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Regulation
of Gene Expression
in Eukaryotes
Key Questions
The MSL complex enhances gene expression on the X chromosome. The MSL complex
(indicated by orange coloring) binds only to the X chromosome in male Drosophila. This image
is an indirect immunofluorescence staining of a chromosomal spread from a salivary gland of
a male larva exposed to MSL1 antiserum. [From J. Lucchesi, W. Kelly, and B. Panning, Chromatin
Remodeling in Dosage Compensation, Annu. Rev. Genet. 39, 2005, 615651.]
Outline
he cloning of Dolly, a sheep, was reported worldwide in 1996. Dolly developed
from adult somatic nuclei that had been implanted into enucleated eggs (eggs
with the nuclei removed). More recently, cows, pigs, mice, and other mammals
have been cloned as well with the use of similar technology (Figure 11-1). The successful cloning of Dolly was a great surprise to the scientific community because
the cloning of mammals from somatic cells was thought to be impossible. A reason
for the initial skepticism was that the formation of male and female gametes
(sperm and egg cells) was known to include sex-specific modifications to the
respective genomes that resulted in sex-specific patterns of gene expression. As
such, Dolly is symbolic of how far we have progressed in understanding aspects of
eukaryotic gene regulation such as the global control of gene expression exemplified by gamete development. However, for every successful clone, including Dolly,
there are many more, perhaps hundreds of embryos that fail to develop into viable
progeny. The extremely high failure rate underscores how much remains to be
deciphered about eukaryotic gene regulation.
385
386
387
EUKARYOTIC
Activator
protein
RNA pol
TATA
Promoter
Operator
Coding
region
Ground state: on
Enhancer
Transcription
factors
Repressor
protein
RNA pol II
TATA
Repressed state: off
Active state: on
388
polymerase II, which transcribes mRNAs, was the focus of Chapter 8 and
will be the only polymerase discussed in this chapter.
2. RNA transcripts are extensively processed during transcription in
eukaryotes; the 5 and 3 ends are modified and introns are spliced out.
3. RNA polymerase II is much larger and more complex than its bacterial
counterpart. One reason for the added complexity is that RNA polymerase II
must synthesize RNA and coordinate the special processing events unique
to eukaryotes.
Multicellular eukaryotes may have as many as 25,000 genes, severalfold more
than the average bacterium. Moreover, patterns of eukaryotic gene expression can
be extraordinarily complex. That is, there is great variation among genes in when a
gene is on (transcribed) or off (not transcribed) and in how much transcript needs
to be made. For example, one gene may be transcribed only during early development and another only in the presence of a viral infection. Finally, the majority of
the genes in a eukaryotic cell are off at any one time. On the basis of these considerations alone, eukaryotic gene regulation must be able to
1. ensure that the expression of most genes in the genome is off at any one
time while activating a subset of genes; and
2. generate thousands of patterns of gene expression.
As you will see later in the chapter, mechanisms have evolved to ensure that
most of the genes in a eukaryotic cell are not transcribed. Before considering how
genes are kept transcriptionally inactive, we will focus on the second point: How
are eukaryotic genes able to exhibit an enormous number and diversity of expression patterns? The machinery required for generating so many patterns of gene
transcription in vivo has many components, including both regulatory proteins
and cis-acting regulatory sequences. The first set of proteins comprises the large
RNA polymerase II complex and the general transcription factors that you learned
about in Chapter 8. To initiate transcription, these proteins interact with DNA
sequences called promoter-proximal elements near the promoter of a gene. The
second group of protein components consists of specific transcription factors that
bind to cis-acting regulatory sequences in the DNA called enhancers or upstream
activating sequences (UASs). These regulatory sequences may be located a
considerable distance from gene promoters. Generally speaking, promoters and
promoter-proximal elements are bound by transcription factors that affect the
expression of many genes. Enhancers are the targets of more specific transcription
factors that control the regulation of smaller subsets of genes. Often, an enhancer
will act in only one or a few cell types in a multicellular eukaryote.
For RNA polymerase II to transcribe DNA into RNA at
a maximum rate, multiple cis-acting regulatory elements
Promoter-proximal elements precede
must play a part. The promoters, promoter-proximal elethe promoter of a eukaryotic gene
ments, and enhancers are all targets for binding by different
GC-rich
trans-acting DNA binding proteins. Figure 11-3 is a schematic
mRNA
box
representation of the promoter and promoter-proximal seCCAAT
TATA
GGGCGG
quence elements. The binding of RNA polymerase II to the
200 bp
100 bp
30 bp
promoter does not produce efficient transcription by itself.
Transcription requires the binding of general transcription
Promoter-proximal
Promoter
factors to additional promoter-proximal elements that are
elements
commonly found within 100 bp of the transcription initiation
site
of
many
(but
not
all)
genes. One of these elements is the CCAAT box, and often
FIGURE 11-3 The region upstream of
another is a GC-rich segment farther upstream. The general transcription factors
the transcription start site in higher
that bind to the promoter-proximal elements are expressed in most cells, and so
eukaryotes contains promoter-proximal
elements and the promoter.
they are available to initiate transcription at any time. Mutations in these sites can
389
3.5
3.0
1.0
GCCACACCC
GGCCAATC
ATATAA
FIGURE 11-4 Point mutations in the promoter and promoter-proximal elements hinder
transcription of the -globin gene. Point mutations throughout the promoter region were
analyzed for their effects on transcription rates. The height of each line represents the
transcription level relative to a wild-type promoter or promoter-proximal element (1.0).
Only the base substitutions that lie within the three elements shown change the level of
transcription. Positions with black dots were not tested. [From T. Maniatis, S. Goodbourn, and
J. A. Fischer, Regulation of Inducible and Tissue-Specific Gene Expression, Science 236, 1987, 1237.]
390
Fusion
(n)
(n)
(2n)
a /a
Mitosis
+
Meiosis
Ascus
( n)
(n )
a /a
(2n)
a /a
(2n)
a (n)
a (n)
Mitosis
Mitosis
(n )
(n) a
Culture
colony
Culture
colony
The life cycle of bakers yeast. The nuclear alleles MATa and MATa
determine mating type.
391
the level of gene expression in the biochemical pathway. In yeast cells growing in
media lacking galactose, the GAL genes are largely silent. But, in the presence of
galactose (and the absence of glucose), the GAL genes are induced. Just as for the
lac operon, genetic and molecular analyses of mutants have been key to understanding how the expression of the genes in the galactose pathway is controlled.
The key regulator of GAL gene expression is the Gal4 protein, a sequencespecific DNA-binding protein. Gal4 is perhaps the best-studied transcriptional regulatory protein in eukaryotes. The detailed dissection of its regulation and activity has been a source of several key insights into the control of transcription in
eukaryotes.
UDP-galactose
In the presence of galactose, the GAL1, GAL2, GAL7, and GAL10 genes are induced
1000-fold or more. In GAL4 mutants, however, they remain silent. Each of these
four genes has two or more Gal4-binding sites located 5 (upstream) of its promoter. Consider the GAL10 and GAL1 genes, which are adjacent to each other and
transcribed in opposite directions. Between the GAL1 transcription start site and
the GAL10 transcription start site is a single 118-bp region that contains four Gal4binding sites (Figure 11-6). Each Gal4-binding site is 17 base pairs long and is
bound by one Gal4 protein dimer. There are two Gal4-binding sites upstream of
the GAL2 gene as well, and another two upstream of the GAL7 gene. These binding sites are required for gene activation in vivo. If they are deleted, the genes are
silent, even in the presence of galactose. These regulatory sequences are enhancers
that are also referred to as upstream activating sequences. The presence of enhancers located at a considerable linear distance from a eukaryotic genes promoter is typical.
Gal10
UDP-glucose
Gal7
Glucose-1-phosphate
Glycosis
Gal4
Chr II
GAL7
GAL10
UAS
GAL1
Chr XII 5
UAS
FIGURE 11-6 The Gal4 protein activates target genes through upstream-activating-sequence
(UAS) elements. The Gal4 protein has two functional domains: a DNA-binding domain (red
square) and an activation domain (orange oval). The protein binds to specific sequences
upstream of the promoters of Gal-pathway genes. Some of the GAL genes are adjacent (GAL1,
GAL10), whereas others are on different chromosomes. The GAL1 UAS element contains four
Gal4-binding sites.
GAL2
UAS
392
393
Active
Gal4
GAL1
ON
UAS
394
Mediator
TFIID
RNA polymerase II
TBP
TATA
domain, and, through this binding, it recruits the TFIID complex and, in turn, RNA polymerase II to the promoter (Figure
11-9). The affinity of this interaction correlates well with
Gal4s potency as an activator. Gal4 also interacts with the
large Mediator complex, which directly interacts with RNA
polymerase II to recruit it to gene promoters. The Mediator
complex is an example of a coactivator, a term applied to a
protein or protein complex that facilitates gene activation by
a transcription factor but that itself is neither part of the transcriptional machinery nor a DNA-binding protein.
The ability of activators to bind to upstream DNA
sequences and to interact with proteins that bind directly or
indirectly to promoters helps to explain how transcription
can be stimulated from more distant regulatory sequences
(see Figure 11-9).
GAL genes
395
(b)
Short region of
DNA double helix
2 nm
Nucleosomes:
the basic unit
of chromatin
11 nm
Chromatin fiber
of packed
nucleosomes
30 nm
nucleosome positions can shift on the DNA from cell to cell and over the life cycle
of an organism. Transcription might be repressed when the promoter and flanking
sequences are wound up in a nucleosome and inaccessible to RNA polymerase II.
Activation of transcription would thus require the blocking nucleosome to be reorganized by nudging the histones or removing them entirely. Conversely, when gene
repression is necessary, histone octamers may shift into a position that prevents
transcription. The changing of nucleosome position is referred to as chromatin
remodeling. Now, chromatin remodeling is known to be an integral part of
eukaryotic gene expression, and great advances are being made in determining the
underlying mechanism(s) and the regulatory proteins taking part. Here, again,
genetic studies in yeast have been pivotal.
Chromatin remodeling
exposes regulatory
sequences
Nucleosome
remodeling
396
Gal4 also binds to the SWISNF complex and recruits the chromatin-remodeling
complex to activated promoters. Yeast strains containing a defective SWISNF
complex show a reduced level of Gal4 activity. Why might an activator use multiple
activation mechanisms? There are at least two reasons understood at present. The
first is that the accessibility of target promoters may change at different stages of
the cell cycle or in different cell types (in multicellular eukaryotes). For example,
during mitosis, when chromatin is more condensed, genes are less accessible. At
that stage, Gal4 must recruit the chromatin-remodeling complexes, whereas, at
other times, such recruitment might not be required to activate gene expression.
A second reason is that many transcription factors act in combinations to control gene expression synergistically. We will see shortly that this combinatorial
synergy is a result of the fact that chromatin-remodeling complexes and the transcriptional machinery are recruited more efficiently when multiple transcription
factors act together.
H4
Acetylation
Methylation
H2A
H3
H2A
H3
H4
397
NH3
Amino group at end
of lysine side chain
CoA
S
C
CH3
Acetyl CoA
N
H
Acetyl group
CH3
Note that the reaction is reversible, which means that acetyl groups can be
added and removed from the same histone residue. With 44 histone lysine residues
available to accept acetyl groups, the presence or absence of these groups can carry
a tremendous amount of information. For this reason, the covalent modification of
histone tails is said to be a histone code. Scientists coined the expression histone
code because the covalent modification of histone tails is reminiscent of the genetic
code. For the histone code, information is stored in the patterns of histone modification rather than in the sequence of nucleotides. With more than 150 known histone modifications, there are a huge number of possible patterns and their effects
on chromatin structure and transcriptional regulation are just beginning to be deciphered. To add to this complexity, the code is likely not interpreted in precisely the
same way in all organisms. For now, lets see how the acetylation of histone amino
acids influences chromatin structure and gene expression.
Evidence had been accumulating for years that the histones associated with the
nucleosomes of active genes are rich in acetyl groups (said to be hyperacetylated),
whereas inactive genes are underacetylated (hypoacetylated). The enzyme responsible for adding acetyl groups, histone acetyltransferase (HAT), proved very
difficult to isolate. When it was finally isolated and its protein sequence deduced, it
was found to be an ortholog of a yeast transcriptional activator called GCN5 (meaning that it was encoded by the same gene in a different organism). Thus, the conclusion was that GCN5 is a histone acetyltransferase. It binds to the DNA in the regulatory regions of some genes and activates transcription by acetylating nearby
histones. Various protein complexes that are recruited by transcriptional activators
are now understood to possess a HAT activity.
How does histone acetylation facilitate changes in gene expression? There
appear to be at least two mechanisms for doing so. First, the addition of acetyl
groups to specific histone residues can alter the interaction in a nucleosome
between the DNA and a histone octamer so that the octamer is
more likely to slide along the DNA to a new position. Second,
Histone deacetylation can turn
histone acetylation, in conjunction with other histone modificaoff gene transcription
tions, influences the binding of regulatory proteins to the DNA.
Gal4
The bound regulatory protein may take part in one of several
functions that either directly or indirectly increase the freTup1
quency of transcription initiation.
Mig1
GAL1
Like other histone modifications, acetylation is reversible,
OFF
and histone deacetylases (HDATs) also have been identified.
UAS
Mig1
Such proteins play key roles in gene repression. For example, in
site
the presence of galactose and glucose, the activation of GAL
FIGURE 11-13 Recruitment of a
genes is prevented by the Mig1 protein. Mig1 is a sequence-specific DNA-binding
repressing complex leads to repression
repressor that binds to a site between the UAS element and the promoter of the
of transcription. In the presence of
GAL1 gene (Figure 11-13). Mig1 recruits a protein complex called Tup1 that conglucose, GAL1 transcription is repressed
tains a histone deacetylase and that represses gene transcription. The Tup1 comby the Mig1 protein, which binds to a
plex is an example of a corepressor, which faciliates gene repression but is not
site between the UAS and the promoter
itself a DNA-binding repressor. The Tup1 complex is also recruited by other yeast
of the GAL1 gene. Mig1 recruits the Tup1
repressors, such as MAT2 (see page 400), and counterparts of this complex are
repressing complex, which recruits a
histone deacetylase, turning gene
found in all eukaryotes.
398
DNAbending
proteins
The development of a complex organism requires that transcription levels be regulated over a wide range. Think of a regulation mechanism as more like a rheostat
than an on-or-off switch. In eukaryotes, transcription levels are made finely
adjustable by the clustering of binding sites into enhancers. Several different transcription factors or several molecules of the same transcription factor may bind to
adjacent sites. The binding of these factors to sites that are
Enhanceosomes help recruit
the correct distance apart leads to an amplified, or superthe transcriptional machinery
additive, effect on activating transcription. When an effect
is greater than additive, it is said to be synergistic.
The binding of multiple regulatory proteins to the
multiple binding sites in an enhancer can catalyze the formation of an enhanceosome, a large protein complex that
acts synergistically to activate transcription. In Figure 11-14,
you can see how architectural proteins bend the DNA to
promote cooperative interactions between the other DNACBP
binding proteins. In this mode of enhanceosome action,
transcription is activated to very high levels only when all
RNA pol II
the proteins are present and touching one another in just
the right way.
To better understand what an enhanceosome is and
how it acts synergistically, lets look at a specific example.
399
example shown in Figure 11-15. One characteristic of enhancers is that they can activate transcription when they are
located at great distances from the promoter (>50 kb), either
upstream or downstream from a gene or even in an intron.
Enhanceosome
nuc 1
nuc 2
The enhanceosome forms
a binding site for GCN5, which
binds and adds acetyl
groups to nuc 1, 2.
GCN5
GCN5
complex
RNA pol II
CBP
SWI
SNF
RNA pol II
CBP
TBP
400
MAT locus, but their protein products are required for mating. One group of structural genes is expressed only in the ` cell type (`-specific genes), and another set is
expressed only in the a cell type (a-specific genes). The different alleles of the
MAT locus encode different regulatory proteins that control which of these sets of
structural genes is expressed in each cell type. In addition, a regulatory protein not
encoded by the MAT locus, called MCM1, plays a key role in regulating cell type.
The simplest case is the a cell type (Figure 11-16a). The MATa locus encodes a
single regulatory protein, a1. However, this regulatory protein has no effect in haploid cells, only in diploid cells. In a haploid a cell, the regulatory protein MCM1
turns on the expression of the structural genes needed by an a cell, by binding to
regulatory sequences within a-specific gene promoters.
a1
2
Expressed
regulatory
proteins
a-specific
genes
a1
MCM1
MCM1
2
MCM1
a1
1
MCM1
ON
OFF
MCM1
OFF
MCM1
-specific
genes
OFF
ON
OFF
MCM1
a1 2
Haploid-specific
genes
ON
(a) a cell
ON
(b) cell
OFF
(c) a/ cell
In an ` cell, the `-specific structural genes must be transcribed, but, in addition, the MCM1 protein must be prevented from activating the a-specific genes.
The DNA sequence of the MATa allele encodes two proteins, 1 and 2, that are
produced by separate transcription units. These two proteins have different regulatory roles in the ` cell, as can be demonstrated by analyzing their DNA-binding
properties in vitro (Figure 11-16b). The 1 protein binds in concert with the MCM1
protein to a discrete DNA sequence controlling several `-specific genes. Thus, 1 is
an activator of `-specific gene expression. The 2 protein represses transcription of
the a-specific genes. It binds as a dimer, with MCM1, to sites in DNA sequences
located 5 of a group of a-specific genes and acts as a repressor.
In a diploid yeast cell, all three regulatory proteins encoded by the MAT locus
are expressed (Figure 11-16c). What is the result? The a1 protein encoded by MATa
has a part to play at last. The a1 protein can bind to 2 and alter its binding specificity such that the a12 complex does not bind to a-specific genes. Rather, the
a12 complex binds to a different sequence found upstream of another set of
genes, called haploid specific, that are expressed in haploid cells but not diploid
cells. In diploid cells, then, 2 exists in two forms: (1) as an 2MCM1 complex that
represses a-specific genes and (2) in a complex with a1 that represses haploid-spe-
401
ON
OFF
Promoter 2
Enhancer Enhancerblocking
insulator
Promoter 1
cific genes. The different binding partners determine which specific DNA sequences are bound and which genes are regulated by each 2-containing complex.
The regulation of different sets of target genes by the association of the same transcription factor with different binding partners plays a major role in the generation
of different patterns of gene expression in different cell types within multicellular
eukaryotes.
Enhancer-blocking insulators
A regulatory element, such as an enhancer, that can act over tens of thousands of
base pairs could interfere with the regulation of nearby genes. To prevent such
promiscuous activation, regulatory elements called enhancer-blocking insulators
have evolved. When positioned between an enhancer and a promoter, enhancerblocking insulators prevent the enhancer from activating transcription at that promoter. Such insulators have no effect on the activation of other promoters that are
not separated from their enhancers by the insulator (Figure 11-17). Several models
have been proposed to explain how an insulator could block enhancer activity only
when placed between an enhancer and a promoter. Many of the models, like the
one shown in Figure 11-18, propose that the DNA is organized into loops containing
ON
Promoter 1
Promoter 2
OFF
Enhancer
402
active genes. According to this model, insulators act by moving a promoter into a
new loop, where it is shielded from the enhancer.
As you will see next, enhancer-blocking insulators are a fundamental component of a phenomenon called genomic imprinting.
NH2
C
Maternal allele
N3
C2
O
CTCF
Igf2
OFF
ICR
Paternal allele
M M M M
Igf2
ON
>50 Kb
ICR
5 C Methyltransferase
6C
N3
C2
O
5C
6C
CH3
Cytosine
>50 Kb
NH2
403
bind to the ICR and the enhancer can activate Igf2 transcription (recall that
enhancers can act at great distances). The enhancer cannot activate H19, however,
because the methylated region extends into the H19 promoter. The methylated
promoter cannot bind proteins needed for the transcription of H19.
Thus, we see how an enhancer-blocking insulator (in this case, CTCF bound to
part of the ICR) prevents the enhancer from activating a distant gene (in this case,
Igf2). Furthermore, we see that the CTCF-binding site is methylated only in chromosomes derived from the male parent. The methylation of the CTCF-binding site
prevents CTCF binding in males and permits the enhancer to activate Igf2.
Note that parental imprinting can greatly affect pedigree analysis. Because the
inherited allele from one parent is inactive, a mutation in the allele inherited from
the other parent will appear to be dominant, whereas, in fact, the allele is expressed because only one of the two homologs is active for this gene. Figure 11-20
shows how a mutation in an imprinted gene can have different outcomes on the
phenotype of the organism if inherited from the male or from the female parent.
Many steps are required for imprinting (Figure 11-21). Soon after fertilization, mammals set aside cells that will become their germ cells. Imprints are
removed or erased before the germ cells form. Without their distinguishing mark
of DNA methylation, these genes are now said to be epigenetically equivalent. As
these primordial germ cells become fully formed gametes, imprinted genes receive the sex-specific mark that will determine whether the gene will be active or
silent after fertilization.
Unusual inheritance
of imprinted genes
No mutations
B
M
B
ICR
B
M
B
ICR
OUTCOME UNAFFECTED
B
M
Male
Homologous
chromosomes
Female
Igf2
H19
B
ICR
OUTCOME AFFECTED
Igf2
H19
Primordial
germ cells
1 Imprints
erased
Primordial
germ cells
2 Imprints
initiated
Gametes
Sperm
3 Propagation
of imprints
Oocyte
Silent allele
Fertilization and
development
Active allele
404
405
MATa
Active
HMRa
Silent
a mating type
HML
MAT
HMRa
Silent
a mating type
HML
Silent
MATa
HMRa
a mating type
Gene silencing is a very different process from gene repression; silencing is a position effect that depends on the neighborhood in which genetic information is
located. You will learn more about position effects later, in the section on positioneffect variegation in the fruit fly Drosophila melanogaster.
In summary, there are two distinct levels in the control of yeast mating type.
First, the regulation of a DNA rearrangement controls the array of regulatory
products synthesized within the cell. Second, the DNA-binding activities of these
regulatory proteins (a1, 1, and 2) control the batteries of structural genes
expressed within each cell type. These two levels form a hierarchy: the genes of the
first level control the activation of genes on the second level, which in turn control
the activation of the structural genes. These structural genes encode the proteins
having roles in the actual mating process and the biology of each cell type. As we
shall see in regard to animals in Chapter 12, the genetic control of developmental
processes is often hierarchical: networks of regulatory genes set up the cell- and
tissue-specific expression of proteins that mediate cell behavior and function.
406
especially around the centromeres and telomeres (called constitutive heterochromatin), whereas the regions forming euchromatin become less condensed.
Geneticists first suspected a limited role for the influence of chromatin structure on gene regulation early in the history of genetics. At that time, they noticed
that heterochromatic DNA contained few genes, whereas euchromatin was rich
in genes. But what is heterochromatin if not genes? Most of the eukaryotic
genome is composed of repetitive sequences that do not make protein or structural RNAsometimes called junk DNA (see Chapter 14). Thus, the densely
packed nucleosomes of heterochromatin were said to form a closed structure
that was inaccessible to regulatory proteins and inhospitable to gene activity. In
contrast, euchromatin, with its more widely spaced nucleosomes, was proposed to
assume an open structure that permitted transcription. The existence of open
and closed regions of chromatin was also suggested as a reason that recombination
frequencies are 100- to 1000-fold higher in euchromatin compared with heterochromatin. Euchromatin, with its more open conformation, was hypothesized to
be more accessible to proteins needed for DNA recombination.
407
Chromosome
white+
white+ gene
expressed
Centromere
Telomere
Wild-type eye
white+
Red facet
white+ gene
expressed
white+
White facet
white+
silent
gene
Eye is a mixture
of red and white
facets.
Heterochromatin spreads
is able to regulate the expression of genesin this case, determining whether genes
with identical DNA sequence will be active or silenced.
Message Active genes that are relocated to genomic neighborhoods that are
heterochromatic may be silenced if the heterochromatin spreads to the genes.
408
Second-site
mutations that affect
the spreading of
heterochromatin
Drosophila eye
(translocated white+)
Spreading
enhanced.
More white+
are silenced.
E(var)
Spreading
suppressed.
Fewer white+
are silenced.
Su(var)
adds methyl groups to a specific amino acid residue in the tail of histone H3
(called histone H3 methyltransferase or HMTase). One of the reactions catalyzed
by HMTase is shown here:
COO
+
H3N
COO
HMTase
H3N
(CH2)4
HMTase
H3N
(CH2)4
NH3
H3C
COO
HMTase
H3N
(CH2)4
N+
Lysine
COO
Monomethyl lysine
H3C
(CH2)4
N+
H
N+
CH3
Dimethyl lysine
H3C
CH3
CH3
Trimethyl lysine
Proteins similar to HP-1 and HMTase have been isolated in diverse taxa, suggesting
the conservation of an important eukaryotic function.
We have seen that actively transcribed regions are associated with nucleosomes whose histone tails are hyperacetylated and that transcriptional activators
such as GCN5 encode a histone acetytransferase activity. As heretofore discussed,
acetyl marks can also be removed from histones by histone deacetylases. Similarly,
chromatin made up of nucleosomes that are methylated at lysine 9 of H3 (called
H3meK9) and bound up with HP-1 protein contain epigenetic marks that are associated with heterochromatin. Scientists are now able to separate heterochromatin
and euchromatin and analyze differences in histone modifications and bound pro-
409
HP-1
M
HAT
M
Ac
Ac
Heterochromatin
Ac
Euchromatin
Barrier
insulator
Ac
410
Different mechanisms of
dosage compensation
Female
Male
Hypertranscription (Drosophila)
XX
1
X inactivation (mammals)
X
1
XY
1
1
2
1
2
No Y
all progeny cells. (In the germ line, the second X chromosome becomes reactivated
in oogenesis). The inactivated chromosome, called a Barr body, can be seen in the
nucleus as a darkly staining, highly condensed, heterochromatic structure.
Two aspects of X-chromosome inactivation are relevant to a discussion of
chromatin and the regulation of gene expression. First, most of the genes on the
inactivated X chromosome are silenced, and the chromosome has epigenetic
marks associated with heterochromatin including methylation of H3 at lysine 9
and hypermethylation of its DNA. Second, genes on the inactivated chromosome
remain inactive in all descendants of these cells. Because the DNA sequence itself
is unchanged, this heritable alteration is an example of epigenetic inheritance.
Interestingly, although diverse taxa exhibit dosage compensation, the compensation mechanism can differ dramatically. For example, in fruit flies, the expression of genes on the X chromosome is compensated not by inactivating one of the
two Xs in females, but instead, by doubling the expression of the genes on the one
X in the male (Figure 11-27). This mechanism is characterized by the binding of a
RNAprotein complex, called MSL, along the entire length of the X chromosome
in males (see illustration on page 385). One of the components of the MSL complex is a histone acetyltransferase. Recall that acetylated histones are a main feature of active chromatin. Thus, the function of the MSL complex appears to be to
add acetyl groups to histones. MSL stands for male-specific lethal, and the complex
was so named because genetic screens for mutations lethal to males identified its
components.
Message For most diploid organisms, both alleles of a gene are expressed
independently. X inactivation and genomic imprinting are examples of monoallelic
expression. In these cases, epigenetic mechanisms silence one copy of an entire
chromosome or of a single chromosomal locus, respectively.
Nucleosome
Replication
Newly synthesized
histones, no histone code
Histones with code
411
Summary
them in both the parental and the daughter strands. This process is accomplished
by the random distribution of the old histones from existing nucleosomes to
daughter strands and the delivery of new histones to the replisome. In this way, the
old histones with their modified tails and the new histones with unmodified tails
are assembled into nucleosomes that become associated with both daughter
strands. The code carried by the old histones most likely guides the modification of
the new histones (Figure 11-28).
The inheritance of DNA methylation is better understood. Semiconservative
replication generates daughter helices that are methylated on one of their two
strands (the parental strand). The unmethylated strands are methylated by DNA
methyltransferases that have a high affinity for these so-called hemimethylated
substrates and are guided by the methylation pattern on the parental strand (Figure 11-29). Thus, the information inherent in the histone code and the existing
DNA methylation patterns serve to reconstitute the local chromatin structure that
existed before DNA synthesis and mitosis.
Methylated
DNA
methyltransferase
MCpG
MCpG
GpC
GpCM
MCpG
GpCM
CpG
GpCM
MCpG
GpCM
Summary
Many aspects of eukaryotic gene regulation resemble the regulation of bacterial operons. Both operate largely at the level
of transcription, and both rely on trans-acting proteins that
bind to cis-acting regulatory target sequences on the DNA
molecule. These regulatory proteins determine the level of
transcription from a gene by controlling the binding of RNA
polymerase to the genes promoter.
There are three major distinguishing features of the control of transcription in eukaryotes. First, eukaryotic genes possess enhancers, which are cis-acting regulatory elements located at sometimes great linear distances from the promoter.
Many genes possess multiple enhancers. Second, these enhancers are often bound by more transcription factors than
are bacterial operons. Multicellular eukaryotes must generate
thousands of patterns of gene expression with a limited number of regulatory proteins (transcription factors). They do so
through combinatorial interactions among transcription factors. Enhanceosomes are complexes of regulatory proteins
that interact in a cooperative and synergistic fashion to pro-
412
Key Terms
activation domain (p. 392)
Problems
BASIC PROBLEMS
1. What analogies can you draw between transcriptional
trans-acting factors that activate gene expression in
eukaryotes and the corresponding factors in bacteria?
Give an example.
2. Contrast the states of genes in bacteria and eukaryotes
with respect to gene activation.
3. Predict and explain the effect on GAL1 transcription,
in the presence of galactose alone, of the following
mutations:
a. Deletion of one Gal4-binding site in the GAL1 UAS
element.
b. Deletion of all four Gal4-binding sites in the GAL1
UAS element.
c. Deletion of the Mig1-binding site upstream of GAL1.
d. Deletion of the Gal4 activation domain.
e. Deletion of the GAL80 gene.
413
Problems
Enhancer Promoter
Enhancer
22. You receive four strains of yeast in the mail and the
accompanying instructions state that each strain contains a single copy of transgene A. You grow the four
strains and determine that only three strains express the
protein product of transgene A. Further analysis reveals
that transgene A is located at a different position in the
yeast genome in each of the four strains. Provide an
hypothesis to explain this result.
23. In Neurospora, all mutants affecting the enzymes carbamyl phosphate synthetase and aspartate transcarbamylase map at the pyr-3 locus. If you induce pyr-3
mutations by ICR-170 (a chemical mutagen), you find
that either both enzyme functions are lacking or only
the transcarbamylase function is lacking; in no case is
the synthetase activity lacking when the transcarbamylase activity is present. (ICR-170 is assumed to induce
frameshifts.) Interpret these results in regard to a possible operon.
24. You wish to find the cis-acting regulatory DNA elements responsible for the transcriptional responses of
two genes, c-fos and globin. Transcription of the c-fos
gene is activated in response to fibroblast growth factor
(FGF), but it is inhibited by cortisol (Cort). On the
other hand, transcription of the globin gene is not
affected by either FGF or cortisol, but it is stimulated
by the hormone erythropoietin (EP). To find the cisacting regulatory DNA elements responsible for these
transcriptional responses, you use the following clones
of the c-fos and globin genes, as well as two hybrid
combinations (fusion genes), as shown in diagram 1.
The letter A represents the intact c-fos gene, D represents the intact globin gene, and B and C represent the
c-fosglobin gene fusions. The c-fos and globin exons (E)
and introns (I) are numbered. For example, E3(f) is the
third exon of the c-fos gene and I2(g) is the second
intron of the globin gene. (These labels are provided to
help you make your answer clear.) The transcription
start sites (black arrows) and polyadenylation sites (red
arrows) are indicated.
p E1(f)
A
E2(f)
I1(f)
E3(f)
I2(f)
p
C
I1(g)
I2(g)
D
E1(g)
Diagram 1.
E2(g)
E3(g)
414
FGF
Cort
EP
Clone
A
B
from the introduced genes in response to various treatments are shown; the intensity of these bands is proportional to the amount of transcript made from a particular clone. (The failure of a band to appear indicates
that the level of transcript is undetectable.)
a. Where is the DNA element that permits activation
by FGF?
b. Where is the DNA element that permits repression
by Cort?
C
D
Diagram 2.