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									Modified from http://www.mhhe.com/brooker                                              Fall 2006
BIO 184                                                                              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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Modified from http://www.mhhe.com/brooker                                 Fall 2006
BIO 184                                                                 LECTURE 8

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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Modified from http://www.mhhe.com/brooker                                   Fall 2006
BIO 184                                                                   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

      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.

      The example involves a white and black variegated coat color found in
       certain strains of mice




      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)

      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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Modified from http://www.mhhe.com/brooker                                    Fall 2006
BIO 184                                                                    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-6-
             PD 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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Modified from http://www.mhhe.com/brooker                                              Fall 2006
BIO 184                                                                              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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Modified from http://www.mhhe.com/brooker                                    Fall 2006
BIO 184                                                                    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

                                            Sex Chromosome   Number of
            Phenotype                       Composition      Barr bodies
            Normal female                   XX               1
            Normal male                     XY               0
            Turner syndrome (female) X0                      0
            Triple X syndrome               XXX              2
            (female)
            Klinefelter syndrome            XXY              1
            (male)

         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

         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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Modified from http://www.mhhe.com/brooker                                 Fall 2006
BIO 184                                                                 LECTURE 8

          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%

          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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Modified from http://www.mhhe.com/brooker                                     Fall 2006
BIO 184                                                                     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

          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:

          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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Modified from http://www.mhhe.com/brooker                                     Fall 2006
BIO 184                                                                     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.

      At the cellular level, imprinting is an epigenetic process that can be divided
       into three stages

                 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.

          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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Modified from http://www.mhhe.com/brooker                                      Fall 2006
BIO 184                                                                      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.”

             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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Modified from http://www.mhhe.com/brooker                                     Fall 2006
BIO 184                                                                     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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