Transcriptional Regulation of Eukaryotic Genes

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Transcriptional Regulation of Eukaryotic Genes
?????????
ReferenceGenes IX (Benjamin Lewin)
??? 2008.11.20 PKU
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1. RNA polymerase II2. promoter and
enhancers3. transcription factors
Eukaryotic gene expression is usually controlled
at the level of initiation of transcription.
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RNA polymerase II synthesizes mRNA in the
nucleoplasm (???)
All eukaryotic RNA polymerases have 12
subunits and are aggregates of gt500 kD. Some
subunits are common to all three RNA
polymerases. The largest subunit in RNA
polymerase II has a CTD (carboxy-terminal domain)
consisting of multiple repeats of a heptamer. The
CTD can be highly phosphorylated on serine or
threonine residues this is involved in the
initiation reaction and RNA processing.
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Key Terms A basal (general) factor is a
transcription factor required by RNA polymerase
II to form the initiation complex at all
promoters. Factors are identified as TFIIX, where
X is a number. The basal transcription apparatus
is the complex of transcription factors that
assembles at the promoter before RNA polymerase
is bound. An enhancer is a cis-acting sequence
that increases the utilization of (some)
eukaryotic promoters, and can function in either
orientation and in any location (upstream or
downstream) relative to the promoter.
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Activators and their many targets

• TBP
• TFIID
• TFIIB
• TFIIA
• TFIIH
• TFIIE
• Pol II
• Mediator
• SAGA/Chromatin modifiers
• Many more

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Ordered Assembly vs Pol II Holoenzyme
one-step
multiple-step
TFIID
TFIID

• Holoenzyme --- a supramolecular complex
  comprising Pol II,
• most GTFs, and Mediator/Srb complex
• In yeast, a 2MDa holoenzyme TBP suffices for
  transcription

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Sequential Assembly
Binding of TFIID (TBP 11 TAFs, 800KD) to the
TATA box is the first step in initiation. Other
transcription factors bind to the complex in a
defined order, extending the length of the
protected region on DNA. When RNA polymerase II
binds to the complex, it is ready to initiate
transcription.
TBP TATA binding protein TAFs TBP associated
factors
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25bp
TFIIB binds to DNA and contacts RNA polymerase
near the RNA exit site and at the active center,
and orients it on DNA.
Q prok -10bp vs euk -25bp?
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CTDRNA Pol II C-terminal domain
Phosphorylation of the CTD by the kinase activity
of TFIIH may be needed to release RNA polymerase
to start transcription.
TFIIE and TFIIH are required to melt DNA to
allow polymerase movement. Phosphorylation of
the CTD (by TFIIH and other kinases) is required
for elongation to beginfire the Pol II. The
CTD may coordinate processing of RNA with
transcription.
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The CTD may also be involved in processing RNA
after it has been synthesized. The capping
enzyme binds to the phosphorylated CTD this may
be important in enabling it to modify the 5 end
as soon as it is synthesized. A set of proteins
called SCAFs bind to the CTD, and they may in
turn bind to splicing factors. This may be a
means of coordinating transcription and splicing.
Some components of the cleavage/ polyA
apparatus also bind to the CTD. so RNA
polymerase is all ready for the 3 end processing
reactions as soon as it sets out! CTD may be a
general focus for coupling other processes (mRNA
maturation) with transcription
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Many Transcriptional Activators
i.e. CAAT GC-box
Factors involved in gene expression include RNA
polymerase and the basal apparatus, activators
that bind directly to, co-activators that bind to
both activators and the basal apparatus, and
regulators that act on chromatin structure
(chromatin remodeling complex).
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Modular organization of Transcription Factors

• DNA binding Domain
• Zn fingers, Homeodomains, bHLH, bZip, MADS,
• Oligomerization Domain
• homo/hetero oligomers ---usually dimers, though
  trimers are not uncommon HSF, NF-Y
• Regulatory Domain
• Activation -acidic, Q-rich, P-rich, RNA-binding,
  
• Repression -basic, HDAC binding peptides,
• Other Domains (not in all factors)
• including nuclear localization,
  protein-interaction domains, ligand binding,
  signal-responsive sites

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Zinc Finger
bZIP
Homeodomain
bHLH
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SP1 stimulates transcription in presence of
TAFII110
SV40 early promoter

• GC boxes bound by DNA binding protein SP1
• SP1 recruits TFIID by binding TAFII110
• Partially reconstituted complex (TBP and 3 TAFs)
  in addition to other GTFs, Pol II leads to high
  levels of transcription

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Mediator complex is targeted by an activator
(?????)

• Mediator is a stable complex containing several
  proteins (20-50)
• Mediator binds to the RNA pol II and
  transcription factors (activators or
  repressors) and mediates the regulatory signals
  to pol II

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Tethering the Mediator complex
In yeast SRB complex (suppressors of RNP B).
It contains factors that are necessary for
transcription from many or most promoters. It
provides interaction surfaces for many
transcriptional activators or repressors, thus
mediates both activation and repression of
transcription.

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tat protein of HIV can stimulate transcription
initiation without binding DNA at all The
activating domain of the tat protein can
stimulate transcription if it is tethered in the
vicinity of promoter by binding to the RNA
product (tar sequence) of a previous round of
transcription.
tat
tar
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DNA-binding domain is to bring the activation
domain into the vicinity of the startpoint. And
activation is independent of the means of
tethering.
we can think of DNA-binding (or RNA-binding in
the case of tat) domain as providing a
"tethering" function, whose main purpose is to
ensure that the activation domain is in the
vicinity of the initiation complex. The notion
of tethering is a more general idea that
initiation requires a high concentration of
transcription factors in the vicinity of the
promoter. This may be achieved when activators
bind to enhancers, upstream promoter elements, or
in an extreme case by tethering to a newly-made
RNA product.
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Interchangeable Modules
(Activation domain is interchangeable)
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• Interaction Assays
• Design of Two-hybrid / Three-hybrid /etc

Two-hybrid assay (protein-protein)
separable functional domains
Tri-hybrid assay (protein-RNA)
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• Summary
• The principle that governs the function of all
  activators is that a DNA-binding domain
  determines specificity for the target promoter or
  enhancer.
• The DNA-binding domain is responsible for
  localizing a transcription-activating domain in
  the proximity of the basal apparatus.
• An activator that works directly has a
  DNA-binding domain and an activating domain.
• An activator that does not have an activating
  domain may work by binding a coactivator that has
  an activating domain.
• Several factors in the basal apparatus are
  targets with which activators or coactivators
  interact.
• RNA polymerase may be associated with various
  alternative sets of transcription factors in the
  form of a holoenzyme complex.

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What is the mechanism of activation?

• Two models
• Tethering holoenzyme (recruitment)
• Activating holoenzyme (allosteric)

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In favor of recruitment model
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activators recruit transcription machinery

• Gal4-binding sites are bound by Gal4
• Pre-binding by Gal4 is necessary for
• recruitment of the machinery
• Role in re-initiation as well

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Synergy High levels of transcription induced
by multiple factors

• Transcription factors can enhance transcription
  in a non-linear manner
• Synergisitic activation occurs due to multiple
  contacts with the machinery
• Multiple copies of the same activator also
  induce synergistic activation

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Interferon ß enhancer

• Enhancers often have binding sites for several
  transcription factors
• Transcription factors can bind cooperatively at
  adjacent sites
• Architectural factors (with no regulatory
  domains, i.e. HMG1) can
• assist assembly
• Remarkably increase binding affinity for both
  DNA and machinery

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???????,???????
HMG1
?????????,?????????
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Regulatory mechanism from a distance
Compaction
Sliding
Looping
Why do enhancers act independent of distance and
orientation?
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Two experiments support the looping model
--The essential role of the enhancer is to
increase the concentration of activator in the
vicinity of the promoter---
An enhancer may function by bringing proteins
into the vicinity of the promoter. An enhancer
does not act on a promoter at the opposite end of
a long linear DNA, but becomes effective when the
DNA is joined into a circle by a protein bridge.
An enhancer and promoter on separate circular
DNAs do not interact, but can interact when the
two molecules are catenated.
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(Signal transduction and gene regulation)
How is a gene regulated by signals?
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Each gene contains multiple response elements
The regulatory region of a human metallothionein
gene contains regulator elements in both its
promoter and enhancer. The promoter has elements
for metal induction an enhancer has an element
for response to glucocorticoid. Promoter elements
are shown above the map, and proteins that bind
them are indicated below.
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The activity of a regulatory transcription factor
may be controlled by synthesis of protein,
covalent modification of protein, ligand binding,
or binding of inhibitors that sequester the
protein or affect its ability to bind to DNA.
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Steroid receptors are transcription factors
Steroid receptors are examples of
ligand-responsive activators that are activated
by binding a steroid. There are separate
DNA-binding and ligand-binding domains. The DNA
binding domain is a type of zinc finger that has
Cys but not His residues. They bind to DNA as
dimers. Glucocorticoid and estrogen receptors
each have two zinc fingers, the first of which
determines the DNA target sequence. Binding of
ligand to the C-terminal domain increases the
affinity of the DNA-binding domain for its
specific target site in DNA.
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Receptors for many steroid and thyroid hormones
have a similar organization, with an individual
N-terminal region, conserved DNA-binding region,
and a C-terminal hormone-binding region
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The first finger of a steroid receptor controls
which DNA sequence is bound (positions shown in
red) the second finger controls spacing between
the sequences (positions shown in blue).
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ERE
GRE
Discrimination between GRE and ERE target
sequences is determined by two amino acids at the
base of the first zinc finger in the receptor.
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Activation of Glucocorticoid Receptor (GR)
Nuclear shuttling
(or dexamethasone)
Glucocorticoids regulate gene transcription
by causing their receptor to transport into the
nucleus and bind to an enhancer whose action is
needed for promoter function.
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Glucocorticoid receptor (GR) fusion protein
induction
Dex binds to GR, exposes nucleus localization
signal (NLS), allows fusion protein to transport
into the nucleus to activate transcription
----A good system to activate the function of
nuclear-localized proteins (including
transcription factors)
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Activation Tagging approach in plants
Plant transformation
Genetic screen

• phenotypic characterization of mutants
• locate T-DNA insertion site in Arabidopsis genome
  (how?)
• identify the right gene conferring mutant
  phenotype (how?)
  
  
  
• functional study of the gene

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A chemical-inducible activation tagging vector
pER16 in plants
T-DNA fragment (can integrate into plant genome)
XVE,was assembled by fusion of the DNA-binding
domain of LexA (X), the acidic activation domain
of VP16(V), and the regulatory region of the
human estrogen receptor (E)
???????G10-90??????,XVE ?????????????????,??????
???,??XVE??????????,????????????????,LexA?DNA????
????LexA????,???VP16????????-46 35S
???,????????????????
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Corepressor/ coactivator switch
TR and RAR bind the SMRT corepressor in the
absence of ligand. The promoter is not expressed.
When SMRT is displaced by binding of ligand, the
receptor binds a coactivator complex. This leads
to activation of transcription by the basal
apparatus.
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Summary1) Cis-elements mark the sites of
transcription2) Some bound by GTFs and others by
Regulators (TFs)3) Regulatory TFs are trigged by
cellular signals4) Regulatory TFs can be
positive or negative5) TFs are modular- DBD
binds Enhancers and defines genes that are
targeted- Regulatory domain controls the
expression of the gene(s)6) Simple architecture
of regulatory proteins has led to the creation
of powerful tools (2-hybrid and ATFs)
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????????Roger Kornberg???? ?????????
??????,???????,???????????????????2006????????
????????????????????
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If nucleosomes form at a promoter,
transcription factors (and RNA polymerase) cannot
bind. If transcription factors (and RNA
polymerase) bind to the promoter to establish a
stable complex for initiation, histones are
excluded.
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Chromatin remodeling
The dynamic model for transcription of chromatin
relies upon factors that can use energy provided
by hydrolysis of ATP to displace nucleosomes from
specific DNA sequences.
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two major types of chromatin remodeling complex
SWI/SNF and ISW (imitation SWI)
Chromatin remodeling is undertaken by large
complexes that use ATP hydrolysis to provide the
energy for remodeling. The heart of the
remodeling complex is its ATPase subunit.
Remodeling complexes are usually classified
according to the type of ATPase subunit
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Remodeling complexes can cause nucleosomes to
slide along DNA, can displace nucleosomes from
DNA, or can reorganize the spacing between
nucleosomes.
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A remodeling complex binds to chromatin via
an activator (or repressor)
Remodeling complexes are recruited to promoters
by sequence-specific activators. A genomic
survey suggested that most sites that bind
transcription factors are free of nucleosome.
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Hormone receptor and NF1 cannot
bind simultaneously to the MMTV promoter in the
form of linear DNA, but can bind when the DNA is
presented on a nucleosomal surface. The MMTV
promoter requires a change in rotational
positioning of a nucleosome to allow an activator
to bind to DNA on the nucleosome.
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???????? ?????????????N???,??????H3?H4??????
???????????????20??????,???DNA?????????????
54
??????????
???(?)??? De/Acetylation ??????
Methylation ?????? Phosphorylation ??????
Ubiquitination ?????? ADP-Rybosilation ????Swi/S
nf?????? Swi/Snf complex, which, in vitro, uses
the energy of ATP hydrolysis to disrupt
histone-DNA interactions ??????????????????????,
?????DNA?????,???????????
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Histone modification is a key event
Acetylation of H3 and H4 is associated with
active chromatin, while methylation is associated
with inactive chromatin.
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Sites of post-translational modifications on the
histone tails
Zhang Y., Reinberg D. Genes Dev. 2001152343-2360
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Most modified sites in histones have a single,
specific type of modification, but some sites can
have more than one type of modification.
Individual functions can be associated with some
of the modifications.
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Histone Code
K
K
Acetylation Transcriptional Competence
Methylation Transcriptionally Silenced
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Histone acetylation occurs in two circumstances
Histone acetylation occurs transiently at
replication. Histone acetylation is associated
with activation of gene expression.
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Histone acetyltransferase (HAT) enzymes modify
histones by addition of acetyl groups some
transcriptional coactivators have HAT
activity. A deacetylase is an enzyme that
removes acetyl groups from proteins. Histone
deacetyltransferase (HDAC) enzymes remove acetyl
groups from histones they may be associated with
repressors of transcription.
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????????????? HAT???????????(?SAGA?????GCN5),???H
AT????????????????,?????????(?P300,
ACTR?PCAF?????)
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????????????,??????????????????????(histone
deacetylase, HDAC)??????? ????????(HDAC)???4??  
   (1)???????????RPD3??????,???????????HDAC????HD
AC13?6? 7?9? 18,19,?????????    
(2)??????HDAl????,??HDAC4?5?10????????????HDAl???
?,????????????     (3)???????????????????2(silen
t information regulator 2,SIR 2)????????????,?????
SIR 2????????????,SIR 2???????????????????????????
? (4)???HD2?,?????????
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A repressor complex contains three components a
DNA binding subunit, a corepressor, and a histone
deacetylase.
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HDAC??????????????????HDACl?HDAC2?????????m
Sin3A??????????????????????,??HDAC-mSin3??????????
???,??????????????????????????????Mad/Max?????MeCP
2?p53??
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?????? ?????????????????(histonemethyl
transferase,HMT)???? ????? ?????????????????????
,???????????????????,??????????????,??????????????
??????????????????? ????????????(Lys)????(Arg)???
N???????H3??4?9?27?36?,H4??20?Lys,H3??2?l7?26??H4?
?3?Arg???????????
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????????????????,?????????????,?H3?H4????????????
????? ??????????????????????????,H3?4???????????
??????,??9???27?????????????????,H4K20???????????,
H3K36?H3K79???????????????????,???????????????????
???????DNA???????
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Acetylation of histones activates chromatin, and
methylation of DNA and histones inactivates
chromatin. Methylation of DNA and of histones is
associated with heterochromatin. The two types
of methylation event may be connected.
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Summary of histone acetylation, histone
methylation and DNA methylation
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Promoter activation involves an ordered series of
events
Promoter activation involves binding of a
sequence-specific activator, recruitment and
action of a remodeling complex, and recruitment
and action of an acetylating complex.
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?????????? ????,????????????????,????????????????
??????????????,??????????????,??????????????ADP???
????????????????????????,?????????????????????????
????????????????????????????????,?????????????????
???????   ??,????H2B????????H3K4?H3K79????,???????
?????????????
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Mechanisms of Transcriptional Activation
1) The Components of the Gene Expression
Machinery 2) Ordered versus One-Step
Recruitment 3) Recruitment versus Allosteric
Regulation 4) Nature of the Activation Domain
5) Transcriptional Activators and Chromatin
Remodeling 6) Future of Transcriptional
Regulation
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Future of Transcriptional Regulation A) From
regulation of one gene to the study of all at
once B) Importance of Genomics C) The role of
informatics D) Tools to dissect the immediate
from the secondary E) Expression profiling as a
fundamental biological and a diagnostic
tool F) Understanding the genomic access code