Download Gene Regulation and Genetics

MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
OVERVIEW OF EPIGENETICS
Date:
Time:
Room:
Lecturer:
August 26, 2005 *
9:00 am - 9:50 am *
Berryhill 103
Terry Magnuson
4312 MBRB
[email protected]
843-6475
*Please consult the online schedule for this course for the definitive date and time
for this lecture.
Office Hours: by appointment
Assigned Reading: This syllabus.
Key Concepts vs Supplementary Information: Because this syllabus is meant
to replace the need for a Genetics textbook, it contains a mixture of information
that is critical for you to know and information that serves to illustrate and explain
the key points. I have attempted to emphasize important terms, definitions and
concepts in red.
Basic Principles:
Epigenetics is the organization of the eukaryotic genome within chromatin. This
organization is involved in the regulation of gene expression. Two examples of
epigenetic regulaton that will be dicussed are autosomal imprinting and X-inactivation.
Lecture Objectives:
By the end of this lecture, you should:
understand that autosomal imprinting is parent of origin gene expression regardless of the
sex of the individual.
understand that, in females, one of the X-chromosomes is inactivated in each and every
cell.
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
A. Imprinting: The phenomenon in which there is a differential expression of a gene
depending on whether it was maternally or paternally inherited.
Paternal imprinting means that an allele inherited from the father is not expressed in the
offspring.
Maternal imprinting means that an allele inherited from the mother is not expressed in the
offspring.
B. What is imprinting of genes?
There are two copies of every gene in each cell of the body: one copy comes from the
mother (the "maternal" copy of the gene on the chromosome copy inherited from the
mother) and the other comes from the father (the "paternal" copy of the gene on the
chromosome copy inherited from the father). Usually, the information contained in both
the maternal and paternal copies of the genes are used by the cells to make these
products. In other words, both the maternal and paternal genes are usually active or
"expressed" in the cells. However, it is now known that the expression of about 100 of
the 30,000 or so genes in the cells depends on whether the gene copy was passed down
from the father or the mother. This process, whereby the expression of a gene copy is
altered depending upon whether it was passed to the baby through the egg or the sperm,
is called imprinting. The term "imprinting" refers to the fact that some chromosomes,
segments of chromosomes, or some genes, are stamped with a "memory" of the parent
from whom it came: in the cells of a child it is possible to tell which chromosome copy
came from the mother (maternal chromosome) and which copy was inherited from the
father (paternal chromosome). Thus, genetic imprinting is the term used when the
expression of a gene depends on whether it is inherited from the mother or the father.
Imprinting "breaks" one of Mendel's laws that genes act the same whether transmitted by
either parent. Imprinting was first discovered in corn: kernels are dark purple if the Red
gene is inherited from the egg but blotchy lavender if the same gene is transmitted
through the pollen. This observation was made in 1910 but not understood.
Imprinting is a process whereby the expression of a gene is affected but the DNA
sequence of the target gene is not altered. It also represents a reversible form of gene
activation that changes from generation to generation. While this is therefore not a
common mechanism controlling gene expression in humans, it is nevertheless an
important one and provides interesting new insights into the mechanisms of gene
expression. Some examples include Igf2, Peg1, Ube3A, Kcnq1, Wt1. Genetic imprinting
occurs in the ovary or testis early in the formation of the eggs and sperm. Some genes are
imprinted to be switched off or inactive only if they are passed down through an egg cell;
others will be inactivated only if they are passed down through a sperm cell. Imprinting
will then occur again in the next generation when that person produces his or her own
sperm or eggs.
Prader-Willi/Angelman syndrome
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
When the medical world first learned about Prader-Willi syndrome in 1956, doctors had
no idea what caused people to have this collection of features and problems that we now
know as PWS. It is only in the past 20 years that researchers have discovered the genetic
changes on chromosome 15 that are responsible for the syndrome.
In 1981, Dr. David Ledbetter and his colleagues reported a breakthrough discovery: They
found that many people with PWS had the same segment of genes missing from one of
their chromosomes. They had discovered the deletion on chromosome 15 that accounts
for more than half of the cases of PWS. Since then, researchers have made a series of
other important discoveries about the genes involved in Prader-Willi syndrome. There are
several genetic forms of this complex disorder, and genetic tests exist that can confirm
nearly every case
Prader-Willi syndrome is caused by lack of expression of paternally contributed gene(s).
The maternal genes are silent.
-Prevalence: 1:12,000- 15,000 (both sexes, all races)
-Major characteristics: hypotonia, hypogonadism, hyperphagia, cognitive impairment,
difficult behaviors
-Major medical concern: morbid obesity
More than one gene is involved in PWS, and these genes are near each other in a small
area the long arm of chromosome 15—in a region labeled 15q11-q13. Scientists still
don’t know exactly how many genes and which specific ones are involved. During the
1980s, scientists puzzled over why some people who seemed to have PWS did not have a
chromosome deletion, and why some people with the chromosome 15 deletion seemed to
have a different condition from PWS.
The next breakthrough came in 1989, when Dr. Robert Nicholls and fellow researchers
announced their discovery that PWS is an example of genetic imprinting. In what
scientists call the Prader-Willi region of chromosome 15 (the area where the deletion
occurs), there are two or more genes that must come from the baby’s father in order to
work. In Prader-Willi syndrome, these critical genes are either missing, or they were not
properly imprinted to be turned "on" in the chromosome that came from the father.
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
Prader-Willi Syndrome
• 75% of PWS cases due to cytogenetic deletion 15q11-q13 of
paternal 15
A
Not expressed
A
A
X
A
Angelman Syndrome: Also in the chromosome 15q11-q13 region is one gene that is
imprinted to be turned "on" only in the mother’s chromosome. When this gene is missing
or not working properly on the mother’s chromosome 15, the result is an entirely
different syndrome called Angelman syndrome (AS). This discovery explained the
mysterious cases of people who had a chromosome 15 deletion but did not have the
characteristics of PWS—their deletion was on the chromosome 15 that came from the
mother. Because the genetic errors happen in the same section of chromosome 15, PWS
and AS are sometimes called "sister" syndromes even though the disorders are not alike.
Clinical characteristics include seizures, gait and movement disorders, hyperactivity,
laughter and happiness, speech and language, mental retardation and developmental
testing and hypopigmentation.
In 1997, a gene within the AS deletion region called UBE3A was found to be mutated in
approximately 5% of AS individuals. These mutations can be as small as 1 base pair.
This gene encodes a protein called a ubiquitin protein ligase, and UBE3A is believed to
be the causative gene in AS. UBE3A is an enzymatic component of a complex protein
degradation system termed the ubiquitin-proteasome pathway. This pathway is located in
the cytoplasm of all cells. The pathway involves a small protein molecule, ubiquitin, that
can be attached to proteins thereby causing them to be degraded. In the normal brain, the
copy of UBE3A inherited from the father is almost completely inactive, so the maternal
copy performs most of the UBE3A function in the brain. Inheritance of a UBE3A
mutation from the mother causes AS; inheritance of a UBE3A mutation from the father
has no detectable effect on the child. In some families, AS caused by a UBE3A mutation
can recur in more than one family member.
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
Angelman Syndrome
• Angelman syndrome: severe
MR with limited speech; ataxic
gait; spontaneously happy
affect; seizures
• 70% of AS cases due to
cytogenetic deletion 15q11-q13
of maternal 15
B
B
B
X
B
Not expressed
Question: How can two alleles be identical (DNA sequence) but one be expressed and
the other not.
DNA methylation is an epigenetic mechanism that plays an important role in
mammalian gene control and represents a general mechanism for maintaining repression
of transcription. Genes unnecessary for any given cell's function can be tagged with the
methyl groups. The number and placement of the methyl tags provides a signal saying
that the gene should not be expressed. There are proteins in the cell that specifically
recognize and bind the tagged C's, preventing expression of the gene. As would be
expected from something important in determining which genes are used, DNA
methylation is essential for the normal development and functioning of organisms. This
has been shown by engineering mice that can't make the enzymes that put the methyl tags
on DNA, called methyltransferases. These mice die before birth. Problems with the DNA
methylation machinery also cause developmental diseases in people. People with
mutations causing abnormal function of one of the DNA methyltransferase enzymes
called Dnmt3b have a disease called ICF syndrome. These people have abnormal
immune systems and other genetic problems. Similarly, abnormalities in one of the
proteins recognizing and binding mC (called MeCP2) develop Rett syndrome, a form of
mental retardation affecting young girls. Hence, we cannot develop and function
normally without DNA methylation.
Abnormal DNA methylation plays an important role in other developmental diseases as
well. With most genes, it probably does not matter that both copies of the gene (the one
from the mother plus the one from the father) are both active. This has been shown to be
due to a failure in the establishment of the normal pattern of methyl group tags that
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
blocks the activity of one of the copies of the gene. Diseases caused by this type of
methylation problem include Prader-Willi syndrome and Angelman's syndrome.
Abnormal placement of the DNA methylation tags also develops with aging. The tags can
decrease in number in some genes, and increase in others, causing inappropriate
decreases or increases in the activity of the genes affected. The changes in the placement
of the methyl tags may be responsible for a variety of changes in cellular function that
occur during aging. There is also evidence that abnormal placement of the methyl tags
may contribute to the development of human lupus.
Very frequent abnormal increases or decreases in DNA methylation tags are found in
most human cancers and contribute to their development. If the genes affected by
abnormal methylation tagging happen to be involved in regulating cell proliferation,
uncontrolled cell division can occur, and this uncontrolled cell growth is the problem
underlying cancer. Scientists are trying to change the abnormal tags or the effects of these
tags as one treatment for cancer and to use these tagging differences in early diagnosis of
cancer and in monitoring various treatments of cancer.
CpG islands: small stretches of DNA that are characterized by more than 50% CpG’s.
The typical CpG content is around 20% because of deamination of 5-methylcytosine. The
CpG islands can extend over hundreds of nucleotides and there are approximately 45,000
in the human genome. Over half of the human genes are predicted to be associated with
CpG islands and in case of genes showing widespread expression, associated CpG islands
are almost always found at the 5’ ends of genes, usually in the promoter region, often
extending into the first exon.
CpG’s within islands tend to be undermethylated where genes are expressed and
methylated in tissues where the gene is not expressed. There are dramatic changes in
methylation during development. Male and female germ cells have unique patterns of
methylation, almost all of which become demethylated in the preimplantation embryo.
De novo methylation occurs in the early postimplantation embryo.
Imprinted genes show differential methylation: often the inactive allele is methylated at
CpG islands and the active gene is not (there are exceptions where the reverse is true).
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
p57Kip2 5’ 293bp (35CGs; -556 to -163)
Maternal: active
Paternal: inactive
CpG Island
P
M
What are the mechanisms that control methylation patterns?
Distal Chromosome 7 Imprinted Domain
M
Nao1/4
P
Nao1/4
Kip2
tssc3
tssc5
mash2
mtr
Kcnq1 (Kvlqt1)
Lit1
tssc4
H19
th
tapa1
mtr
th
ins
Igf2
tapa1
Imprinting Control Region (ICR)
Delete ICR from paternal chromosome imprint is lost & everything is expressed
P
Nao1/4
tssc3
Kip2
tssc5
Lit1
mtr
mash2
Kcnq1 (Kvlqt1)
tssc4
M
P
th
ins
Igf2
H19
tapa1
Oocyte
Sperm
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X
methylated
CpG
islands
MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
KvDMR1 - ICR (34CpGs):
T
Maternal
G
Paternal
T
T
T
T
T
T
T
G
G
G
G
G
G
G
M
P
Gametic imprint
M
P
Oocyte
Embryo
Sperm
CTCF
methylated
CpG
islands
Spread of methylation to establish embryonic imprint
Covalent Modification of Histone Tails: Another form of epigenetic modification
involves methylation, acetylation and ubiquination of nucleosome histones. For example,
a polycomb group complex known as PRC2 methylates lysine 27 on histone H3 and this
chromatin modification has been shown to be important for genetic imprinting.
X-Chromosome Inactivation is an example of epigenetic regulation of a whole
chromosome.
Females are born with two copies of the X chromosome in their cells and therefore their
cells contain two copies of the X chromosome genes. On the other hand, males have only
one copy of the X chromosome in their cells so they only have one copy of the X
chromosome genes.
X-Chromosome
Contains about 5% of the haploid genome.
Genes encode house keeping and specialized functions.
Completely conserved in gene content between species.
It does not encode sex determination or differentiation.
In females one of the X-chromosomes is inactivated in each
and every cell. [known since 1961]
In order to correct this potential imbalance between males and females the cells have a
system to ensure that only one copy of most of the X chromosome genes in a female cell
are "active". Most of the other X chromosome copy is "inactivated" or switched off. This
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MagnusonAugust 26, 2005
Epigenetics
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mechanism ensures that only the genes located on the "active" copy of the X
chromosome can be used by the cell to direct the production of proteins.
In mammals, the X chromosome undergoing the inactivation event is a random choice
from one cell to another. Once an X chromosome has been inactivated, it remains
inactivated in the daughter cells derived from the original cell. This results in a "mosaic
effect" or a "patchwork effect" in the individual and can be demonstrated in humans and
other mammals.
Demonstration of X-Inactivation
Cells are taken from a female who is heterozygous for two different X-linked enzyme
polymorphisms. She makes two, different forms (A and B) of the glucose-6-phosphate
dehydrogenase (G6PD) enzyme and two, different forms (1 and 2) of the
phosphoglycerol kinase (PGK) enzyme. In each case, the different forms of the enzymes
are distinguishable by their speed of separation in gel electrophoresis. A plug of
fibroblast tissue was removed from the woman and individual cells were cloned such that
each clone of cells was derived from a single cell. Each clone was then assayed for the
type of G6PD and PGK present. The data is shown in the figure below along with a
diagram of her two X chromosomes. Clearly, some clones (1, 4, 6, 7, and 9) have only
G6PD A and PGK 2 and some clones (2, 3, 5, and 8) have only G6PD B and PGK 1).
These combinations reflect the linkage patterns of the X chromosomes of the female as
shown in the figure below at the right.
X inactivation affects most of the genes located on the X chromosome but not all. Some
genes on the X chromosome have their "partner" or pair on the Y chromosome, such as
the gene called ZFK that codes for a protein that is possibly involved in the production of
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MagnusonAugust 26, 2005
Epigenetics
9:00 – 9:50 am
both egg and sperm cells. In male cells, therefore, two copies of these genes would be
active in the cell: one on the X and one on the Y chromosome. So in order for the same
number of active genes to be operating in females, these special genes on the X
chromosome are not switched off so that females also have two copies of these genes
available for the cell to use. In addition, one gene called XIST that is thought to control
the inactivation process itself, is not switched off.
Women who are "carriers" of the faulty genes on their X chromosomes involved in
conditions such as Duchene muscular dystrophy and hemophilia, will have some cells in
their body in which the faulty gene is activated and in others the correct copy of the gene
will be activated. The usual random process of X inactivation means that these women
would not show any symptoms due to the faulty gene as there would be enough cells with
the correct copy of the gene to produce the necessary protein. However, rarely, some
women have more cells where the X chromosome carrying the faulty gene is active so
that these women do show some of the symptoms of these disorders. In these rare cases
the X - inactivation has been "skewed" rather than random.
In other rare cases, women have a structural change of one of their X chromosomes: their
X chromosome may be missing a small part (deleted) or rearranged in some way. Usually
it is this abnormal X chromosome that is inactivated rather than the correct copy. While
this may appear to be a protective mechanism by the cell, it is more likely that the cells
where the correct copy is inactivated do not survive because the deleted or abnormal X
chromosome would be missing segments of important genes.
In other rare cases, women have a special type of chromosome rearrangement in their
cells called a translocation where the X chromosome is attached to one of the numbered
chromosomes (autosomes). In the cells of these women, on the other hand, it is the
correct copy of the X chromosome that is usually inactivated, rather than the translocated,
abnormal X. If the translocated chromosome was inactivated, not only would the process
"switch off" the X chromosome genes but also those on the autosome that were attached
to it. The cells in which the translocated chromosome was inactivated would be missing a
large number of important genes located on the autosome and would be unlikely to
survive.
The mechanism leading to X-inactivation is known to involve a gene called Xist which is
active on the X that will be inactivated and inactive on the X that remains active. Xist
RNA coats the chromosome that will be inactivated to initiate the process. Chromatin
remodeling proteins are then recruited, histones are modified and the CpG islands of
many of the promoters of the genes on the inactive X are methylated.
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