then this
empty egg is reconstituted with either one sperm and one oocyte pronucleus (normal
complement), two sperm pronuclei and no oocyte pronucleus (androgenetic complement),
or two oocyte pronuclei and no sperm pronuclei (gynogenetic complement). After
intrauterine implantation of the embryo or embryoids in pseudo pregnant mice,
they differentially develop along lines remarkably homologous to the germ cell
tumors in humans according to parental origin of nuclear genome (Figure 5).
Figure 5 : Mouse germ cell pronuclear transplant
experiments convincingly demonstrate a different agenda for sperm- versus
egg-derived nuclear genomes during development. Development in the absence
of a sperm-derived genome (middle column) shows fairly good development
of the embryo proper but failed development of the trophoblast lineage.
Development in the absence of an egg-derived genome (right column) shows
failed development of the embryo proper but exuberant trophoblast growth.
Figure by permission, Nature.
Following neoplastic teratoma and hydatidiform mole, the first
human clinical syndromes recognized to result from imprinted loci were Prader-Willi
syndrome and Angelman syndrome as reported in 1989 (Nicholls et al., 1989).
These studies revealed that identical genetic deletions as well as uniparental
disomy for a domain on 15q resulted in markedly different clinical phenotypes
depending on the parental origin of the deletion/disomy.
Figure 6: Recognition of imprinted inheritance
of Prader-Willi and Angelman syndromes. Nicholls et al. reasoned that
parentally imprinted gene(s) reside in human 15q11-13.
The idea that maternally-and paternally-derived alleles of certain
genes function differently in the cell was further confirmed when the first
distinct imprinted genes were identified. These were the genes coding for insulin
like growth factor 2 (IGF2) and for its receptor, the mannose 6-phosphate/IGF2
receptor (M6P/IGF2R) (Barlow et al., 1991; Dechiara et al., 1991). IGF2 is a
critical fetal growth factor, while the M6P/IGF2R targets IGF2 for degradation
and therefore suppresses fetal growth. Heterozygous mice that harbor null alleles
of these genes have different phenotypes, depending on the sex of the parent
from which they inherited the null allele. Genetic and molecular analyses in
mice showed that IGF2 is expressed uniquely from the paternally-inherited allele,
while M6P/IGF2R is expressed from the maternally-inherited allele.
Figure 7: A mutant maternally-derived allele of
Igf2r results in a malformed mouse embryo with placental overgrowth. Image
from Wylie et al., AJP, 2003.
The monoallelic expression of these and other imprinted genes,
in a parent-of-origin-dependent manner, differs from the post-zygotic monoallelic
expression of certain genes involved in olfaction and immunity. At present,
some 4 score genes are known to be imprinted, and it is estimated that mammalian
genomes may contain several hundred imprinted genes in total (Luedi PP et al.,
2005.). In addition to identifying and validating the various imprinted genes,
a major focus of current research in this field is to understand how and why
some alleles "remember" their parental lineage long after pronuclear
fusion in the zygote, while the majority of alleles "forget" from
which parent they were inherited. This entails dissecting the unique physical
chromatin structure and epigenetic DNA modifications, as well as the enzymatic
processes that propagate them.
Figure 8. Human imprinting
map, showing genomic distribution of known imprinted genes and clinical
syndromes. Figure from http://greallyoffice.aecom.yu.edu/.
The relative diminished expression from one parental locus is
sufficient to create a pathologic phenotype in heterozygous mutant animals in
which the imprint gene null allele is inherited through the dominant/expressing
parent. Similarly, in human uniparental disomies that encompass imprinted loci,
diminished expression from imprinted loci is often syndromic. In fact, one strategy
for identifying imprinted genes is based upon UPD genotype-phenotype correlations.
Thus, the diminished gene expression from the stifled parental
biologically insufficient to support a healthy phenotype, and imprinted gene
mutations are usually dominant when they affect the expressed allele. Feedback
regulation of transcription at imprinted loci does not allow sufficient upregulation
of transcription from the silenced allele, and organisms do not have recourse
to the silenced otherwise wild-type allele in the event that the expressed allele
Figure 9: Pedigree of imprinted maternally expressed
phenotype. The phenotype is expressed only when the mutant allele is inherited
from the mother. Thus, mutant imprinted alleles can remain masked when
they are paternally inherited, but clinically re-appear in one-half of
children of carrier daughters.
Clinical human diseases and syndromes stemming from the unique
vulnerabilities of imprinted loci include : gestational trophoblastic disease,
syndrome, Prader-Willi syndrome, Angelman syndrome, Silver-Russell syndrome,
transient neonatal diabetes, social-cognitive defects in Turner syndrome, and
multiple neoplasias associated with loss of imprinting at oncogene loci. OMIM
(On-line Mendelian (!) Inheritance in Man) database of the NCBI (United States
National Center for Biotechnology Information) contains detailed entries on
many imprinted genes and syndromes.
In summary therefore, a mammalian individual's DNA contains information
about the parental origin of numerous genes and, for these parentally-differentiated
loci, improper balancing of allelic sex may have pathological effects.
Epigenetic programming loosely refers to any modification to DNA
that is imposed after DNA polymerase assembles the primary DNA sequence. Heritable
epigenetic modifiers include physical as well as spatio-temporal programming
of DNA, and candidate epigenetic markings capable of gene imprinting include
cytosine methylation (Reik et al., 1987; Mayer et al., 2000; Figure 10), histone
acetylation and other modifications, replication timing asynchrony, chromatin
structure and nuclear localization. Molecular dissection of the Prader-Willi/Angelman
syndrome imprinted domain on 15q provides a good example of the physical epigenetic
modifications that can regulate an imprinted domain (reviewed by Soejima and
Wagstaff, 2005; Figures 11 and 12).
Figure 10: Immunostaining
for 5-methyl cytosine in zygotes reveals a remarkable global methylation
differentiation between the maternally- versus paternally-inherited chromosomes
following fertilization. In particular, the paternally inherited chromosomes
appear nearly completely demethylated beginning 6-8 hours after fertilization,
while the maternal chomosome methylation persists.
Figure 11: 15mat imprinted domain: Physical examination
of the imprinted domain on maternally inherited chromosome 15 reveals
DNA cytosine methylation, histone H3 tail methylation at lysine 9, recruitment
of histone deacetylating enzymes, and deacetylated histones. These features
are typical of closed, transcriptionally inactive chromatin, creating
a functional knockout of multiple genes in the domain (center panel),
including an antisense transcript to UBE3A. Silencing of asUBE3A permits
expression of UBE3A from the maternally inherited chromosome.
Figure 12: 15pat: The physical
structure of the chromosome 15 PWS/AS domain inherited from the father
is distinct from that from the mother. There is absent DNA cytosine 5-methylation,
and tails of histones H3 and H4 are lysine 4-methylated and acetylated
(H3-K4me and H4-Ac), respectively. There is recruitment of histone acetyltransferase
(HAT) to the domain on the paternal chromosome. These features are typical
of open, transcriptionally active chromatin. There is a "virtual"
deletion of some genes in cis, including UBE3A and ATP10C.
The behavior/expression of imprinted genes does not depend on
the sex of the individual in which those genes reside, but on the sex of the
parent from which the particular allele was inherited. In diploid somatic cells
of an individual mammal, maternal and paternal alleles co-exist, but in the
case of an imprinted gene, normally only one allele is functionally active.
Propagation of this situation means that each DNA replication must be followed
by self-templated imprint maintenance. The alternate stage in the life cycle
of imprinted alleles occurs in the germ line. Here the imprints manifest in
somatic cells are erased and an appropriate sperm-specific or egg-specific imprint
is established on all gametic alleles, presumably by gonad-specific factors
that reprogram the alleles. The testis-specific transcription factor BORIS regulates
imprint establishment in the male germ line, while a female germ line specific
imprint regulatory molecule has yet to be identified. When a new individual
is generated by fusion of an egg and a sperm, the situation found in the parents
is recreated. Thus, imprints cycle between periods of maintenance and establishment.
Genomic imprinting represents a violation of Mendelian principles
of inheritance, one of which stipulates that the dominance of one genetic allele
over another is an inherent function of the alleles themselves, and does not
depend upon the parent of origin of the allele. For example, Mendel observed
the patterns of dominance and recessiveness for such traits as flower color
and seed shape were independent of whether the dominant trait derived from pollen
or ovum. Such observations may indicate a resistance of genetic alleles to environmental
influences, such as the different climatic or cellular environments in which
the male and female germ cells are propagated. While parental imprinting does
not invalidate the results of Mendel's work, it does constitute a significant
inheritance mechanism not observed by Mendel (Figure 6). By contrast, genomic
imprinting provides positive evidence that genomes can show heritable functional
plasticity dependent on allele environment; such a concept of genetic inheritance
was favored by Lamarck and discredited through much of the Twentieth Century.
Figure 13: Genomic imprinting, in which some genetic
traits are determined by the parent-specific germ cell milieu, violates
Gregor Mendel's (left panel) principles of inheritance; by contrast imprinting
supports, or at least takes the edge off some of the anathema heaped on
Jean-Baptiste Lamarck's (right panel) concept of inheritance.
The consequences of imprinting are potentially disastrous since,
for imprinted genes, animals have effectively abandoned the 'safety net' provided
by diploidy and have shut-off a perfectly good gene copy. This drawback has
spawned much philosophical debate over why imprinting could have possibly evolved,
and furthermore, why it has been maintained throughout the mammalian radiation.
One model proposes that imprinting evolved precisely to prevent parthenogenesis,
and that the imprinting of a few loci is a small price to pay to guarantee functional
diploidy in all other genes. A second model proposes that imprinting evolved
as a consequence of the action of the host defense system against parasitic
foreign DNA and that the presence of imprinted genes in mammalian genomes represents
the shutting-off of "innocent by-standers". Note that these two models
suggest that imprinting is an adaptive mechanism beneficial to the survival
of the species. They also assign an insignificant role to imprinting as a mechanism
to control gene dosage. One prediction of the conflict hypothesis that - that
imprinting is limited to viviparous animals - has been tested and the results
support the hypothesis.
Probably the most widely accepted model of imprinting evolution
is known as the conflict hypothesis (Haig and Graham, 1991; Moore and Haig,
1991). The conflict hypothesis views imprinting not as a beneficial adaptation
of the species but as the deleterious consequence of a reproductive scenario
involving polygamy, viviparity, and substantial maternal investments in the
offspring, in the absence of a similar level of investment by the father. According
to the conflict model, once viviparity arose among mammalian ancestors, natural
selection acting upon asymmetric parental investments in diploid offspring operates
on two conflicting strategies. On the one hand it is to the male's advantage
that his offspring extract a maximum amount of nutrients from the mother, for
he is unlikely to mate with that female again, and this should maximize his
reproductive success and that of his offspring. On the other hand it is to the
female's advantage to ration her investment in any given offspring, thereby
conserving her resources for herself and her future offspring. According to
the conflict hypothesis, therefore, imprinting arose due to a genetic tug-of-war
between the parents that is played out in the offspring, through antagonistic
efforts to control gene dosage.
Figure 14: According the conflict hypothesis, genomic
imprinting results from an interparental tug-of-war over the resources
allocated the fetus by the mother during intrauterine gestation. The potential
for conflicts between polygamous viviparous mammals is highlighted by
the killing of lion young by non-paternal males (left). From the epigenomic
perspective, the paternal epigenome can conflict with the maternal epigenome
over offspring nutrient availability during intrauterine gestation Such
conflicts are insufficient in oviparous animals such as monotreme mammals
to drive the deleterious imprinted silencing of genes.
Other predictions of this hypothesis are that imprinting occurs
principally at fetal growth regulatory loci, that paternal epigenotypes drive
expression of pro-growth genes while maternal epigenotypes suppress growth,
and that such interparental conflicts exist especially under the reproductive
physiology of viviparity.
Figure 15: The phylogenetic distribution of genomic
imprinting of IGF2R in birds, egg-laying mammals, marsupial mammals, and
placental mammals (Killian et al., 2001). Black lines: not imprinted,
ancestors not imprinted; green: imprinted, maternally expressed; red:
imprinting lost. Blue lines refer to presence or absence (dashed) of putative
IGF2R intron 2 imprint control element, for more information please see
Figure 16: The potential
roles of placentation and viviparity in the evolution of imprinting have
been investigated through the phyloepigenetic analysis of IGF2 and M6P/IGF2R
imprinting in birds, monotreme mammals, and marsupials. To date, genomic
imprinting has only been demonstrated in viviparous mammals, supporting
the conflict hypothesis.
Elucidating the phenomenon of imprinting has provided much insight
into epigenetic regulation of development and cancer, but also helps explain
centuries-old biological observations. Mule breeders 3 millennia ago observed
that a horse mare crossed with a jack donkey yields a mule, whereas a horse
stallion crossed with a jennet donkey produces a hinny, which has shorter ears,
a thicker mane and tail, and stronger legs than the mule; thus indicating parental
sex-dependent influence on phenotype. Although ancestral donkey crossers would
likely have no problem with the concept and reality of parental genomic imprinting,
imprinting more recently carries an iconoclastic aura, evidence of the powerful
influence exerted by Gregor Mendel's writings; indeed, the phenomenon of imprinting
has been classified within the realm of non-Mendelian genetics, as if Mendel's
laws represent the Platonic ideal of genetic behavior.
Figure 17: No hinnies in
Washington. Following is an account of the origin of the mule industry
in the United States, as per the archives of the U.S. Library of Congress.
In the late Eighteenth Century there were no mules in the United States,
but George Washington had become interested in them after learning of
their unique attributes as work animals. The requisite male donkeys needed
to breed mules must have also been scarce, for Spain had a virtual monopoly
on the ass industry and it was illegal to export ass from the Spanish
territories. Washington made an inquiry with the U.S. ambassador to Spain,
and in 1785 King Charles III of Spain sent a large jack donkey to George
Washington as a gift. The donkey was named "Royal Gift" and
became the father of the mule industry in the U.S. It is interesting to
note the male sex of the donkey sent to Washington, which is required
in order to breed a true mule. Thus, technically speaking, because of
genomic imprinting, there were no hinnies only mules in early Washington.
Of course, female donkeys must have been eventually obtained in order
to propagate a breeding donkey population.