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Chapter 17 Gene regulation in
• Many eukaryotic genes have more regulatory
binding sites and are controlled by more regulatory
proteins than are typical bacterial genes.
• An enhancer binds regulators responsible for
activating the gene at the given time and place.
• Insulators block activation of the promoter by
activators bound at the enhancer.
• A silencer binds regulators responsible for
repressing the gene at the given time and place.
Activator have separate DNA binding and activating
functions. The two surfaces are very often on separate
domains of the protein.
Gal4 binds to four sites located 275 bp upstream of GAL1. When
bound there, in the presence of galactose, Gal4 activates
transcription of the GAL1 gene 1000-fold.
Domain swap experiments
A reporter gene
consists of binding sites
for the yeast activator
inserted upstream of the
promoter of a gene
whose expression level
is readily measured.
Lex A, a bacterial
• In some cases, DNA binding domain and
activating domains are even carried on
separate polypeptides, and form a complex
on DNA.
• Herpes virus activator VP16 interacts with
the Oct1 DNA-binding protein found in
cells infected by this virus.
The two hybrid assay
Eukaryotic regulators use a range of DNAbinding domains, but DNA recognition
involves the same principles as found in
• There is no fundamental difference in the ways
DNA-binding proteins from different organisms
recognize their sites.
• Eukaryotic regulators often bind as dimers and
recognize specific DNA sequences using an αhelix
inserted into the major groove.
• Some regulators in eukaryotes bind DNA as
Homeodomain is a class of helix-turn-helix DNA-binding domain
and recognizes DNA in essentially the same way as those bacterial
proteins. They were discovered in Drosophila where they control
many basic developmental programs.
Zinc Containing DNA-binding domains are various different
forms of DNA-binding domain that incorporate a zinc atom(s). A
classically defined zinc finger protein: TFIIIA
Leucine zipper motif combines dimerization and DNA-binding
surfaces within a single structural unit. Leucine-zipper-containing
proteins often form heterodimers as well as homodimers.
Helix-loop-helix proteins: The two helices are separated by a
flexible loop that allows them to pack together. Leucine zipper
and HLH proteins are often called basic zipper and basic HLH
proteins: the region of the αhelix that binds DNA contains basic
amino acid residues.
Activating regions are not welldefined structures
• Activating regions are grouped on the basis
of amino acid content.
• Acidic activating region: Gal 4
• Glutamine-rich activating region: Sp1
• Proline-rich activating region: CTF-1
Recruitment protein complexes to genes
by eukaryotic activators
• The activators recruit polymerase indirectly in two
ways: the activator interacts with parts of the
transcription machinery other than polymerase;
activators can recruit nucleosome modifiers that
alter chromatin in the vicinity of a gene and
thereby help polymerase bind.
• The eukaryotic transcriptional machinery contains
numerous proteins in addition to RNA polymerase.
• Many of these proteins come in preformed
complexes such as the Mediator and the TFIID
In vivo, transcription Initiation requires
additional Proteins, including the
mediator complex
• The high, regulated levels of transcription in vivo
require the Mediator Complex, transcriptional
regulatory proteins and in many cases,
nucleosome-modifying enzymes.
• Mediator is associated with the CTD “tail” of the
large polymerase subunit through one surface,
while presenting other surfaces for interaction
with DNA-bound activators.
Different Mediator subunits to bring polymerase to different genes.
The need for nucleosome modifiers and remodellers also differs at
different promoters.
Mediator consists of many subunits, some
conserved from yeast to human
• There are various forms of
Mediator, each containing
subsets of Mediator
• A complex consisting of
Pol II, Mediator, and a
some of the general
transcription factors can
be isolated from cells as a
single complex in the
absence of DNA---RNA
Pol II holoenzyme.
Activation of transcription initiation in eukaryotes by
recruitment of the transcription machinery
Activation of transcription through direct tethering of mediator
to DNA. The Gal 1 gene can be activated equally well by a
fusion protein containing the DNA-binding domain of the
bacterial protein Lex A fused directly to a component of the
Mediator Complex.
Box 17-2 Chromatin Immunoprecipitation
Recruitment can be visualized using ChIP
Activators also recruit nucleosome
modifiers that help the transcription
machinery bind at the promoter
• Recruitment of nucleosome modifiers can
help activate a gene inaccessibly packaged
within chromatin.
• Histone acetyltransferases (HATs): add
chemical groups to the tails of histone
• ATP-dependent activity of SWI/SNF:
remodel the nucleosomes
Local alterations in chromatin structure directed by activators
Action at a distance: Loops and Insulators
• Many eukaryotic activators- particularly in higher eukaryotes- work from
a distance.
• IHF, an architectural protein, binds to DNA and by bending the DNA IHF
helps the DNA-bound activator reach RNA polymerase at the promoter.
• Specific elements called insulators control the actions of activators.
Appropriate regulation of some groups of
genes requires locus control regions
• A group of regulatory elements collectively called the locus control region,
or LCR, is found 30-50 kb upstream of the whole cluster of globin genes.
• The simplest explanation is that regulatory proteins binds to the LCR and
recruit chromatin modifying complexes to the region.
Regulation by LCRs
Signal integration and combinatorial
• Activators work together synergistically to
integrate signals
• When multiple activators work together, they do
so synergistically. That is, the effect of two
activators working together is greater (usually
much greater) than the sum of each of them
working alone.
• Synergy can also result from activators helping
each other bind under conditions where the
binding of one depends on binding of the other.
Cooperative binding of activators
Signal integration
• The HO gene, expressed only in mother cells when yeast divides by
budding, is controlled by two regulators; one recruits nucleosome
modifiers and the other recruits mediator.
• If both activators are present and active, the action of SWI5 enables
SBF to bind and activate the transcription of the HO gene.
SWI5 can recruit nucleosome
modifiers (histone acetyl
SBF recruits Mediator
Signal integration
• Cooperative binding of
activators at the human interferon gene
• Viral infection triggers
three activators: NFB,
IRF, and jun/ATF.
• The structure formed by
these regulators bound to
the enhancers is called an
• HMG-1 has an
architectural role in the
The human -interferon enhancersome
Combinatorial control lies at the heart of the complexity and
diversity of eukaryotes
Combinatorial control of the mating-type Genes from Saccharomyces cerevisiae
Transcriptional repressors
• Repressors can recruit nucleosome
modifiers, they compact the chromatin or
remove groups recognized by the
transcriptional machinery, for example:
histone deacetylases.
Ways in which eukaryotic repressor work
In the presence of glucose, Mig1 binds and switches off
the Gal 1 genes. Mig1 recruits a “repressing complex”
containing the Tup1 protein.
Tup1 recruits histone deacetylase
Signal transduction and the control
of transcriptional regulators
• Signals are often communicated to transcriptional
regulators through signal transduction pathways
• The initiating ligand is typically detected by a
specific cell surface receptor.
• The most common way in which information is
passed through signal transduction pathways is via
Two signal transduction pathways from mammalian cells:
Signal control the activators of eukaryotic
transcriptional regulators in a variety of ways
• In eukaryotes, transcriptional regulators are
not typically controlled at the level of DNA
binding (though there are exceptions).
Regulators are instead usually controlled in
one of two basic ways:
1. Unmasking an activating region
2. Transport into and out of the nucleus
The yeast activator Gal 4 is regulated by the Gal 80 protein
Unmasking an activating region
Transport into and out of the nucleus
Activators and repressors sometimes
come in pieces
• The activator can come in pieces: the DNA-binding
domain and activating region can be on separate
• Glucocorticoid receptor (GR) can either activate or
repress transcription depending on the nature and
arrangement of its DNA-binding sites at a given
• The terms” co-repressor” and “ co-activator ” are
often applied to any auxiliary protein which is
neither part of the transcriptional machinery nor
itself a DNA-binding regulator, but which is
nevertheless involved in transcriptional regulation.
Gene “silencing” by modification of
histone and DNA
• Gene silencing is a position effect- a gene is
silenced because of where it is located, not in
response to a specific environmental signal.
• The most common form of silencing is associated
with a dense form of chromatin called
heterochromatin; it appears dense compared to
other chromatin.
• Transcription can also be silenced by methylation
of DNA by enzymes called DNA methylases.
Silencing in yeast is mediated by deacetylation and methylation
of histones: the final 1-5 kb of each chromosome is found in a
folded, dense structure. SIR 2, 3 and 4 are silent information
regulators, and Sir 2 is a histone deacetylase.
Binds to the repeated sequences
Rap1 recruits SIR2
Unacetylated tails then bind SIR3
and 4
Methylation of the tail of Histone H3 blocks the binding of the Sir2
• In high eukaryotes and in the yeast,
silencing is typically associated with
chromatin containing histones that are not
only deacetylated but methylated as well.
Histone modification and the histone
code hypothesis
• Histone code: different patterns of modifications on
histone tails can be read to mean different things.
• Multiple modifications at several positions in the histone
tails are possible, the examples of H3 and H4, together
with H2A and B.
• Lysine 9 on the tail of histone H3: different modification
states of this residue have different meaning. Acetylation
of it is associated with actively transcribed genes. When
lysine 9 is unmodified, it is associated with silenced
regions. This lysine can be methylated then binds proteins
that establish and maintain a heterochromatic state.
DNA methylation is associated with
silenced genes in mammalian cells
Switching a gene off through DNA methylation and histone
DNA-binding proteins (such as MeCP2)
Then recruit histone deacetylases and
histone methylases or chromatin
remodeling complex
•Imprinting: there are a few cases that one copy of a gene is
expressed and the other is silent.
Some states of gene expression are inherited
through cell division even when the initiating
signal is no longer present
• The inheritance of gene expression patterns, in the
absence of either mutation or the initiating signal,
is called epigenetic regulation.
• Nucleosome and DNA modifications can provide
the basis for epigenetic inheritance.
• Methylated nucleosomes in daughter molecules
recruit protein bearing chromodomains to
methylated the adjacent nucleosomes.
•DNA methylation is reliably inherited, so-called maintenance
methylases modify hemimethylated DNA.
•Patterns of DNA methylation can be maintained through cell