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BIO 184
Fall 2006
LECTURE 8
Lecture 8:
EPIGENETIC INHERITANCE
A tortoise-shell cat. The cat’s unique coat pattern is the result of X inactivation and thus
usually only affects females. Rare tortoise-shell Tom cats have unusual genetic conditions
such as an XXY karyotype. http://messybeast.com/tricolours.htm
I. What Is Epigenetic Inheritance?
Epigenetic inheritance refers to a pattern of inheritance in which a modification
occurs to a nuclear gene or chromosome that temprarily alters gene expression
 However, the modification, unlike a mutation, is not permanent and
therefore the alteration in gene expression is also not permanent
 The “epigenetic mark” can be added and removed
Epigenetic changes are caused by DNA and chromosomal modifications
 These can occur during oogenesis, spermatogenesis or early embryonic
development
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II. Dosage Compensation
One common type of epigenetic marking occurs during embryogenesis in female
mammals.
 The epigenetic mark “turns off” one of the X chromosomes that the
female has inherited from her parents
 The purpose of inactivating one of the X chromosomes is dosage
compensation
o offset differences in the number of active sex chromosomes
and therefore the levels of expression of X-linked genes in the
male and female genomes
Dosage compensation has been studied extensively in mammals, Drosophila and
Caenorhabditis elegans. Depending on the species, dosage compensation occurs via
different mechanisms.
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LECTURE 8
In 1949, Murray Barr and Ewart Bertram identified a highly condensed structure
in the interphase nuclei of somatic cells in female cats but not in male cats
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This structure became known as the Barr body (See Brooker, Figure 7.3a).
In 1960, Susumu Ohno correctly proposed that the Barr body is a highly
condensed X chromosome
In 1961, Mary Lyon proposed that dosage compensation in mammals occurs
by the inactivation of a single X chromosome in females
Liane Russell also proposed the same theory at about the same time
The mechanism of X inactivation, also known as the Lyon hypothesis, is
schematically illustrated in Brooker, Figure 7.4.
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The example involves a white and black variegated coat color found in
certain strains of mice
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A female mouse has inherited two X chromosomes
o One from its mother that carries an allele conferring white coat color
(Xb)
o One from its father that carries an allele conferring black coat color
(XB)
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During X chromosome inactivation, the DNA becomes highly compacted
o Most genes on the inactivated X cannot be expressed
o When this inactivated X is replicated during cell division, both copies
remain highly compacted and inactive
o In a similar fashion, X inactivation is passed along to all future
somatic cells that are derived from the original parent cell that
underwent the X inactivation event
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BIO 184
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Fall 2006
LECTURE 8
Another example of variegated coat color is found in tortoise-shell cats (see
photograph on first page of this set of lecture notes).
o “Torties” are heterozygous for a coat color gene located on the X
chromosome. The gene locus is called “O” and there are two alleles:
 “O” codes for an enzyme that converts black pigment to orange
 “o” codes for an inactive form of the enzyme (“null allele”)
o Male cats cannot be “torties” (unless they are unusual genetic
mutants) because they have only one X chromosome’
 A male cat with the genotype XOY is ginger
 A male cat with the genotype XoY is black
 A female cat with the genotype XOXO is ginger
 A female cat with the genotype XoXo is black
o Other cat genes (located on autosomes) are involved in diluting the
coat color (e.g. from black to gray), for white spotting, striping, etc.
III. Experimental Proof for the Lyoin Hypothesis
In 1963, Ronald Davidson, Harold Nitowsky and Barton Childs set out to test the
Lyon hypothesis at the cellular level
To do so they analyzed the expression of a human X-linked gene
 The gene encodes glucose-6-phosphate dehydrogenase (G-6-PD), an enzyme
used in sugar metabolism
o Biochemists had found that individuals vary with regards to the G-6PD enzyme
o This variation can be detected when the enzyme is subjected to
agarose gel electrophoresis (which we will perform this week in lab)
 One G-6-PD allele encodes an enzyme that migrates very quickly
and is therefore called the “fast” enzyme
 Another allele encodes an enzyme that migrates slowly and is
therefore called the “slow” enzyme
 The two types of enzymes have minor differences in their
structures but these do not significantly affect G-6-PD function
(neutral alleles)
The diagram at the top of the next page illustrates how the enzyme forms are
assayed.
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BIO 184
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Fall 2006
LECTURE 8
Thus heterozygous adult females produce both types of enzymes
Hemizygous males produce either the fast or the slow type
According to the Lyon hypothesis, an adult female who is heterozygous for the
fast and slow G-6-PD alleles should express only one of the two alleles in any
particular somatic cell and its descendants, but not both.
To test this, Davidson, Nitowsky, and Childs ran the experiment show in Figure
7.6, Brooker. Their actual data (shown below) supported the Lyon Hypothesis.
Lane 1, all cells from the female combined; Lanes 2-10 clonal populations of individual cells
from the female. Each clonal population only expresses one of the enzyme forms, not both.
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LECTURE 8
IV. X Inactivation Depends on Xic, Xist, TsiX and Xce
Researchers have found that mammalian cells can “count” their X chromosomes and
allow only one of them to remain active
 Additional X chromosomes are converted to Barr bodies
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Phenotype
Sex Chromosome
Composition
Number of
Barr bodies
Normal female
XX
1
Normal male
XY
0
Turner syndrome (female) X0
0
Triple X syndrome
(female)
XXX
2
Klinefelter syndrome
(male)
XXY
1
This helps explain why the phenotypes associated with X chromosome
aneuploidies tend to be less severe than autosomal aneuloidies
The genetic control of inactivation is not entirely understood at the molecular level
 However, a short region on the X chromosome termed the X-inactivation
center (Xic) plays a critical role
o For inactivation to occur, each X chromosome must have a Xic
region
See Figure 7.7, Brooker
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The Xic region contains a gene named Xist (for X-inactive specific
transcript)
o The Xist gene is only expressed on the inactive X chromosome
o It does not encode a protein
 It codes for a long RNA, which coats the inactive X
chromosome
 Other proteins will then bind and promote chromosomal
compaction into a Barr body
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LECTURE 8
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A second region termed the X chromosome controlling element (Xce)
affects the choice of the X chromosome to be inactivated
o This choice occurs during embryonic development and is maintained
in all subsequent cell divisions
o A female heterozygous for different Xce alleles will have a skewed
X-inactivation
 The X chromosome that carries a strong Xce allele is more
likely to remain active than one with a weak Xce allele
 The degree of skewing, however, is rarely more than 70% to
30%
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A gene designated TsiX also plays a role in chromosome choice
 It is located in the Xic region
 It is expressed only during early embryonic development
 It encodes an RNA complementary to Xist RNA termed antisense
RNA (where Xist RNA is the sense RNA)
 Tsix antisense RNA is believed to bind to Xist sense RNA and
inhibit its function
 In other words, TsiX RNA prevents X chromosome inactivation
The process of X inactivation can be divided into three stages:
o Initiation
 One of the X chromosomes is targeted to be inactive
o Spreading
 The chosen X chromosome is inactivated
o Maintenance
 The inactivated X chromosome is maintained as such during
future cell divisions
See Figure 7.8, Brooker
A few genes on the inactivated X chromosome are expressed in the somatic cells
of adult female mammals
o These genes escape the effects of X inactivation. They include
 Xist
 Pseudoautosomal genes
 Dosage compensation in this case is unnecessary because
these genes are located both on the X and Y
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LECTURE 8
V. Genomic Imprinting
A second form of epigenetic inheritance is genomic imprinting, in which expression
of a gene depends on whether it is inherited from the male or the female parent
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Imprinted genes follow a non-Mendelian pattern of inheritance
Depending on how the genes are “marked”, the offspring expresses
either the maternally-inherited or the paternally-inherited allele but not
both
o This is termed monoallelic expression
Consider the following example in mice:
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The Igf-2 gene encodes a growth hormone called insulin-like growth
factor 2
o A functional Igf-2 gene is necessary for a normal size
o Imprinting results in the expression of the paternal but not the
maternal allele
 The paternal allele is transcribed into RNA
 The maternal allele is not transcribed
o Igf-2m is a mutant allele that yields a defective protein
 This may cause a mouse to be dwarf depending on whether it
inherits the mutant allele from its father or mother
The following cross involving this mutation yields a surprising result:
Normal male (Igf-2 Igf-2) X mutant female (Igf-2m Igf-2m)
ALL Igf-2, Igf-2m
(all normal size)
mutant male (Igf-2m Igf-2m) X normal female (Igf-2 Igf-2)
ALL Igf-2, Igf-2m
(all dwarf)
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LECTURE 8
When a gene is imprinted, it matters which parent supplies the mutant allele! In
this case, the female parent “turns off” the Igf-2 locus in all of her eggs, while the
male parent leaves it “on.”
Therefore, the offspring will always express the paternal genotype at this locus
regardless of what they received from their mother!
Figure 7.10 in Brooker shows how the system works in mice and how the “imprint”
is maintained from one generation to the next.
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At the cellular level, imprinting is an epigenetic process that can be divided
into three stages
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Establishment of the imprint during gametogenesis
Maintenance of the imprint during embryogenesis and in the adult
somatic cells
Erasure and reestablishment of the imprint in the germ cells
Thus, genomic imprinting is permanent in the somatic cells of an animal
 However, the marking of alleles can be altered from generation to
generation
 It may involve
o A single gene
o A part of a chromosome
o An entire chromosome
VI. Imprinting and DNA Methylation
Genomic imprinting involves a chemical marking process called methylation.
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A methyl (-CH3) group is added to cytosines in the DNA
Usually occurs in regions needed for proper regulation and expression of
the gene
 Are called differentially methylated regions (DMRs)
o They are methylated either in the oocyte or sperm but not both
o For most genes, methylation at a DMR results in inhibition of
gene expression
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LECTURE 8
o Therefore, imprinting is usually described as a process that
silences gene expression by preventing gene expression
See Figure 7.11b, Brooker.
To date, imprinting has been identified in dozens of mammalian genes
 The human genome is less imprinted than that of most other mammals;
it appears to have lost its imprinting marks at several loci
VII. WHY IMPRINT?
The biological significance of genomic imprinting is still a matter of speculation,
but the theory with the most support is called the “Genetic Conflict Theory.”
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Proposes that males and females have different “fitness strategies”
that play out at the molecular level
Particularly important in species that practice “polyamory” (e.g. cats,
mice), in which the female carries multiple fetuses per pregnancy, all
of which may have been fathered by different males
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LECTURE 8
Each male wishes to maximize the size of his own embryo and its
access to the female’s resources
o Therefore, the male leaves “on” genes involved in promoting
embryo growth, like Igf-2, but “turns off” (imprints via
methylation) genes involved in reducing embryo growth
The female wants all of the embryos to survive equally well (they all
contain her genes) and to protect her own resources for future
pregnancies
o Therefore, the female imprints genes involved in promoting
embryo growth but she leaves “on” genes involved in reducing
embryo growth
Evidence for the Genetic Conflict Theory:
1. Many of the imprinted gene loci code for proteins involved in embryo
growth.
2. Mouse fetuses created from the fusing of two egg nuclei are tiny while
those created from the fusing of two sperm nuclei (in an egg cytoplasm)
are grossly overgrown. (Neither survive to term.)
3. Humans appear to have lost imprinting at many of the loci imprinted by
other mammals, possibly because humans have evolved toward single
pregnancies
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