CONTENTS
Main Page Dynamic Development
The Foundations of Developmental
Biology
Gametogenesis
From Sperm and Egg to Embryo
Genetic Regulation of Development
Organizing the Multicellular
Embryo
Generating Cell Diversity
Dynamic Development at a
Glance
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The Developmental Biology Journal
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Developmental Biology Tutorial |
Genomic Imprinting
C. Cristofre Martin
Fels Institute
Temple University
3307 N. Broad Street
Philadelphia, PA 10140
Genomic imprinting can be loosely defined as the gamete-of-origin dependent
modification of phenotype. That is, the phenotype elicited from a locus
is differentially modified by the sex of the parent contributing that particular
allele. This process results in a reversible gamete-of-origin specific marking
of the genome that ultimately produces a functional difference between the
genetic information contributed by each parent. In mammals, the term genomic
imprinting has been restricted to describing mono-allelic gene expression
or the inactivation of either the maternal or paternal allele of a particular
locus.
Similarly, dominance modification involves the production of phenotypic
differences in the degree of penetrance of a particular locus (Fisher, 1928).
This epigenetic modification is produced by the action of other, distinct
modifier loci, most commonly acting in trans. Genomic imprinting
is thought to be a particular sub-type of dominance modification (Sapienza,
1990), whereby imprinting would result when one or more of these modifiers
is sex-linked and would therefore produce a dosage difference.
Imprinting-like phenomena have been observed in a wide range of phyla from
both the plant and animal kingdoms. Some manifestations of this gamete-of-origin
dependent modification include: parental dominance in hybrid plants (Heslop-Harrison,
1990); paternal chromosome elimination in Sciara (Metz, 1938; Crouse
et al., 1971); inactivation of the paternal genome by heterochromatization
in the scale insects (coccids) (Nur, 1990); parent-of-origin specific modification
of position effect variegation in Drosophila (Spofford, 1976); phenotypic
differences in progeny produced from interspecific crosses in fish (Whitt
et al., 1977) and between donkey and horse; preferential inactivation of
the paternally derived X chromosome in marsupials and rodent extraembryonic
tissues (VandeBerg et al., 1987); parent-of-origin dependent switching
of yeast mating types (Klar, 1987); and allelic exclusion of immunoglobulin
genes (Holliday, 1990). Probably the most extensively studied and best understood
examples of genomic imprinting are the gamete-of-origin dependent modifications
of transgene methylation and expression in mice (Swain et al., 1987;
Reik et al., 1987; Sapienza et al., 1987) and more recently
in a line of transgenic zebrafish (Martin and McGowan, 1995a; 1995b). It
is not at all clear yet whether all of the above phenomena are occurring
via a similar molecular mechanism. That a parent-of-origin effect occurs
in such a wide range of organisms suggests that either there is strong evolutionary
conservation of this phenomenon or that it arose independently a number
of times. The final result in all cases is an individual that appears to
be functionally hemizygous at an imprinted locus. Some of these functionally
hemizygous loci would be the result of imprinting by the male parent and
some would be the result of imprinting by the female parent (Figure 1).
Figure 1: Two examples of a hypothetical imprinted gene
responsible for body color. (LEFT) In this example the pigment gene is maternally
imprinted (maternal allele is inactivated). Matings between a male who possesses
the allele for pigment and a female who possesses the allele for no pigment
produces offspring that show only the pigmented phenotype. In this example,
the mother's allele is imprinted and inactivated in the offspring. Therefore,
the only actively-expressing allele is the father's pigment allele, which
is not imprinted in the offspring. (RIGHT) In this example the pigment gene
is paternally imprinted (paternal allele is inactivated). Matings between
a male who possesses the allele for pigment and a female who possesses the
allele for no pigment produces offspring that show only the pigmented phenotype.
In this example, the father's allele is imprinted and inactivated in the
offspring. Therefore, the only actively expressing allele is the mother's
no pigment allele, which is not imprinted in the offspring. (Figure courtesy
of Ross McGowan, Dept. Zoology, University of Manitoba).
Because there is monoallelic parent-of-origin specific expression from an
imprinted autosomal locus, genomic imprinting is counter to classical Mendelian
genetic theory, which states that there is equal inheritance of parental
traits. An imprinted mutant allele would appear to be recessive when inherited
from one sex, because it would be inactive (and consequently invisible),
whereas it would be the only active allele when inherited from the other
sex and, therefore, appear to be dominant (Figure 2).
Figure 2: Schematic representation of the phenotypic
effects of maternal imprinting of a mutant allele. Darkened body indicates
individual that is mutant for the hypothetical imprinted locus. A cross
is used to indicated the imprinted/inactive allele. (CENTER) Both parents
are homozygous for the normal allele at the imprinted locus. Although only
one allele is active (the paternal copy) in the offspring produced from
these parents, it must be a normal allele and therefore all offspring will
have a normal phenotype. (LEFT) The mother is homozygous mutant at the imprinted
locus, and the father is normal. Since this hypothetical locus is maternally
imprinted, the maternal mutant copy will be inactivated in their offspring
and the paternal normal copy will be the only active allele. The offspring
will be phenotypically normal, and the mutant allele will appear to be a
recessive mutation. (RIGHT) The mother is homozygous normal at the imprinted
locus, and the father is homozygous mutant. The maternal normal allele is
imprinted and inactivated in the offspring of these parents. The only allele
that is active is the mutant paternal copy. Therefore, all offspring produced
from these parents will display the mutant phenotype, and the mutant allele
will appear to be a dominant mutation. (Figure courtesy of Ross McGowan,
Dept. Zoology, University of Manitoba).
As stated previously, the genome imprinting phenomenon has been most intensively
studied in mammals, particularly mice. One type of epigenetic modification
in mammals involves the addition of a methyl group to position 5 of cytosine
and occurs most frequently at CpG dinucleotides. This type of DNA modification
is significant because, in general, methylated DNA sequences are transcriptionally
inactive. Furthermore, the methylation of DNA residues can be stably preserved
through the replication process by the action of maintenance methylases,
which use the replicated hemimethylated DNA as a template (Razin and Riggs,
1980). It has, therefore, been suggested that DNA methylation may be the
epigenetic marking responsible for the imprinting phenomenon in mammals,
or at least reflects the imprinted state of a locus produced during gametogenesis
(reviewed in Sasaki et al., 1993). It is logical to assume that the
epigenetic differences observed in the paternal and maternal genetic contributions
would be established during the only time when the two genomes are segregated;
i.e., during gametogenesis.
If methylase differences exist in the gametes, one would expect to find
different DNA methylation patterns in sperm and eggs. These differences
should be perpetuated at least through early development when the final
somatic tissue methylation pattern is established. Monk et al. (1987)
and Sanford et al. (1987) demonstrated in mice that sperm DNA is
more methylated than oocyte DNA. In the mouse, a period of overall demethylation
has been observed from fertilization, lasting until approximately the blastocyst
stage, followed by extensive de novo methylation. This period begins
at gastrulation and ultimately produces methylation levels higher than those
observed in either of the gametes (reviewed in Monk, 1990). Similar changes
in the methylation phenotype of a variety of transgenes in mice have been
observed during development (Chaillet et al., 1991; Ueda et al.,
1992).
A number of laboratories have demonstrated a gamete-of-origin influence
on the level of methylation of a variety of transgenes in mice (Hadchouel
et al., 1987; Reik et al., 1987; Sapienza et al., 1987;
Swain et al., 1987). With one exception, methylation tends to increase
after passage of a transgene locus through the female germline and decrease
after passage through male gametogenesis. In a number of cases an accompanying
effect has been found with respect to expression of the transgene: Expression
is decreased after passage through a female and increased by passage through
a male (Swain et al., 1987; McGowan et al., 1989; Allen et
al., 1990). A mosaic pattern of transgene expression has also been found
in several lines of transgenic mice, which suggests that the imprinted state
may not be retained in all cells (McGowan et al., 1989) or that only
a sub-set of early stem cells is affected by the imprint.
Several endogenous genes, as well as regions of many mouse chromosomes (Cattanach
and Beechey, 1990), have been shown to be imprinted. To date only a small,
but still growing, list of mammalian genes has been demonstrated unequivocally
to be imprinted (Table 1).
Estimates of the number of imprinted genes in the mammalian genome range
from less than 100 to greater than 200. Like the imprinted transgenes, these
endogenous genes show high levels of DNA methylation at the imprinted/inactive
allele. At present, no consensus sequences for imprinted loci have been
identified at/or surrounding these imprinted genes. However, the imprint
imposed at these loci is a very specific and localized process. The imprinted
genes H19 (maternal) and Igf2 (paternal) are tightly linked
to each other and localized to the same region of mouse chromosome 7 but
are imprinted in the opposite direction.
The pronuclear transplantation experiments of McGrath and Solter (1984)
and Surani et al. (1984; 1986) using mice have unequivocally demonstrated
that there is an absolute requirement for a genetic contribution from both
sexes in order for development to proceed normally; i.e., maternal
and paternal contributions are not equivalent. Embryos containing only maternal
contributions develop minimal extraembryonic tissues (trophectoderm), whereas
a poorly developed embryo proper is characteristic of embryos containing
only paternal genomes. The importance to the mammalian embryo of both maternal
and paternal genomes is most apparent in the very low viability of mammalian
parthenogenotes. The timing of developmental arrest and the phenotype of
parthenogenetic mouse embryos is highly variable and is most dependent on
the genetic background of the embryos. Parthenogenetic embryos produced
from one inbred strain of mice may only develop to blastula or gastrula
stages, whereas parthenogenetic embryos produced from another strain of
mice might develop to the 40 somite stage. Understanding of the role of
genomic imprinting in development has been hampered by the lack of a distinct/consistent
embryonic phenotype in parthenogenetic embryos and the lack of a precise
role for the presently known imprinted genes in development.
Considerable interest has been generated by the realization that the penetrance
and severity of many complex human diseases can be affected by the sex of
the parent contributing the allele responsible for the disease. The parent
of origin effects associated with a disease suggest that imprinting may
be involved. Some of these putative imprinted diseases include Huntington's
disease, cystic fibrosis, Prader-Willi and Angelman syndromes (reviewed
in Hall, 1990; Sapienza and Hall, 1995). Parent of origin effects are also
seen for birth defects such as spina bifida (Chatkupt et al., 1992)
and certain cancers. The role of imprinting in the development of cancers
such as Wilm's tumour, rhabdomyosarcomas and osteosarcomas has been particularly
well studied. By examining DNAs collected from tumors and normal tissues,
it has been revealed that the development and proliferation of cancerous
tumors may be associated with a loss of imprinting at a number of different
loci. Two examples of these imprinted loci are: Igf2 (insulin-like
growth factor), which is involved in beta-cell tumorigenesis, hepatoblastoma,
Wilm's tumor and rhabdomyosarcomas (Christofori et al., 1995; Li
et al., 1995; Pedone et al., 1994) and the gene H19,
which has been shown to be involved in hepatoblastoma, lung cancer, Wilm's
tumor and bladder carcinoma (Li et al., 1995; Kondo et al.,
1996; Ariel, 1995). The development of these cancers is thought to be due
to the relaxation of the normal monoallelic expression or 'imprinted' status
of these genes resulting from the loss of DNA methylation at the imprinted
allele.
Numerous investigators have presented theories regarding the evolutionary
origins/advantages of genomic imprinting. Few of these theories have been
able to satisfy all the disparate information from our present understanding
of the phenomenon, such as: 1) broad phylogenetic distribution, 2) the different
types of imprinted genes, and 3) both paternal and maternal imprinting.
Chandra and Nanjundiah (1990) put forward several postulates describing
in a general way the kinds of influences that could produce imprinting and
suggest several roles for imprinting: 1) imprinted and non-imprinted alleles
of a locus may confer different phenotypes; 2) modifiers of imprinting (so-called"imprintor
genes") may have pleiotropic effects such that they are selected for
reasons other than their action on the imprinted locus; and 3) imprinting
could have co-evolved with other traits. Holliday (1990) suggests that the
function of genomic imprinting is to produce functionally haploid gene sets
in order for the cell's regulatory machinery to be able to 'fine tune' itself
on just a single gene copy. It was further implied that imprinting would
also serve to prevent possible detrimental cross-talk between maternal and
paternal transcripts at developmentally important regulatory loci. Barlow
(1993) has suggested that genome imprinting as a mechanism of gene regulation
originated from the use of DNA methylation as a host defense mechanism.
The same processes that inactivate incoming viruses in oocytes might also
result in the inactivation of some of the organism's own (maternal) loci.
Recently, Thomas (1995) proposed that imprinting acts as a mechanism of
surveillance for chromosome loss and thus protects against cancers and mono-
and trisomic embryos. If imprinted loci are scattered throughout the genome,
loss of a chromosome or portion of a chromosome would result in the absence
of an active copy of an allele producing probable detrimental effects. Other
theories are based solely on genomic imprinting as it occurs is mammals.
McGrath and Solter (1984) noted that there is a phenotypic difference in
the developing embryos of mouse andro- and gynogenotes. More specifically,
androgenetic embryos show poorly developed embryos and excessive trophectoderm
(extraembryonic supportive tissue), whereas gynogenetic embryos have well
developed embryos and poorly developed trophectoderm. These "imprinting"
phenotypes of andro- and gynogenetic embryos seem to suggest the possibility
of the male using imprinting to maximize maternal input to the embryo (Haig
and Westoby,1989). Finally, Varmuza and Mann (1994) suggested that imprinting
in mammals may be a means of protecting the female from ovarian germ cell
tumors. Imprinting would prevent the development of malignant trophoblast
disease because of the inviability of unfertilized eggs and inability of
parthenogentically activated oocytes to implant and develop.
It has been the intention of this review to provide a basic understanding
of the process of genomic imprinting (further review of the listed references
will provide a much greater appreciation of this very complex process).
Although genomic imprinting has been intensively studied in the past few
years, it is still a very new field. Our present knowledge of imprinting
is limited to the identification of imprinted genes and to only the very
fundamental factors that contribute to the process. Researchers are now
focusing their efforts on identifying the trans- acting modifiers
of imprinting -(i.e., "imprintor" genes), understanding
the role of DNA conformation and chromatin structure surrounding imprinted
loci (ie., cis elements) and on determining the role of imprinting
in development (establishment of the imprint) and human disease.
Learning Objectives
- Define genomic imprinting.
- What is meant by the expression "parent of origin expression"?
- Diagram a cross showing the phenotypes and genotypes of a gene that
is only active if it is derived from the maternal parent. Do the same for
a gene that is only active if it is derived from the male parent.
- What modification of DNA is thought to be responsible for genomic imprinting?
- What is the evidence favoring this kind of modification?
- Discuss the pronuclear transplantation experiments of McGrath and Solter.
- What may be some consequences of the loss of imprinting? Give examples.
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