Promoters
Epigenetics 2014 by Nigel Atkinson
The University of Texas at Austin
A) Lecture - Promoters
1) Expectations
You should be able to answer the following: What are promoters? What is a core promoter? How are
they recognized (who does it? and a bit about how it is done)?
2) Important elements for transcription by RNA polymerase II
RNA polymerase II transcribes protein-encoding genes
DNA elements
promoter, core promoter, tss (transcription start site)
proximal promoter? ~250 bp upstream.
enhancer
silencer
insulator
3) Promoter recognition
The meaning of core promoter, proximal promoter elements, enhancers?
b) How is a promoter is recognized?
4) RNA polymerase is a complex enzyme that interacts with a large number of proteins.
What are transcription
factors?
• In normal cells, anything other than RNA
polymerase or histones that binds a
promoter or DNA regulatory element that
increases or reduces the rate of
transcription initiation.
Promoters & regulatory
sequences
•
Core Promoter - a DNA sequence that specifies where
transcription should start and which way it will go.
•
•
By itself it does not have to actually specify that transcription
actually occurs. - exceptions exist
Regulatory sequences are sequences that determine how much,
and when to transcribe a gene.
•
By itself, it does not specify where transcription should start
or which way it should go. - exceptions exist.
•
Enhancer and silencers are DNA elements
Promoters & regulatory
sequences
• What do people mean when they say
promoter?
• What do people mean when they say
proximal promoter?
• Be sure that you understand the difference
between how these are currently used and
the concept of the core promoter.
Eukaryotic RNA polymerase II
is complex
•
For protein encoding genes it is RNA polymerase II that is
performing transcription
• 12 proteins
• 3 are evolutionarily related to prokaryotic
• Five subunits are common to all nuclear polymerases
Eukaryotic RNA polymerase II
is complex
pink - essential for function
yellow - common to all three eukaryotic polymerases
Transcriptional
Regulation
• Mammals regulate ∼25,000 genes
• Many with multiple promoters
• DNA being regulated is wrapped in
chromatin
• Combinatorial control
Core promoters used by
RNAP II
•
•
•
-40 to +50
•
In vivo they are inactive or expressed very
weakly. Need exogenous stimulation
(exceptions exist). May have much more
activity in vitro.
Preinitiation complex assembles here
Determines start site and direction of
transcription
•
•
•
TFIID
TFIID=TBP + 8-10
TAFIIS
TATA Inr
A
TFIID
B
TFIID binds minor
groove
of the TATA box.
A
TFIID A
B
DAB complex
Pol II
TFIIF - ATP
dependent
helicase activity
2 proteins,
also reduces
affinity of
polymerase for
nonpromoter DNA
Pol II
B F
F
Pol II
Pol II
BTFIID
F
A
mediator
B F
H
mediator
Holoenzyme
E
H
E
Pol II E
A
BTFIID
F
H mediator
Complete
TFIID A
B
TFIID
DABTFIID
complexTFIID
Upstream
element
Not
shown
- CpG
islands
TFIIB
TFIIE, IH, RNApolI
TFIID
TFIIA
SP1
Pol II
F
+1
TFIIB
<-- upstream
TFIIA
Pol II
TFIIE, IIH, RNAPolII
3ʼ
SP1
Upstream
TATA
INR
BRE
DCE II
DCE I
BRE TATA
upstream elements
-31 to -26
-2 to +4
-37 to-32
DPE DCE DCE
INR
I
II
Pol II
+16 to +21
+6 to -11
-2 to +4
TATA A/T A A/Tcore promoter
Py Py AN T/A Py Py
Proximal Promoter
TATA: TATA box
INR: Initiator element
+16 to +21
A
DCEIII
Pol I
+32 to +34
+28 to +30
CTTC
CTTC
mediator
proximal promoter elements
BRE: TFIIB binding element
+6 to +32
+11
to +34
DPE
+28 to +30
BTFIID
F
G/C G/C G/C CGCCC
B F
DCE III
-31 to -26
-37 to -32
•
•
•
•
•
TFIID
IIB
downstream -->
5ʼ
TFIID
Core promoter
B F
AGC
RG A/T CGTG
Holoenz
E
H
DCE: Downstream core element
DPE: downstream promoter element
Pol II E
A
BTFIID
F
H mediator
Complete
TFIID
TFIID
TFIIB
TFIIE, IH, RNApolI
TFIIA
SP1
TFIID
+1
<-- upstream
downstream -->
5ʼ
Upstream
TATA
3ʼ
INR
BRE
DCE II
DCE I
DCE III
DPE
-31 to -26
-2 to +4
-37 to-32
+16 to +21
+6 to -11
+32 to +34
+28 to +30
core promoter
proximal promoter elements
A few common combinations
TATA or a DPE usually not both
TATA with DCE
TATA INR
INR DPE
Not shown:
MTE stands for "motif ten element"
Found at +18 to +29
Found in Drosophila
Has not yet been shown to be important in mammals.
MTE requries INR
TATA MTE is common
MTE DPR is common
MTE can substitute for TATA and INR
Figure 3. Signatures of active promoters. A nucleosome free region (NFR) surrounds the transcriptional start site (TSS) in the core
promoter, which may contain core promoter elements, including BRE, TATA, Inr, MTE, DPE and others (positions are relative to the +1
TSS within the Inr; please see detailed explanation of these elements in the main text and in Table 1). The nucleosomes flanking the NFR
contain the histone variant H2A.Z, while other nucleosomes contain normal H2A and other histone proteins that are subject to various
modifications. Histone acetylation peaks just downstream
of the promoter,
while methylation of histone
3 lysine 4 is present in a gradient,
TFIID
TFIID
TFIID
from trimethylation (H3K4me3) at the promoter, to di- and then monomethylation (H3K4me2, H3K4me1) with increasing distance from
the promoter into the transcribed region. This diagram is
a composite TFIIE,
of features
in yeast, fly and mammalian systems; it is
TFIIB
IH,determined
RNApolI
representative of some important characteristics of promoters identified in large-scale studies.
SP1
TFIIA
+1
the sequence motifs responsible for this critical step in chem.qmul.ac.uk/iubmb/misc/naseq.html) was deter<-- upstream
-->
gene regulation, revealing a collection of short regulamineddownstream
by comparison
of 5a flanking regions in several
tory DNA sequence elements conserved across species. organisms
[31].
The
TATA-box
is located approximately
5ʼ
3ʼ
While the first core promoter element has been known for 25–30 bp upstream of the transcription start site in most
almost 30 years, additional novel sequence elements have eukaryotes, though in yeast it is found slightly further upbeen discovered recently, emphasizing the importance of stream [32]. It is typically recognized by the TATA bindcontinued research of these Upstream
regulatory sequences.TATA
Most ing
subunit of the general transcription
INRprotein (TBP)
DCE
DCE III
II
of the canonical core promoter elements have
been
thorfactor
TFIID
[33],
though
additional related
but distinct
BRE
DCE I
DPE
oughly reviewed elsewhere [2], but it is useful to describe
proteins can also recognize this element [34].
-31 to -26
their general features here (see Table 1) in light of recent The initiator element (Inr; YYANWYY) immediately
-2 to +4
+16 to +21
+32 to +34
genome-wide analyses of these elements. Note that there surrounds the transcription start site [35] and is found in
-37 to-32
+6 to -11
+28 to +30
are no ‘universal’ core promoter elements; the sequences promoters containing or lacking a TATA-box. While the
described below are found in only a subset of promoters, Inr can stimulate transcription independently of a TATAand the origins and functional consequences of the result- box, these two elements act synergistically when found
ing core promoter diversity are a topic of current study.
together [36]. This element is recognized by the TAF1
core promoter
The first core promoter element identified was the TATA- and TAF2 subunits of TFIID [37].
box, whose consensus
sequence (TATAWAAR; degener- The downstream promoter element (DPE; RGWYV) [38]
proximal promoter elements
ate nucleotides according to IUPAC code, http://www. is typically found in TATA-less promoters and functions
with the Inr as a downstream counterpart to the TATAbox [39]. The DPE is located at +28 to +32 relative to
Table 1. Summary of sequence and frequency of core promoter ele- the TSS, with this exact spacing critical to optimal tranments.
scription [40]. Like the TATA-box and Inr, this element is
recognized by TFIID, likely the TAF6 and TAF9 subunits,
Core
Position Consensus
Frequency in proelement relative
sequence**
moters
but not TBP [41]. There is evidence that the presence of a
to TSS*
TATA-box or DPE in a promoter can influence its interacFlies
Vertebrates
tions with enhancers [42] and transcriptional activation or
TATA
approx.
TATAWAAR
33–43% 10–16%
repression [43], suggesting multiple regulatory mecha–31 to
nisms acting at the core promoter.
–26
The TFIIB recognition element (BRE; SSRCGCC) conInr
–2 to +4 YYANWYY
69%
55%
sists of the 7 bp immediately upstream of the TATA-box,
DPE
+28 to
RGWYV
40%
48%
and as its name suggests, it is bound by transcription fac+32
tor IIB [44]. The BRE has been shown to both stimulate
BRE
approx.
SSRCGCC
–
12–62%
and repress transcriptional activity [45].
–37 to
–32
The motif ten element (MTE; CSARCSSAACGS) was
MTE
+18 to
CSARCSSAACGS 8.5%
–
identified in a computational survey of Drosophila pro+29
moters [46], located +18 to +29 downstream of the TSS
and overlapping slightly with the 5a-end of the DPE. The
* The TSS is assigned to position +1.
** Degenerate nucleotides represented using IUPAC codes.
MTE requires Inr and functions synergistically with the
TFIID
TFIID
TFIIB
TFIIE, IH, RNApolI
TFIIA
SP1
TFIID
+1
<-- upstream
downstream -->
5ʼ
Upstream
TATA
3ʼ
INR
BRE
DCE II
DCE III
DCE I
DPE
-31 to -26
-2 to +4
-37 to-32
+16 to +21
+6 to -11
+32 to +34
+28 to +30
core promoter
proximal promoter elements
Why so many elements?
core promoters. The use of DPE- and TATA-specific activators would
enable the construction of more sophisticated and effective connections between enhancers and promoters (Fig. 5).
There exist many variants on the
core promoter sequence. Why?
TBP-related factors (TRFs) and transcriptional regulation
There is diversity not only in core promoter elements but also in
the basal transcription machinery. This concept is nicely exemplified
in studies of the TBP-related factors (TRFs) (for reviews, see: Jones,
2007; Müller et al., 2007; Reina and Hernandez, 2007; Torres-Padilla
and Tora, 2007). There are three TRFs, which are generally termed
TRF1, TRF2, and TRF3.
• 1. May assemble different pre-initiation complex
with specific features required for cell-specific
regulation. One example is tuning between
enhancers and promoters.
Juven-Gershon, T. & Kadonaga, J.T.
(2010) Regulation of gene expression
via the core promoter and the basal
transcriptional machinery. Dev Biol,
339, 225-229.
Fig. 5. A simplified, hypothetical diagram of activation by DPE- and TATA-specific
factors. Transcription factors bind to enhancers but only activate transcription from
promoters with the appropriate core promoter elements. The core promoter containing
both TATA and DPE motifs can be activated by either DPE- or TATA-specific activators.
Conclu
The
two im
of gen
and fu
contrib
compl
essent
gene r
be stu
Altern
with c
Fig. 6.
complex
bind to
establish
There exist many variants on the
core promoter sequence. Why?
• 2. Different core promoters might bind the
same basal factors more or less tightly.
Why is this important?
What are transcription
factors?
• In normal cells, anything other than RNA
polymerase or histones that binds a
promoter or DNA regulatory element that
increases or reduces the rate of
transcription initiation.
General transcription
factors
• Interact directly with core promoter.
Determine site of initiation and the
direction of transcription. TFIIA, TFIIB,
TFIID, TFIIF, TFIIH and Mediator.
• In eukaryotes, RNA polymerase
holoenzyme cannot recognize promoters
by itself.
• The general (aka basal) transcription
factors recognize the core promoter.
•
•
•
TFIID=TBP +
8-10 TAFIIS
TFIID
A
TFIID binds minor
groove
of the TATA box.
TFIIF - ATP
dependent
helicase activity
2 proteins,
also reduces
affinity of
polymerase for
nonpromoter DNA
TATA Inr
TFIID
B
A
TFIID A
B
DAB complex
Pol II
Pol II
B F
F
Pol II
Pol II
BTFIID
F
A
mediator
B F
H
mediator
Holoenzyme
E
H
E
Pol II E
A
BTFIID
F
H mediator
Complete
DAB complex
Pol II
Pol II
•
•
•
•
F
TFIID
TFIIH - kinase that
phosphorylates
YSPTSPS
(CTD domain)
Unphosphorylated
RNAP
= RNAPIIA
= initiation specific
Phosphorylated
RNAP
= RNAPIIO
= for chain elongation
TFIIH also has
helicase
activity
B F
TATA Inr
Pol II
A
A
BTFIID
F
TFIID A
B
Pol II
B F
H
Pol II
Pol II
Pol II E
A
BTFIID
F
H
Complete
B F
F
Pol II
Pol II
BTFIID
F
A
mediator
B F
E
H
mediator
Holoenzyme
E
H
H
Holoenzyme
E
TFIID A
B
DAB complex
E
Pol II E
A
BTFIID
F
H mediator
Complete
Upstream
element
TFIID
TFIID
TFIIB
TFIIE, IH, RNApolI
TFIID
TFIIA
SP1
TFIID
+1
TFIIB
<-- upstream
TFIID
IIB
downstream -->
TFIIA
5ʼ
TFIID
TFIIE, IIH, RNAPolII
3ʼ
SP1
Upstream
TATA
BRE
upstream elements
-31 to -26
INR
DCE II
DCE I
BRE TATA
-2 to +4
-37 to-32
DPE DCE DCE
INR
I
II
+6 to +32
+11
to +34
+16 to +21
-31 to -26
+6 to -11
-37 to -32
DCE III
-2 to +4
+16 to +21
CTTC
proximal promoter elements
TFIID
+32 to +34
+28 to +30
CTTC
TATA A/T A A/Tcore promoter
Py Py AN T/A Py Py
•
DCEIII
+28 to +30
G/C G/C G/C CGCCC
Proximal Promoter
DPE
Core promoter
AGC
RG A/T CGTG
TFIID: What is TFIID?
TFIID=TBP + 8-10 TAFIIS
TAF250
TA
TAF110
F60
40
30!
80
TFIID
TBP
30"
TAF150
TFIID: TBP can bend
DNA
• Crystal structure
of TBP suggested
a saddle.
• TBP DNA co-
crystal
indicated that not
like a saddle on a
horse.
TFIID: Who recognizes what?
• TATA-less
promoters
are not recognized
by TBP
• TAFII250 & TAFII150
impart ability to
recognize Inr & DPE
• Other TF (Sp1)
necessary for TATA,
Inr & DPE-less
promoters
TAFs can have enzymatic
activity
• TAFII250 - Histone acetyl transferase
activity that acetylates lysine residues of
histones. This can lead to remodeling of the
chromatin.
• TAFII250 - Protein kinase activity that
phosphorylates itself and TFIIF, TFIIA and
TFIIE. Thought to modulate activity of
initiation complex.
Enhancers
Enhancers are DNA elements
that regulate promoter activity
TFIID has many targets for interactions
Enhancers are DNA elements that
regulate core promoter activity
Promoter Clearance
Kinase activity of TFIIH activates polymerase for
elongation.
ATP-dep DNA helicase activity of TFIIH is required
for promoter clearance.
Enhancers - how do they
work?
Proteins bind these DNA elements and then do at least of the following
Stabilization of the pre-initiation complex
Help hold it down - many hands model
TFIID alone has many places where interactions can take place
Activate enzymatic activities within the pre-initiation complex
eg. TAFII250 HAT or TFIIH kinase
Bend the DNA - eg. lef1
Prepare the area <--- REALLY IMPORTANT THIS CONTAINS MOST OF THE
EPIGENETICS MECHANISMS THAT WE WILL DISCUSS
This can make the binding sites visible
Changes to histones can stabilize the preinitiation complex
Changes to histones can help recruit the preinitiation complex
What do I mean by the word recruit?
eg. Some preparations can include modifications of histones. These can be attractive to
important transcription factors. That is they can increase the local concentration of the
transcription factor. eg. Alter the histones so that needed proteins such as TFIID
concentrate in the area.
Silencers - how do they
work?
Proteins bind these DNA elements and then do the opposite.
Enhancers and silencers
These are names. The names are chosen because it is the first thing that an
element is observed to do. It is common that later one finds out that the enhancer
is used to suppress transcription in a different cell.
My point is that biology is not restrained by the names that we give things. Many,
many people forget this.