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Chapter 16
Lecture Outline
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INTRODUCTION

 Chapter 15 considered two general aspects of

eukaryotic gene regulation
 1. Regulatory transcription factors may activate or inhibit
genes
 2. Changes in chromatin structure affect gene expression

 Chapter 16 will consider two additional types of

gene regulation
 1. Epigenetic regulation
 2. RNA modifications

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16.1 OVERVIEW OF
EPIGENETICS
 Epigenetics is the study of mechanisms that lead to
changes in gene expression that can be passed
from cell to cell and are reversible, but do not
involve a change in the sequence of DNA
 Epigenetic inheritance involves epigenetic changes
that are passed from parent to offspring
 An example is genomic imprinting described in Chapter 5

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 Different types of molecular change underlie

epigenetic regulation (see Table 16.1)
 DNA methylation
 Chromatin remodeling
 Covalent histone modification
 Localization of histone variants
 Feedback loops

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 Epigenetic changes may be targeted to genes by

transcription factors or by noncoding RNAs (see Figure
16.1)

Figure 16.1 Establishing epigenetic modifications. 16-6

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cis- and trans-Epigenetic Changes

 Epigenetic changes may by cis- or trans-
 Cis-epigenetic changes are maintained at a
specific site
 For example, a cis-epigenetic change may affect only
one copy of gene but not the other copy
 Trans-epigenetic changes are maintained by
diffusible factors, such as transcription factors
 A trans-epigenetic change affects both copies of a
gene

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cis-Epigenetic Changes
 cis-epigenetic changes are maintained during
cell division (see Figure 16.2)

Figure 16.2 Pattern of transmission of a cis-epigenetic

mechanism that maintains an epigenetic modification. 16-8

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Distinguishing cis- versus trans-

Epigenetic Changes
 cis- and trans-epigenetic changes can be
distinguished via cell fusion experiments (refer
to Figure 16.3)

Figure 16.3 The use of cell-fusion experiments to distinguish

cis- and trans-epigenetic mechanisms. 16-9

Two General Categories of Epigenetic

Gene Regulation
 Epigenetic gene regulation may occur as a
programmed developmental change or be caused by
environmental agents

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16.2 EPIGENETICS AND

DEVELOPMENT
 Development involves a series of genetically
programmed stages in which a fertilized egg becomes
an embryo and eventually an adult

 Many changes that occur during development are

maintained by epigenetic regulation

 Three examples
 Genomic imprinting
 X-chromosome inactivation
 Formation of specific cell types and tissues
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Genomic Imprinting
 As described in Chapter 5, genomic imprinting is a form of
gene regulation in which an offspring expresses the copy of a
gene from one parent but not both; in mammals, only the Igf2
gene inherited from the father is expressed
 The Igf2 gene is de novo methylated during sperm formation
but not during egg formation (refer to Figure 16.4)
 The methylation occurs at two sites: the imprinting control
region (ICR) and a differentially methylated region (DMR)
 Methylation inhibits the binding of a protein called the CTC-
binding factor, which allows the Igf2 gene to be stimulated by
a nearly enhancer.
 In contrast, CTC-binding factor binds to the unmethylated
gene and inhibits transcription by forming a loop
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Genomic Imprinting

Figure 16.4 The molecular mechanism of Igf2 imprinting.

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X-Chromosome Inactivation
 X-chromosome inactivation (XCI) occurs during
embryogenesis in female mammals (see Chapter 5)
 A portion of the X chromosome call the X inactivation center
(XIC) plays a key role. Prior to XCI, the Tsix gene is
expressed on both X chromosomes
 The XIC encodes two genes, Xist and Tsix, which are
transcribed in opposite directions

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X-Chromosome Inactivation
 Prior to X-chromosome inactivation, the Tsix gene is
expressed on both X chromosomes (see Figure 16.5)
 During embryogenesis, the X chromosomes pair up and a
symmetry break causes the pluripotency factors to move to
one X chromosome, which remains active. The other X
chromosome expresses the Xist gene
 The Xist RNA binds to XIC and then spreads to both ends of
the X chromosome
 The Xist RNA recruits proteins to this X chromosome that
cause it to become more compact and be inactive with regard
to the expression of most genes; however, some genes on
this chromosomes may be expressed to some degree
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Figure 16.5b The process of X-chromosome inactivation.

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Figure 16.5b The process of X-chromosome inactivation.

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Development of
Specific Cell Types
 Epigenetic changes occur during embryonic development
that are remembered during subsequent cell divisions
 For example, an embryonic cell may undergo epigenetic changes that
will cause its future daughter cells to become muscle cells
 A specific cell type, such as a muscle cell, will activate specific genes
and repress others

 Two types of competing protein complexes are key

regulators of epigenetic changes during development that
produce specific cell types and tissues
 Trithorax group (TrxG)- involved with gene activation
 Polycomb group (PcG)- involved with gene repression
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Development of
Specific Cell Types
 With regard to the polycomb group complex, there are two
types: PRC1 and PRC2
 Though the mechanism may vary from gene to gene,
repression may begin by the binding of PRC2 to a polycomb
response element. This leads to trimethylation of lysine 27
on histone H3. (See Figure 16.6.)
 PRC1 is then recruited to the gene and may inhibit
transcription in three ways
1. Chromatin compaction
2. Covalent modification of histones
3. Direct interaction with a transcription factor

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Figure 16.6a A simplified model of epigenetic silencing of a gene by

polycomb group complexes. 16-19

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Figure 16.6b A simplified model of epigenetic silencing of a gene by

polycomb group complexes. 16-19

16.3 EPIGENETICS AND

ENVIRONMENTAL AGENTS
 Many environmental agents have been shown to
cause epigenetic changes.
 These include dietary effects as well as toxins in the
environment. Examples include
 Dietary effects on the Agouti gene in mice
 Toxins that contribute to cancer

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Epigenetics and Diet

 The Agouti gene in mice promotes the synthesis of
yellow fur pigment.
 In one strain of mice, a transposable element
carrying a promoter is inserted upstream from the
Agouti gene; this is called the Avy allele

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Epigenetics and Diet

 Strains of mice carrying the Avy allele show a range
of coat colors, from yellow to pseudo-agouti

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Epigenetics and Diet

 When pregnant mice were fed a diet that contained
chemicals that tend to increase DNA methylation, the
offspring tended to be have darker fur.

Figure 16.7c Dietary effects on coat color in mice.

 This result is consistent with the idea that DNA

methylation inhibits the Agouti gene
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Epigenetics and Diet

 The darkness of the coat color correlated with the
level of DNA methylation of CpG islands within the
transposable element

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Epigenetics and Toxins

 Toxins in the environment may alter gene expression
and promote cancer by causing epigenetic changes.
 Epigenetic changes could increase the expression of
oncogenes or decrease the expression of tumor
suppressor genes.
 Three common epigenetic changes associated with
cancer include
 DNA methylation
 Covalent histone modification
 Chromatin remodeling

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16.4 REGULATION OF RNA

PROCESSING AND TRANSLATION
 So far, we have discussed various mechanisms
that regulate the level of gene transcription

 In eukaryotic species, it is also common for gene

expression to be regulated at the RNA level

 Refer to Table 16.4

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Alternative Splicing
 One very important biological advantage of introns in
eukaryotes is the phenomenon of alternative splicing
 Alternative splicing refers to the phenomenon that
pre-mRNA can be spliced in more than one way
 Alternative splicing produces two or more polypeptides
with different amino acid sequences
 In most cases, large sections of the coding regions are the
same, resulting in alternative versions of a protein that
have similar functions
 Nevertheless, there will be enough differences in amino
acid sequences to provide each polypeptide with its own
unique characteristics
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Alternative Splicing
 The degree of splicing and alternative splicing
varies greatly among different species

 Baker’s yeast contains about 6,300 genes

 ~ 300 (i.e., 5%) encode mRNAs that are spliced
 Only a few of these 300 have been shown to be alternatively spliced

 Humans contain ~ 22,000 genes

 Most of these encode mRNAs that are spliced
 It is estimated that about 70% are alternatively spliced
 Note: Certain mRNAs can be alternatively spliced to produce dozens
of different mRNAs

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Alternative Splicing
 Figure 16.8 considers an example of alternative
splicing for a gene that encodes a-tropomyosin
 This protein functions in the regulation of cell contraction
 It is found in
 Smooth muscle cells (uterus and small intestine)
 Striated muscle cells (cardiac and skeletal muscle)
 Also in many types of nonmuscle cells at low levels

 The different cells of a multicellular organism regulate

contractibility in subtly different ways
 One way to accomplish this is to produce different forms of
a-tropomyosin by alternative splicing

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Found in the mature

mRNA from all cell types

Not found in all

mature mRNAs

These alternatively spliced versions of a-tropomyosin vary in

function to meet the needs of the cell type in which they are found

Figure 16.8 Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
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Alternative Splicing
 Alternative splicing is not a random event
 The specific pattern of splicing is regulated in a given cell

 It involves proteins known as splicing factors

 These play a key role in the choice of splice sites

 One example of splicing factors are the SR proteins

 At their C-terminal end, they have a domain that is rich in
serine (S) and arginine (R)
 It is involved in protein-protein recognition
 At their N-terminal end, they have an RNA-binding domain

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 The spliceosome recognizes the 5’ and 3’ splice

sites and removes the intervening intron
 Refer to Chapter 12

 Splicing factors modulate the ability of spliceosomes

to recognize or choose the splice sites

 This can occur in two ways

 1. Some splicing factors inhibit the ability of a spliceosome
to recognize a splice site
 Refer to Figure 16.9a
 2. Some splicing factors enhance the ability of a
spliceosome to recognize a splice site
 Refer to Figure 16.9b

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Alternative
splicing

Splice S p lic e
5′ 3′ 5′ 3′ 5′ 3′ junctions 5′ 3′ 5′ 3′ 5′ 3′
ju n c t i o n s

5′ 1 2 3 4 3′ 5′ 1 2 3 4 3′

The spliceosome A splicing repressor prevents the

recognizes all the Splicing recognition of a 3′-splice junction.
splice junctions. repressor The next 3′-splice junction that
precedes exon 3 will be chosen.

5′ 1 2 3 4 3′ 5′ 1 3 4 3′

All 4 exons are contained Exon 2 is skipped and not

within the mRNA. included in the mRNA.

(a) Splicing repressors

Known as
exon skipping

Figure 16.9a The role of splicing factors during alternative splicing

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These 2 splice junctions Alternative

are not readily recognized splicing
by the spliceosome.

Splice Splice
5′ 3′ 5′ 3′ 5′ 3′ junctions 5′ 3′ 5′ 3′ 5′ 3′ junctions

5′ 1 2 3 4 3′ 5′ 1 2 3 4 3′

The spliceosome only Splicing

The binding of splicing enhancers
recognizes 4 of the enhancer
promotes the recognition of
6 splice junctions. poorly recognized junctions. All 6
junctions are recognized.

5′ 1 2 4 3′ 5′ 1 2 3 4 3′

Exon 3 is not included in the mRNA. Exon 3 is included in the mRNA.

(b) Splicing enhancers

Figure 16.9b The role of splicing factors during alternative splicing

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Stability of mRNA
 The stability of eukaryotic mRNA varies considerably
 Several minutes to several days or even months

 The stability of mRNA can be regulated so that its

half-life is shortened or lengthened
 This will greatly influence the mRNA concentration
 And consequently gene expression

 Factors that can affect mRNA stability include

 1. Length of the polyA tail
 2. Destabilizing elements
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 1. Length of the polyA tail

 Most newly made mRNA have a polyA tail that is about
200 nucleotides long
 It is recognized by polyA-binding protein
 Which binds to the polyA tail and enhances stability
 As an mRNA ages, its polyA tail is shortened by the
action of cellular exonucleases
 The polyA-binding protein can no longer bind if the polyA
tail is less than 10 to 30 adenosines long
 The mRNA will then be rapidly degraded by exo- and
endonucleases

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 2. Destabilizing elements
 Found in mRNAs that have short half-lives
 These elements can be found anywhere in the mRNA
 However, they are most commonly at the 3’ end between the stop
codon and the polyA tail
AU-rich element (ARE)
Recognized and bound by cellular proteins
These proteins influence mRNA degradation

ARE PolyA tail

Stop codon
mRNA Start codon
5′ AUG AUUUA AAAAAAAA 3′

5′-UTR 3′-UTR
Protein binding
5’-untranslated region to ARE
3’-untranslated region
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Double-stranded RNA
and Gene Silencing
 Double-stranded RNA can silence the expression of
certain genes
 This discovery was made from research in plants and the
nematode Caenorhabditis elegans

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Experiment 16A: Double-stranded

RNA and Gene Silencing
 Evidence for mRNA degradation via double-stranded
RNA came from studies in C. elegans
 Injection of antisense RNA (i.e., RNA complementary to a
specific mRNA) into oocytes silences gene expression
 Surprisingly, injection of double-stranded RNA was 10 times more
potent at inhibiting the expression of the corresponding mRNA
 Also, the effects of antisense RNA persisted for a very long time
 This led Andrew Fire and Craig Mello to investigate how
injection of RNA inhibits mRNA

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The Goal

 Understand how the experimental injection of

RNA was responsible for the silencing of
particular mRNAs

Achieving the Goal

 Refer to Figure 16.11

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Experimental level Conceptual level
1. Make sense and antisense mex-3 RNA Sense RNA
in vitro using cloned genes for mex-3 Promoter
with promoters on either side of the Add RNA mex-3 RNA
Gene. RNA polymerase and nucleotides polymerase and gene polymerase
are added to synthesize the RNAs. nucleotides to
Sense cloned genes.
RNA

Antisense RNA

2. Inject either mex-3 antisense RNA or a Antisense RNA or a Antisense RNA

mixture of mex-3 sense and antisense mixture of sense and Promoter]
RNA into the gonads of C. elegans. This antisense RNA
RNA is taken up by the eggs and early
embryos. As a control, do not inject any
RNA.
Single row of eggs

Add labeled
probe.
3. Incubate and then subject early embryos
to in situ hybridization. In this method, Labeled probe
a labeled probe is added that is
complementary to mex-3 mRNA. If cells Embryo
express mex-3, the mRNA in the cells
will bind to the probe and become mex-3 mRNA
labeled. After incubation with a labeled
probe, the cells are washed to remove
unbound probe.

4. Observe embryos under the

microscope.

Figure 16.11
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The Data

Figure 16.11
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Interpreting the Data

mex-3 is expressed at high

levels in the control, lower levels
in cells injected with antisense
RNA and completely degraded
in cells injected with double-
stranded RNA. This shows that
double-stranded RNA is more
potent at silencing gene
expression than antisense RNA.
They termed this phenomenon
RNA interference or RNAi

Figure 16.11
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RNA Interference Is Mediated

by Micro RNAs
 microRNAs (miRNAs) or short-interfering RNAs
(siRNAs) cause RNA interference
 miRNAs are encoded by genes in eukaryotic organisms
 miRNA genes do not encode a protein
 Give rise to small RNA molecules, typically 21 to 23 nucleotides
 Not usually a perfect match to mRNAs
 siRNAs forms from two RNA molecules that form a double
stranded region
 Usually a perfect match or close to a perfect match to specific
mRNAs

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RNA Interference Is Mediated

by Micro RNAs
 In humans, over 1000 genes encoding miRNAs
have been identified
 A proposed mechanism for RNAi is shown in
Figure 16.12

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Mechanism of RNA
Interference

Figure 16.12
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Benefits of RNA Interference

 Presents a newly identified form of gene regulation

 May offer a defense mechanism against certain
viruses
 RNA viruses that have a double-stranded RNA genome
 RNA viruses that produce a double-strand RNAs during
their reproductive cycle
 May play a role in silencing certain transposable
elements
 Random insertion may place an element near a cellular promoter
which will produce a silencing RNA

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Iron Assimilation and Translation

 Regulation of iron assimilation provides an example
of how the translation of specific mRNAs is
modulated

 Iron is an essential element for the survival of living

organisms
 It is required for the function of many different enzymes

 The assimilation of iron is depicted in Figure 16.13

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Fe3+

Protein that carries iron

Transferrin
through the bloodstream Transferrin receptor

Endocytosis Endocytic vesicle

Fe3+
binds to
cellular
enzymes

A hollow spherical protein Fe3+

Iron (Fe3+)
is released
Prevents toxic buildup of Fe3+ Fe3+ into cytosol
too much iron in the cell
Fe3+ stored
within ferritin
Ferritin

Figure 16.13
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 Iron is a vital yet potentially toxic substance

 Mammalian cells have evolved an interesting way to
regulate iron assimilation

 An RNA-binding protein known as the iron regulatory

protein (IRP) plays a key role
 It influences both the ferritin mRNA and the transferrin
receptor mRNA

 This protein binds to a regulatory element within the mRNA

known as the iron response element (IRE)
 An IRE is found in the 5’-UTR of ferritin mRNA
 An IRE is also found in the 3’-UTR of transferrin receptor mRNA

 Regulation of iron assimilation is shown in Figure 16.14

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 Translation of ferritin mRNA is inhibited when iron

levels are low due to the binding of IRP
 At high iron levels, iron binds to IRP, which is
released from the IRE; translation can occur

Figure 16.14 (a) Regulation of ferritin mRNA

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Increased stability of
mRNA is degraded and
mRNA means more
cannot be translated
translation

Figure 16.14 (b) Regulation of transferrin receptor mRNA

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