Download Slide 1

Chapter 32
The Control of Gene Expression in eukaryotes
•Background and introduction
1
•Background and introduction
Gene regulation in eukaryotes is significantly
more complex than in prokaryotes in several
ways.
1.The genomes being regulated are significantly larger.
The E. coli genome consists of a single,circular
chromosome containing 4.6 Mb. This genome encodes
approximately 2000 proteins.
Saccharomyces cerevisiae (baker’s yeast), 16
chromosomes ranging in size from 0.2 to 2.2 Mb, The
yeast genome totals 12 Mb and encodes
approximately 6000 proteins.
Human cell 23 pairs of chromosomes ranging in size
from 50 to 250 Mb. Approximately 23,000 genes are
present within the 3000 Mb of human DNA.
2
•Background and introduction
2. Prokaryotic genomic DNA is
relatively accessible, eukaryotic DNA is
packaged into chromatin, a complex
between the DNA and a special set of
proteins.
The existence of stable cell types is
due to differences in the epigenome,
differences in chromatin structure, and
covalent modifications of the DNA, not
in the DNA sequence itself.
An electron micrograph of chromatin
showing its “beads on a string” character.
3
•Different organs(cells) express different subset of genes
4
•Background and introduction
3. Eukaryotic genes are not generally organized into operons.
Instead, genes that encode proteins for steps within a given pathway
are often spread widely across the genome. This characteristic
requires that other more complex mechanisms function to regulate
genes in a coordinated way.
5
Chromatin is made up of nucleosomes which are complexes of DNA
and histones
Eukaryotic DNA is tightly bound to a group of small basic proteins called
Histones which constitute half the mass of a eukaryotic chromosome. The
entire complex of a cell’s DNA and associated protein is called chromatin.
Histone octamer: The nucleosome core is formed of two H2A-H2B
dimers and a H3-H4 tetramer.
Histone octamer
An electron micrograph of chromatin
6
showing its “beads on a string” character.
DNA wraps around histone octamers to form nucleosomes
The four types of histone that make up the protein core are homologous
and similar in structure.
The nucleosome core is formed of two H2A-H2B dimers and a H3-H4
tetramer.
7
The eight histones in the core are arranged into a (H3)2(H4)2 tetramer
and (H2A–H2B)2 dimers.
The DNA forms a left-handed superhelix as it wraps around the outside
of the histone octamer(left-handed superhelical ramp). The protein core
forms contacts with the inner surface of the DNA superhelix at many points,
particularly along the phos-phodiester backbone and the minor groove.
Each histone has an amino-terminal tail that extends out from the core
structure. These tails are flexible and contain a number of lysine and
arginine residues. Covalent modifications of these tails play an essential
role in regulating gene expression.
Left handed
superhelix
8
The DNA double helices are wound around histone core and further
packed into higher-order chromatin structure
The winding of DNA around the nucleosome core contributes to the
packing of DNA by decreasing its linear extent.
•An extended 200-bp stretch of DNA would have a length of about 680 Å.
Wrapping this DNA around the histone octamer reduces the length to
approximately 100 Å along the long dimension of the nucleosome. Thus
the DNA is compacted by a factor of 7.
•Human chromosomes in metaphase, which are highly condensed, are
compacted by a factor of 104.
The nucleosomes are arranged in a helical array
approximately 360 Å across, forming a series of
stacked layers approximately 110 Å apart . The
folding of these fibers of nucleosomes into loops
further compacts DNA.
9
32.2 Transcription Factors Bind DNA and Regulate Transcription
Initiation
Eukaryotic transcription factors are different in several ways:
1. DNA-binding sites in prokaryotes are usually quite close to promoters,
those in eukaryotes can be farther away from promoters and can exert
their action at a distance.
2. Most prokaryotic genes are regulated by single transcription factors,
and multiple genes in a pathway are expressed in a coordinated fashion
because such genes are often transcribed as part of a polycistronic
mRNA. In eukaryotes, the expression of each gene is typically controlled
by multiple transcription factors, and the coordinated expression of
different genes depends on having similar transcription-factor-binding
sites in each given gene.
3. In prokaryotes, transcription factors usually interact directly with RNA
polymerase. In eukaryotes, some transcription factors interact directly
with RNA polymerase, whereas others interact with other proteins
associated with RNA polymerase and still others act by modifying the
chromatin structure
10
32.2 Transcription Factors Bind DNA and Regulate
Transcription Initiation
Eukaryotic transcription factors consist of several domains:
The DNA-binding domain binds to regulatory sequences that can
either be adjacent to the promoter or at some distance from it. Most
commonly, transcription factors include additional domains that help
activate transcription.
When a transcription factor is bound to the DNA, its activation
domain promotes transcription by interacting with RNA polymerase
II, by interacting with other associated proteins, or by modifying the
local structure of chromatin.
11
A range of DNA-binding structures are employed by
eukaryotic DNA-binding proteins
Structural motifs for DNA binding domains:
1.Homeodomain: The structure of this domain and its mode of
recognition of DNA are very similar to those of the prokaryotic helixturn-helix proteins. In eukaryotes, homeodomain proteins often form
heterodimeric structures, sometimes with other homeodomain
proteins, that recognize asymmetric DNA sequences
A heterodimer motif formed
from two different DNAbinding domains, each
based on a homeodomain.
each homeodomain has a
helix-turn-helix motif with
one helix inserted into the
major groove of DNA
12
Homeodomain structure
A range of DNA-binding structures are employed
by eukaryotic DNA-binding proteins
Structural motifs:
2.Basic-leucine zipper ( bZip)
proteins : A pair of long a helices.
Part 1: Basic region that lies in the
major groove of the DNA and makes
contacts responsible for DNA-site
recognition.
Part2: Coiled-coil structure with its
partner. These units are often stabilized
by appropriately spaced leucine residues,
these structures are often referred to as
leucine zippers
Coiled-coil
structure(part II)
Part I
This heterodimer comprises two basicleucine zipper proteins(Here only dimer
shown).
The basic(alkaline ) region lies in the
major groove of DNA.
13
Basic-leucine zipper
Structural motifs:
3. (Cys2His2) zinc-finger domains : This DNA-binding unit
comprises tandem sets of small domains, each of which binds a
zinc ion through conserved sets of two cysteine and two histidine
residues.
DNA-binding domain comprising three zinc-finger domains (shown in
yellow, blue, and red) is shown in a complex with DNA. Each zinc-finger
domain is stabilized by a
bound zinc ion (shown in
green) through interactions
with two cysteine
residues and two histidine
residues.
Three domains wraps
around the DNA in the
major groove by domain
string.
14
Zinc-finger domains
Activation domains (interacting with other proteins)
The activation domains generally recruit other proteins that
promote transcription. In some cases, these activation domains
interact directly with RNA polymerase II or closely associated
proteins (indirectly). The activation domains act through
intermediary proteins that bridge between the transcription factors
and the polymerase.
Mediator, a large complex of protein
subunits, acts as a bridge
between transcription factors
activation domains and RNA
polymerase II.
These interactions help recruit and
stabilize RNA polymerase II near
specific genes that are then
transcribed.
DNA
binding
domain
Activation
domain
15
Mediator
Common features to activation domains
1.They are often redundant; that is, a part of the activation domain
can be deleted without loss of function.
2. They are modular and can activate transcription when paired
with a variety of DNA-binding domains.
3. Activation domains can act synergistically: two activation
domains acting together create a stronger effect than either
domain acting separately.
Transcrptional repressor
like activators, transcriptional repressors act in many cases by altering
chromatin structure.
16
Multiple transcription factors interact with eukaryotic
regulatory regions
Several general transcription factors join with RNA polymerase II to
form the basal transcription complex to initiate transcription at a
low frequency.
For a gene to achieve a higher rate of mRNA synthesis, additional
transcription factors must bind to other sites that can be near the
promoter or quite distant. This is achieved by combinatorial
control.
Each factor recruits other proteins to build up large complexes that
interact with the transcriptional machinery to activate transcription.
Its advantage is that a given regulatory protein can have different
effects, depending on what other proteins are present in the same
cell.
17
Enhancers can stimulate transcription in specific cell
types
Transcription factors can often act even if their binding sites lie at a
considerable distance from the promoter. These distant regulatory
sites in DNA are called enhancers .
Enhancers function by serving as binding
sites for specific transcription factors. An
enhancer is effective only in the specific cell
types in which appropriate regulatory
proteins are expressed.
Protein A
Enhancer binding sites
Mechanisms:
Influence transcription initiation by
perturbing the local chromatin structure to
expose a gene or its regulatory
sites rather than by direct interactions with
RNA polymerase
Protein A
Protein B
18 C
Protein
Experiment to test enhancers’ fucntion
Enhancer controlling the muscle isoform of
creatine kinase. This enhancer includes
three binding site for regulatory
proteins( transcription factors) which are
necessary for the enhancer’s function.
Protein A
Protein A
Experiment:
Inserting this enhancer near a gene (galactosidase) not normally expressed in
muscle cells.
Protein B
Protein C
The result showed that -galactosidase was
expressed at high levels in muscle cells of
zebrafish embryo but not in other cells.
The embyo was treated with X-Gal which
was catalyzed by -galactosidase into blue
19
product.
-galactosidase in a zebrafish embryo.
Application of transcription factors
----Induced pluripotent stem cells (iPS) can be generated by introducing
four transcription factors into differentiated cells
Pluripotent stem cells have the ability to differentiate into many
different cell types on appropriate treatment. Isolated cells derived
from embryos show a very high degree of pluripotency. And
researchers identified dozens of genes in embryonic stem cells that
contributed to this pluripotency.
Shinya Yamanaka demonstrated that just four genes out of identified
before could induce pluripotency in already-differentiated skin
cells(2006 on mouse cells, 2007 on human cells).
Finally, Yamanaka introduced genes encoding four transcription
factors into skin cells (fibroblasts). Then,the fibroblasts dedifferentiated into cells that appeared to have characteristics very
nearly identical with those of embryonic stem cells. That means
human can make the already-differentiated cells back to embryonic
status with puripotent properties by treatment with related
transcription fators.
20
Application of transcription factors
----Induced pluripotent stem cells (iPS) represent powerful new
research tools and a new class of therapeutic agents
Patient’s fibroblasts could be readily isolated and converted into iPS
cells. These iPS cells could then be treated to differentiate into a desired
cell type that could then be transplanted into the patient.
For example, such an approach might be used to restore a particular
class of nerve cells that had been depleted by a neurodegenerative
disease.
21
32.3 The Control of Gene Expression Can
Require Chromatin Remodeling
Chromatin structure plays a major role in controlling eukaryotic gene
expression. Relaxing of the chromatin is necessary for gene expression.
DNA that is densely packaged into chromatin is less susceptible to
cleavage by the nonspecific DNA-cleaving enzyme DNase I. Regions
adjacent to genes that are being transcribed are more sensitive to
cleavage by DNase I than are other sites in the genome, suggesting that
the DNA in these regions is less compacted than it is else-where and
more accessible to proteins. We call these sites hypersensitive sites.
22
32.3 The Control of Gene Expression Can
Require Chromatin Remodeling
Hypersensitive sites are cell-type specific and developmentally regulated.
Eg.
Hemoglobin genes in the precursors of erythroid cells from 20-hour-old
chicken embryos are insensitive to DNase I. However, when hemoglobin
synthesis begins at 35 hours, regions adjacent to these genes become
highly susceptible to digestion.
In tissues such as the brain that produce no hemoglobin, the globin genes
remain resistant to DNase I throughout development and into adulthood.
It is a necessary for gene expression that chromatin structure change to
relax conformation.
23
Experiments clearly revealed the role of chromatin
structure in regulating access to DNA binding sites
Genes required for galactose utilization in
yeast are activated by a transcription factor
called GAL4, which recognizes DNA binding
sites with two 5’-CGG-3’ sequences on
complementary strands separated by 11 base
pairs. Approximately 4000 potential GAL4
binding sites of the form 5’-CGG(N)11CCG-3’
are present in the yeast genome, but only 10 of
them regulate genes necessary for galactose
metabolism.
How is GAL4 targeted to only a small fraction of
the potential binding sites?
GAL4 binding sites
(zinc-based domains,
24
not zinc-finger motif)
Experiments clearly revealed the role of chromatin
structure in regulating access to DNA binding sites
Chromatin immunoprecipitation (ChIP)
25
Experiments clearly revealed the role of chromatin
structure in regulating access to DNA binding sites
ChIP results for analysis GAL4 binding sites
Results: Only approximately 10 of the 4000 potential GAL4 sites
are occupied by GAL4 when the cells are grow-ing on galactose;
More than 99% of the sites appear to be blocked, presumably by
the local chromatin structure.
Conclusion: not like in prokaryotes all sites appear to be equally
accessible, chromatin structure shields a large number of the
potential binding sites in eukaryotic cells. Chromatin structure is
altered in active genes compared with inactive ones.
26
Steroids and related hydrophobic molecules pass through
membranes and bind to DNA-binding receptors
Estrogen is one of hydrophobic
molecules which can pass through
membranes and bind to DNA-binding
receptors (Estrogen receptors).
Estrogen receptors are members of a
large family of proteins that act as
receptors for a wide range of
hydrophobic molecules, including other
steroid hormones, thyroid hormones,
and retinoids.
The human genome encodes
approximately 50 members of this family,
often referred to as nuclear hormone
receptors.
27
Nuclear hormone receptors have a similar mode of action
On binding of the signal molecule (a ligand ), the ligand–receptor
complex modifies the expression of specific genes by binding to
control elements in the DNA.
Estrogen receptors bind to specific DNA sites (referred to as
estrogen response elements or EREs) that contain the
consensus sequence 5’-AGGTCANNNTGACCT -3’.
As expected from the symmetry of this sequence (double chain
shows rotatory symmetry), an estrogen receptor binds to such
sites as a dimer.
28
Nuclear hormone receptors family has a similar structural features
There are two highly conserved domains: a DNA-binding
domain and a ligand-binding domain
DNA-binding domain:
1. Position: toward the Center
2. Consist of a set of zincbased domains
3. Function: bind to specific
DNA sequences by virtue of
an a helix that lies in the
major groove in the specific
DNA complexes formed by
estrogen receptors
29
Nuclear hormone receptors family has a similar structural features
Ligand-binding domain :
1. Position: C terminal
2. Entirely consist of three
layers of  helices
3. Function as ligand binding
in a hydrophobic pocket
that lies in the center of this
array of helices
4. Changing conformation
when ligand binds to
30
Nuclear hormone receptors family has a similar structural features
Ligand-binding domain
How does ligand binding lead to changes
in gene expression?
The binding of ligand
alter the DNA-binding
properties of the
receptor? Like Lac
repressor?
No
31
Nuclear hormone receptors regulate transcription by
recruiting coactivators to the transcription complex
Coactivators
Because ligand binding does not alter the ability of nuclear
hormone receptors to bind DNA, investigators sought to determine
whether specific proteins might bind to the nuclear hormone
receptors only in the presence of ligand. Such searches led to the
identification of several related proteins called coactivators.
p160 family(named from their size): SRC-1 ( steroid receptor
coactivator-1), GRIP-1 ( glucocorticoid receptor interacting
protein-1), and NcoA-1 (n uclear hormone receptor coactivator-1).
How to recruit coactivator for nuclear hormone receptor?
32
Coactivators recruitment
The binding of ligand to a nuclear hormone receptor
induces a conformational change (  helix folds into a
groove on the side of the structure on ligand binding) in the
ligand-binding domain. This change in conformation
generates favorable sites for the binding of a coactivator
Coactivator recruitment
33
Coactivators application in drug design
------Steroid-hormone receptors as targets for drugs
Molecules such as estradiol that bind to a receptor and trigger
signal-ing pathways are called agonists.
eg: Athletes sometimes take natural and synthetic agonists
of the androgen receptor to enhance the development of lean
muscle mass.
Other molecules bind to nuclear hormone receptors but do not
effectively trigger signaling pathways. Such compounds are
called antagonists.
eg. Tamoxifen and raloxifene are used in the treatment
and prevention of breast cancer, because some breast tumors
rely on estrogen-mediated pathways for growth.
34
Coactivators application in drug design
------Mechanism for Tamoxifen anti-breast cacner drugs
Tamoxifen binds to the same site as
estradiol does. However,
tamoxifen has a group that extends out
of the normal ligand-binding pocket,as
do other antagonists. These groups
block the normal conformational
changes (helix 12 cannot pack in it usual
position to form a groove for coactivator
binding) induced by estrogen.
So tamoxifen blocks the binding of
coactivators and inhibits the activation of
gene expression.
35
How do coactivators modulate transcriptional
activity?
Chromatin structure is modulated through covalent
modifications of histone tails
Much of the effectiveness of coactivators appears to result from
their ability to covalently modify the amino-terminal tails of
histones as well as regions on other proteins.
eg. some of the p160 coactivators and the proteins that they
recruit catalyze the transfer of acetyl groups from acetyl CoA to
specific lysine residues in these amino-terminal tails of histone.
Histone acetyltransferases
(HATs)
36
Histone acetyltransferases
(HATs)
The histone tails are readily extended; so they can fit into the
HAT active site and become acetylated
The amino-terminal tail of histone
H3 extends into a pocket in which a
lysine side chain can accept an
acetyl group from acetyl CoA bound
in an adjacent site.
37
The consequences of histone acetylation by HATs
1. Lysine bears a positively charged ammonium group at neutral pH.
The addition of an acetyl group generates an uncharged amide
group. This change dramatically reduces the affinity of the tail for
DNA and modestly decreases the affinity of the entire histone
complex for DNA, loosening the histone complex from the DNA
2. The acetylated lysine residues interact with a specific
acetyllysine-binding domain (bromodomain) that is present in many
proteins that regulate eukaryotic transcription
38
Bromodomain and Bromodomain-containing
Proteins
Bromodomain
Bromodomain, comprises approximately
110 amino acids that form a four-helix
bundle containing a peptide-binding site at
one end.
This four-helix-bundle domain binds
peptides containing acetyllysine.
Notice that an acetylated peptide
from histone H4 is bound in the
structure.
Structure of a
bromodomain
39
Bromodomain-containing Proteins
Bromodomain-containing proteins are components of two large
complexes essential for transcription
1. TAFs(TATA-box-binding protein associated factors)
TAF1 contains a pair of bromodomains which are oriented
such that each can bind one of two acetyllysine residues at
positions 5 and 12 in the histone H4 tail. Thus, acetylation of
the histone tails provides a mechanism for recruiting other
components of the transcriptional machinery
2. Chromatin-remodeling complexes
Utilize the free energy of ATP hydrolysis to shift the positions of
nucleosomes along the DNA and to induce other
conformational changes in chromatin. Histone acetylation can
lead to a reorganization of the chromatin structure, potentially
exposing binding sites for other factors.
40
Bromodomain-containing Proteins
Chromatin-remodeling complexes
Chromatinremodeling
complexes
Chromatin remodeling
41
Summary for histone acetylation on gene
regulation
Histone acetylation can activate transcription through a combination of
three mechanisms by:
1. Reducing the affinity of the histones for DNA.
2. Recruiting other components of the transcriptional machinery.
3. Initiating the remodeling of the chromatin structure.
42
Acetylation is not the only modification of histones
and other proteins in gene-regulation processes.
The relation between various
histone modifications and
their roles in controlling gene
expression is sometimes
referred to as “the histone
code”.
K: lysine
R: Arginine
S:Serine
43
32.4 Eukaryotic Gene Expression Can Be Controlled
at Posttranscriptional Levels
Just as in prokaryotes, gene expression in eukaryotes can be
regulated subsequent to transcription. We shall consider two
examples.
The first is the regulation of genes taking part in iron metabolism
through key features in RNA secondary structure, similar in many
ways to prokaryotic posttranscriptional regulation.
The second provides an entirely new mechanism. Certain small
regulatory RNA molecules allow the regulation of gene expression
through interaction with a range of mRNA molecules.
44
Genes associated with iron metabolism are
translationally regulated in animals
Iron is an essential nutrient ['nju:triənt], required for the synthesis of
hemoglobin, cytochromes, and many other proteins. Excess iron can
be quite harmful to cells.
Animals have evolved sophisticated systems for the accumulation of
iron in times of scarcity and for the safe storage of excess iron for
later use.
RNA secondary structure plays a role in the regulation of iron
metabolism in eukaryotes.
Key proteins include transferrin, a transport protein that carries iron
in the serum, transferrin receptor, a membrane protein that binds
iron-loaded transferrin and initiates its entry into cells, and ferritin,
an impressively efficient iron-storage protein found primarily in the
liver and kidneys.
45
Genes associated with iron metabolism are
translationally regulated in animals
Ferritin and transferrin-receptor expression levels are reciprocally
related in their responses to changes in iron levels.
When iron is scarce, the amount of transferrin receptor increases and
little or no new ferritin is synthesized.
Interestingly, the extent of mRNA synthesis for these proteins does
not change correspondingly. Instead, regulation takes place at the
level of translation.
46
Ferritin
Ferritin mRNA includes a stem-loop structure termed an iron-response
element (IRE) in its 5’ untranslated region. This stem-loop binds a 90-kd
protein, called an IRE-binding protein (IRP), that blocks the initiation
of translation as the IRE binds IRP on the 5’ side of the coding region.
When the iron level increases, the IRP binds iron as a 4Fe-4S cluster.
The IRP bound to iron cannot bind RNA, because the binding sites for
iron and RNA substantially overlap. Thus, in the presence of iron, ferritin
mRNA is released from the IRP and translated to produce ferritin, which
stores the excess iron.
47
Transferrin
Several IRE-like regions are located in 3’ untranslated region on
transferrin-receptor mRNA. Under low-iron conditions, IRP binds
to these IREs. However, given the location of these binding sites (not 5’),
the transferrin-receptor mRNA can still be translated.
When the iron level increases and the IRP no longer binds transferrinreceptor mRNA, freed from the IRP, transferrin-receptor mRNA is rapidly
degraded. Thus, an increase in the cellular iron level leads to the
destruction of transferrin-receptor mRNA and, hence, a reduction in the
production of transferrin-receptor protein.
48
Small RNAs regulate the expression of many
eukaryotic genes
Genetic studies of development in C. elegans revealed that a gene
called lin-4 encodes an RNA molecule 61 nucleotides long that can
regulate the expression of certain other genes. The 61-nucleotide lin-4
RNA does not encode a protein, but rather is cleaved into a 22nucleotide RNA that possesses the regulatory activity. This discovery
was the first view of a large class of regulatory RNA molecules that are
now called microRNAs or miRNAs.
The key to the activity of microRNAs and their specificity for particular
genes is their ability to form Watson–Crick base-pair-stabilized
complexes with the mRNAs of those genes.
49
Small RNAs regulate the expression of many
eukaryotic genes
The miRNAs bind to members of a class of proteins called the Argonaute
family to form Argonaute–miRNA complexes which can then bind
mRNAs that have sequences complementary to the miRNAs. Once
bound, such an mRNA can be cleaved by the Argonaute–miRNA
complex through the action of a magnesium-based active site.
The miRNAs serve as guide RNAs that determine the specificity of the
Argonaute complex
50
MicroRNA–Argonaute
complex
The miRNA (shown in red) is bound by
the Argonaute protein. Notice that
the miRNA serves as a guide that
binds the RNA substrate (shown in
gray) through the formation of a double
helix. Two magnesium ions are shown
in green.
51
Summary for miRNA in gene regulation
This mode of gene regulation is nearly ubiquitous in eukaryotes.
Each miRNA can regulate many different genes because many
different target sequences are present in each mRNA.
60% of all human genes are regulated by one or more miRNAs
The microRNA pathway has tremendous implications for the
evolution of gene-regulatory pathways.
52