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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 15
GENE REGULATION IN
EUKARYOTES
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INTRODUCTION

Gene regulation is necessary to ensure

1. Expression of genes in an accurate pattern during the
various developmental stages of the life cycle


2. Differences among distinct cell types


Some genes are only expressed during embryonic stages,
whereas others are only expressed in the adult
Nerve and muscle cells look so different because of gene
regulation rather than differences in DNA content
Figure 15.1 describes the levels of gene expression
that are regulated in eukaryotes
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Figure 15.1
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15.1 REGULATORY
TRANSCRIPTION FACTORS

General (basal) transcription factors



Required for the binding of the RNA pol to the core
promoter and its progression to the elongation stage
Are necessary for basal transcription
Regulatory transcription factors


Serve to regulate the rate of transcription of nearby
genes
They influence the ability of RNA pol to begin
transcription of a particular gene
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
Regulatory transcription factors recognize cis
regulatory elements located near the core
promoter

These sequences are known as response
elements, control elements or regulatory
elements

A regulatory protein that increases the rate of
transcription is termed an activator


The sequence it binds is called an enhancer
A regulatory protein that decreases the rate of
transcription is termed a repressor

The sequence it binds is called a silencer
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Figure 15.2
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Figure 17-5
Copyright © 2006 Pearson Prentice Hall, Inc.
Figure 17-4
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Reporter assay in transgenic mouse
Structural Features of Regulatory
Transcription Factors

Transcription factor proteins contain regions, called
domains, that have specific functions



One domain could be for DNA-binding
Another could provide a binding site for effector molecules
A motif is a domain or portion of it that has a very
similar structure in many different proteins
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The recognition helix recognizes and makes contact
with a base sequence along the major groove of DNA
Hydrogen bonding between an a-helix and nucleotide
bases is one way a transcription factor can bind to DNA
Figure 15.3
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Composed of one a-helix and
two b-sheets held together by
a zinc (Zn++) metal ion
Two a-helices intertwined
due to leucine motifs
Alternating leucine residues in
both proteins interact (“zip up”),
resulting in protein dimerization
Homodimers are formed by two
identical transcription factors;
Heterodimers are formed by two
different transcription factors
Figure 15.3
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Figure 17-17b
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Figure 17-18
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Enhancers and Silencers

The binding of a transcription factor to an enhancer
increases the rate of transcription


This up-regulation can be 10- to 1,000-fold
The binding of a transcription factor to a silencer
decreases the rate of transcription

This is called down-regulation
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Enhancers and Silencers

Many response elements are orientation
independent or bidirectional


They can function in the forward or reverse orientation
Most response elements are located within a few
hundred nucleotides upstream of the promoter

However, some are found at various other sites



Several thousand nucleotides away
Downstream from the promoter
Even within introns!
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TFIID and Mediator

Most regulatory transcription factors do not bind
directly to RNA polymerase

Two common protein complexes that communicate
the effects of regulatory transcription factors are


1. TFIID

2. Mediator
Refer to Figure 15.4
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A general transcription factor
that binds to the TATA box
Recruits RNA polymerase
to the core promoter


Transcriptional activator recruits TFIID
to the core promoter and/or activates its
function
Thus, transcription will be activated


Transcriptional repressor inhibits TFIID
binding to the core promoter or inhibits
its function
Thus, transcription will be repressed
Figure 15.4
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STOP



Transcriptional activator stimulates the
function of mediator
This enables RNA pol to form a preinitiation
complex
It then proceeds to the elongation phase of
transcription


Transcriptional repressor inhibits the
function of mediator
Transcription is repressed
Figure 15.4
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Regulation of Regulatory
Transcription Factors

There are three common ways that the function of
regulatory transcription factors can be affected

1. Binding of an effector molecule

2. Protein-protein interactions

3. Covalent modification
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The transcription factor
can now bind to DNA
Formation of
homodimers and
heterodimers
Figure 15.5
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Steroid Hormones and
Regulatory Transcription Factors

Regulatory transcription factors that respond to
steroid hormones are termed steroid receptors


The hormone actually binds to the factor
The ultimate effect of a steroid hormone is to affect
gene transcription

Steroid hormones are produced by endocrine glands
 Secreted into the bloodstream
 Then taken up by cells
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Steroid Hormones and
Regulatory Transcription Factors

Cells respond to steroid hormones in different ways

Glucocorticoids


These influence nutrient metabolism in most cells
 They promote glucose utilization, fat mobilization and protein
breakdown
Gonadocorticoids


These include estrogen and testosterone
They influence the growth and function of the gonads
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Heat shock protein
Heat shock proteins
leave when hormone
binds to receptor
Nuclear localization
Sequence is exposed
Formation of a
homodimer
Glucocorticoid
Response Elements
These function as
enhancers
Transcription of target
gene is activated
Figure 15.6
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The CREB Protein

The CREB protein is another regulatory
transcriptional factor functioning within living cells


CREB is an acronym for cAMP response element-binding
CREB protein becomes activated in response to cellsignaling molecules that cause an increase in cAMP


Cyclic adenosine monophosphate
The CREB protein recognizes a response element with the
consensus sequence 5’–TGACGTCA–3’

This has been termed a cAMP response element (CRE)
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Could be a hormone,
neurotransmitter,
growth factor, etc.
Acts as a
second
messenger
Activates
protein
kinase A
Phosphorylated CREB
binds to DNA and
stimulates transcription
Unphosphorylated CREB
can bind to DNA, but
cannot activate RNA pol
Figure 15.7
The activity of the CREB protein
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15.2 CHANGES IN
CHROMATIN STRUCTURE

Changes in chromatin structure can involve
changes in the structure of DNA and/or changes in
chromosomal compaction

These changes include

1.
2.
3.
4.

Refer to Table 15.1



Gene amplification
Gene rearrangement
DNA methylation
Chromatin compaction
Uncommon ways to regulate
gene expression
Common ways to regulate
gene expression
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Chromatin Structure

The three-dimensional packing of chromatin is an
important parameter affecting gene expression

Chromatin is a very dynamic structure that can
alternate between two conformations

Closed conformation



Chromatin is very tightly packed
Transcription may be difficult or impossible
Open conformation


Chromatin is highly extended
Transcription can take place
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Fig. 15.8

Amphibian oocyte lampbrush chromosomes
Figure 17-7
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Experiment 15A: DNase I
Sensitivity and Chromatin Structure

DNase I is an endonuclease that cleaves DNA


It is much more likely to cleave DNA in an open
conformation than in a closed conformation
In 1976, Harold Weintraub and Mark Groudine used
DNase I sensitivity to study chromatin structure


In particular, they focused attention on the b-globin gene
The gene was known to be specifically expressed in
reticulocytes (immature red blood cells)

But not in other cell types, such as brain cells and fibroblasts
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
First, let’s consider the rationale behind Weintraub
and Groudine’s experimental approach

Globin genes are only a small part of the total DNA


They used a radiolabeled cloned DNA fragment (i.e., a
probe) that was complementary to the b-globin gene


Therefore, they had to find a way to specifically monitor the
digestion of the b-globin gene
This was hybridized to the chromosomal DNA to determine
specifically if the chromosomal b-globin gene was intact
Following hybridization, the samples were then
exposed to another enzyme, termed S1 nuclease

This enzyme only cuts single-stranded DNA
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Figure 15.9
Cut with DNase I
Chromatin:
DNAse sensitive
This indicates that the chromosomal
DNA was in an open conformation
It was accessible to DNase I
and was consequently digested
Do not cut with DNase I
Chromatin:
DNAse insensitive
This indicates that the chromosomal
DNA was in a closed conformation
It was inaccessible to DNase I
and was thus protected from digestion
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The Hypothesis

A loosening of chromatin structure occurs when
globin genes are transcriptionally active
Testing the Hypothesis

Refer to Figure 15.10
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Figure 15.10
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Figure 15.10
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Figure 15.10
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The Data
Source of nuclei
% Hybridization of DNA probe
Reticulocytes
25%
Brain cells
>94%
Fibrobalsts
>94%
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Interpreting the Data
Source of nuclei

% Hybridization of DNA probe
Reticulocytes
25%
Brain cells
>94%
Fibrobalsts
>94%
Reticulocytes had a much smaller percentage of hybridization

Therefore, their globin genes were more sensitive to DNase I

The globin genes are known to be expressed in reticulocytes but not in
brain cells and fibroblasts

Therefore, these results are consistent with the hypothesis:

The globin gene is less tightly packed when it is being expressed
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Globin Gene Expression

The family of globin genes is expressed in the
reticulocytes


However, individual members are expressed at different
stages of development
For example:


b-globin  Adult
g-globin  Fetus
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Figure 15.11
Segments of DNA that are deleted in these populations

Thalassemia is a defect in the expression of one or more
globin genes

An intriguing observation of some thalassemic patients is that they
cannot synthesize b-globin even though the gene is perfectly normal
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

A DNA region upstream
of the b-globin gene
was identified as
necessary for globin
gene expression
This region is termed
locus control region
(LCR)


Genes are now accessible
to RNA pol and transcription
factors
It helps in the regulation
of chromatin opening
and closing
It is missing in certain
persons with
thalassemias
Figure 15.12
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Globin Gene Expression

Aside from chromatin packing, a second structural
issue to consider is the position of nucleosomes

In chromatin, the nucleosomes are usually
positioned at regular intervals along the DNA

However, they have been shown to change positions in
cells that normally express a particular gene
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Positioned at regular intervals from -3,000 to + 1,500
Disruption in nucleosome positioning
from -500 to + 200
Figure 15.3
Changes in nucleosome position during the activation of the b-globin gene
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Chromatin Remodeling

As discussed in Chapter 12, there are two common
ways in which chromatin structure is altered

1. Covalent modification of histones

2. ATP-dependent chromatin remodeling
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
1. Covalent modification of histones

Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation
Adds acetyl groups, thereby
loosening the interaction
between histones and DNA
LOOSE
TIGHT
Figure 12.13
Removes acetyl groups,
thereby restoring a
tighter interaction
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Figure 17-10
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
2. ATP-dependent chromatin remodeling

The energy of ATP is used to alter the structure of
nucleosomes and thus make the DNA more accessible
Proteins are members of the
SWI/SNF family
Figure 12.13
These effects may significantly alter
gene expression
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Chromatin Remodeling

An important role for transcriptional activators is to
recruit the aforementioned enzymes to the promoter

A well-studied example of recruitment involves a
gene in yeast that is involved in mating



Yeast can exist in two mating types, termed a and a
The gene HO encodes an enzyme that is required for the
mating switch
Refer to Figure 15.14
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SWI refers to mating type
switching
SAGA is an acronym for
Spt/Ada/GCN5/Acetyltransferase
Genes known to be transcriptionally
regulated by histone acetyltransferase
Figure 15.14
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SBP is an acronym for a
mating type switching cell
cycle box protein)
Figure 15.14
RNA polymerase
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DNA Methylation
(or DNA methylase)
CH3
Only one strand is
methylated
CH3
Both strands are
methylated
CH3
Figure 15.15
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
DNA methylation usually inhibits the transcription of
eukaryotic genes


Especially when it occurs in the vicinity of the promoter
In vertebrates and plants, many genes contain
CpG islands near their promoters

These CpG islands are 1,000 to 2,000 nucleotides long

In housekeeping genes



The CpG islands are unmethylated
Genes tend to be expressed in most cell types
In tissue-specific genes

The expression of these genes may be silenced by the
methylation of CpG islands
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Transcriptional
activator binds to
unmethylated DNA
This would inhibit the
initiation of transcription
Figure 15.16 Transcriptional silencing via methylation
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Figure 15.16 Transcriptional silencing via methylation
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15.3 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 15.2
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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
This phenomenon was termed RNA interference (RNAi)
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Short RNA from the
antisense strand
Thus the expression of
the gene that encodes
this mRNA is silenced
Cellular mRNA is degraded by
endonucleases within the complex
Figure 15.24
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Double-stranded RNA and Gene
Silencing

RNA interference is widely found in eukaryotes

It is believed to

1. Offer a host defense mechanism against certain viruses


Those with double-stranded RNA genomes, in particular
2. Play a role in silencing certain transposable elements

Some of these elements produce double-stranded RNA
intermediates as part of the transposition process
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