Promoters

Epigenetics 2014 by Nigel AtkinsonThe 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 IIRNA polymerase II transcribes protein-encoding genes

DNA elementspromoter, core promoter, tss (transcription start site)

proximal promoter? ~250 bp upstream.enhancersilencerinsulator

3) Promoter recognitionThe 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.

• 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

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 functionyellow - 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

• Preinitiation complex assembles here

• Determines start site and direction of transcription

• In vivo they are inactive or expressed very weakly. Need exogenous stimulation (exceptions exist). May have much more activity in vitro.

• TFIID=TBP + 8-10 TAFIIS

• TFIID binds minor grooveof the TATA box.

• TFIIF - ATP dependenthelicase activity2 proteins,also reduces affinity ofpolymerase for non-promoter DNA

A

TATA Inr

TFIID A

TFIID

TFIID A

B

B

Pol II

B F

Pol II

B F

Pol II

B FPol II

F

E

H

H

TFIID APol II

B FH

Holoenzyme

DAB complex

Complete

TFIID A

E

E

mediatormediator

mediator

• BRE: TFIIB binding element

• TATA: TATA box

• INR: Initiator element

• DCE: Downstream core element

• DPE: downstream promoter element

Upstream element

upstream elements BRE TATA INR DCEI DCEII DCEIIIDPE

G/C G/C G/C CGCCC

TATA A/T A A/T Py Py AN T/A Py Py CTTC RG A/T CGTG

AGC

-37 to -32

-31 to -26

-2 to +4

+6 to +11

+16 to +21

CTTC

+28 to +30

+32 to +34

SP1

TFIID TFIID TFIID

TFIIE, IIH, RNAPolII

TFIIA

TFIIB IIB

Core promoterProximal Promoter

5ʼ 3ʼ

INRDCE

DCE

DPE

DCE IIIII

I

TATABRE

-37 to-32

-31 to -26

-2 to +4

+6 to -11

+16 to +21

+28 to +30

+32 to +34

Upstream

<-- upstream downstream -->

core promoter

+1

proximal promoter elements

TFIID TFIID TFIID

TFIIB TFIIE, IH, RNApolI

TFIIASP1

A

TATA Inr

TFIID A

TFIID

TFIID A

B

B

Pol II

B F

Pol II

B F

Pol II

B FPol II

F

E

H

H

TFIID APol II

B FH

Holoenzyme

DAB complex

Complete

TFIID A

E

E

mediatormediator

mediator

Not shown - CpG islands

A few common combinationsTATA or a DPE usually not bothTATA with DCETATA INRINR DPE

Not shown:MTE stands for "motif ten element"Found at +18 to +29Found in DrosophilaHas not yet been shown to be important in mammals.MTE requries INRTATA MTE is commonMTE DPR is commonMTE can substitute for TATA and INR

5ʼ 3ʼ

INRDCE

DCE

DPE

DCE IIIII

I

TATABRE

-37 to-32

-31 to -26

-2 to +4

+6 to -11

+16 to +21

+28 to +30

+32 to +34

Upstream

<-- upstream downstream -->

core promoter

+1

proximal promoter elements

TFIID TFIID TFIID

TFIIB TFIIE, IH, RNApolI

TFIIASP1

390 N. D. Heintzman and B. Ren Eukaryotic transcriptional promoters

the sequence motifs responsible for this critical step in gene regulation, revealing a collection of short regula-tory DNA sequence elements conserved across species. While the first core promoter element has been known for almost 30 years, additional novel sequence elements have been discovered recently, emphasizing the importance of continued research of these regulatory sequences. Most of the canonical core promoter elements have been thor-oughly reviewed elsewhere [2], but it is useful to describe their general features here (see Table 1) in light of recent genome-wide analyses of these elements. Note that there are no ‘universal’ core promoter elements; the sequences described below are found in only a subset of promoters, and the origins and functional consequences of the result-ing core promoter diversity are a topic of current study.The first core promoter element identified was the TATA-box, whose consensus sequence (TATAWAAR; degener-ate nucleotides according to IUPAC code, http://www.

chem.qmul.ac.uk/iubmb/misc/naseq.html) was deter-mined by comparison of 5 flanking regions in several organisms [31]. The TATA-box is located approximately 25–30 bp upstream of the transcription start site in most eukaryotes, though in yeast it is found slightly further up-stream [32]. It is typically recognized by the TATA bind-ing protein (TBP) subunit of the general transcription factor TFIID [33], though additional related but distinct proteins can also recognize this element [34].The initiator element (Inr; YYANWYY) immediately surrounds the transcription start site [35] and is found in promoters containing or lacking a TATA-box. While the Inr can stimulate transcription independently of a TATA-box, these two elements act synergistically when found together [36]. This element is recognized by the TAF1 and TAF2 subunits of TFIID [37].The downstream promoter element (DPE; RGWYV) [38] is typically found in TATA-less promoters and functions with the Inr as a downstream counterpart to the TATA-box [39]. The DPE is located at +28 to +32 relative to the TSS, with this exact spacing critical to optimal tran-scription [40]. Like the TATA-box and Inr, this element is recognized by TFIID, likely the TAF6 and TAF9 subunits, but not TBP [41]. There is evidence that the presence of a TATA-box or DPE in a promoter can influence its interac-tions with enhancers [42] and transcriptional activation or repression [43], suggesting multiple regulatory mecha-nisms acting at the core promoter.The TFIIB recognition element (BRE; SSRCGCC) con-sists of the 7 bp immediately upstream of the TATA-box, and as its name suggests, it is bound by transcription fac-tor IIB [44]. The BRE has been shown to both stimulate and repress transcriptional activity [45].The motif ten element (MTE; CSARCSSAACGS) was identified in a computational survey of Drosophila pro-moters [46], located +18 to +29 downstream of the TSS and overlapping slightly with the 5 -end of the DPE. The MTE requires Inr and functions synergistically with the

Table 1. Summary of sequence and frequency of core promoter ele-ments.

Core element

Position relative

Consensus sequence**

Frequency in pro-moters

to TSS*

Flies Vertebrates

TATA approx. –31 to –26

TATAWAAR 33–43% 10–16%

Inr –2 to +4 YYANWYY 69% 55%

DPE +28 to +32

RGWYV 40% 48%

BRE approx. –37 to –32

SSRCGCC – 12–62%

MTE +18 to +29

CSARCSSAACGS 8.5% –

* The TSS is assigned to position +1.** Degenerate nucleotides represented using IUPAC codes.

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, 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 of features determined in yeast, fly and mammalian systems; it is representative of some important characteristics of promoters identified in large-scale studies.

5ʼ 3ʼ

INRDCE

DCE

DPE

DCE IIIII

I

TATABRE

-37 to-32

-31 to -26

-2 to +4

+6 to -11

+16 to +21

+28 to +30

+32 to +34

Upstream

<-- upstream downstream -->

core promoter

+1

proximal promoter elements

TFIID TFIID TFIID

TFIIB TFIIE, IH, RNApolI

TFIIASP1

Why so many elements?

5ʼ 3ʼ

INRDCE

DCE

DPE

DCE IIIII

I

TATABRE

-37 to-32

-31 to -26

-2 to +4

+6 to -11

+16 to +21

+28 to +30

+32 to +34

Upstream

<-- upstream downstream -->

core promoter

+1

proximal promoter elements

TFIID TFIID TFIID

TFIIB TFIIE, IH, RNApolI

TFIIASP1

There exist many variants on the core promoter sequence. Why?

activation via the DPE (in the presence or absence of a BREu), weakeractivation via the TATA box in the absence of a BREu, and little or noactivation via the TATA box with a BREu.

Hence, these studies of Caudal and the Hox genes reveal howspeci!c core promoter motifs can play a central role in an importantbiological network. Yet, it is important to consider why Caudal mightact as a DPE-speci!c activator. In a simple sense, it could be imaginedthat DPE speci!city would be useful in the construction of regulatorynetworks. As in the wiring of a printed circuit board, there could beconnections between transcriptional enhancers and their cognatecore promoters. The use of DPE- and TATA-speci!c activators wouldenable the construction of more sophisticated and effective connec-tions between enhancers and promoters (Fig. 5).

TBP-related factors (TRFs) and transcriptional regulation

There is diversity not only in core promoter elements but also inthe basal transcription machinery. This concept is nicely exempli!edin studies of the TBP-related factors (TRFs) (for reviews, see: Jones,2007; Müller et al., 2007; Reina and Hernandez, 2007; Torres-Padillaand Tora, 2007). There are three TRFs, which are generally termedTRF1, TRF2, and TRF3.

TRF1 does not exist in yeast and humans but is present in Droso-phila. In many eukaryotes, including yeast and humans, TBPparticipates in transcription by RNA polymerases I, II, and III.However, in Drosophila, TRF1 is used instead of TBP for RNApolymerase III transcription (Takada et al., 2000).

TRF2 (also known as TLF, TLP, TRF, and TRP) is present in mosteukaryotes and is involved in transcription by RNA polymerase II.TRF2 does not bind to TATA box sequences and cannot replace TBPin vitro. It appears that many genes are regulated by TRF2 instead ofTBP – one such example is the Drosophila histone H1 gene (Isogaiet al., 2007). The TATA-less H1 linker histone gene is in a cluster ofgenes that also includes the four TATA-containing core histone genes,which are transcribed with TBP. These !ndings suggest the use ofdifferent transcriptional mechanisms within a cluster of genes.

TRF3 (also known as TBP2 and TBPL2) appears to be present onlyin vertebrates and is the TRF that is most closely related to TBP. TRF3can bind to TATA boxes and support TATA-dependent transcription(Bártfai et al., 2004; Jallow et al., 2004). TRF3 was found to beimportant for embryonic development (Bártfai et al., 2004; Jallowet al., 2004). In addition, zebra!sh embryos that are depleted ofTRF3 exhibit multiple developmental defects and fail to undergohematopoiesis (Hart et al., 2007).

A particularly striking function of TRF3 was discovered during theanalysis of the differentiation of myoblasts to myotubes (Deato andTjian, 2007; Deato et al., 2008). Myoblasts were found to contain thecanonical TBP-containing TFIID complex; however, upon terminaldifferentiation into myotubes, the TFIID complex was replaced by aTRF3–TAF3-containing complex (Fig. 6). These !ndings suggest thatterminally differentiated cells may employ specialized transcriptionsystems that are dedicated to the particular functions of the cells. Itwill be interesting to see if an analogous effect is observed in thedifferentiation of other cell types.

Conclusions and perspectives

The core promoter and the basal transcriptional machinery aretwo important yet relatively unexplored dimensions in the regulationof gene expression. It is now apparent that diversity in the structureand function of core promoters and basal transcription factorscontributes to developmental processes that lead to organismalcomplexity (Levine and Tjian, 2003). Thus, in the future, it will beessential to consider and to incorporate these factors in the analysis ofgene regulation. For instance, transcriptional enhancers would ideallybe studied in conjunction with their cognate core promoters.Alternatively, a new generation of reporter vectors could be designedwith core promoters that function with both TATA- and DPE-speci!c

Fig. 4. Caudal is a DPE-speci!c activator. Caudal preferentially activates transcriptionfrom DPE-dependent core promoters relative to TATA-dependent core promoters. Inaddition, the presence of a BREu motif upstream of the TATA box further suppresses theability of Caudal to activate transcription. The BREu motif does not affect the ability ofCaudal to activate a DPE-dependent core promoter. The TATA box also does not alter theability of Caudal to activate transcription via the DPE motif (not shown). Results takenfrom Juven-Gershon et al. (2008a).

Fig. 5. A simpli!ed, hypothetical diagram of activation by DPE- and TATA-speci!cfactors. Transcription factors bind to enhancers but only activate transcription frompromoters with the appropriate core promoter elements. The core promoter containingboth TATA and DPE motifs can be activated by either DPE- or TATA-speci!c activators.Transcription levels can be further regulated by the presence of the BREu as well asother core promoter motifs.

Fig. 6. Replacement of the canonical TFIID complex by a TRF3–TAF3-containingcomplex upon terminal differentiation of myoblasts into myotubes. Both complexesbind to TATA box motifs via the TBP or TRF3 subunits. These !ndings exemplify theestablishment of a new basal transcription system upon cell differentiation and suggestthat analogous processes may occur in other cell types. Results taken from Deato andTjian (2007).

228 T. Juven-Gershon, J.T. Kadonaga / Developmental Biology 339 (2010) 225–229

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.

• 1. May assemble different pre-initiation complex with specific features required for cell-specific regulation. One example is tuning between enhancers and promoters.

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 binds minor grooveof the TATA box.

• TFIIF - ATP dependenthelicase activity2 proteins,also reduces affinity ofpolymerase for non-promoter DNA

A

TATA Inr

TFIID A

TFIID

TFIID A

B

B

Pol II

B F

Pol II

B F

Pol II

B FPol II

F

E

H

H

TFIID APol II

B FH

Holoenzyme

DAB complex

Complete

TFIID A

E

E

mediatormediator

mediator

• TFIIH - kinase that phosphorylates YSPTSPS(CTD domain)

• Unphosphorylated RNAP= RNAPIIA= initiation specific

• Phosphorylated RNAP= RNAPIIO= for chain elongation

• TFIIH also has helicaseactivity

A

TATA Inr

TFIID A

TFIID

TFIID A

B

B

Pol II

B F

Pol II

B F

Pol II

B FPol II

F

E

H

H

TFIID APol II

B FH

Holoenzyme

DAB complex

Complete

TFIID A

E

EA

TATA Inr

TFIID A

TFIID

TFIID A

B

B

Pol II

B F

Pol II

B F

Pol II

B FPol II

F

E

H

H

TFIID APol II

B FH

Holoenzyme

DAB complex

Complete

TFIID A

E

E

mediatormediator

mediator

• TFIID

Upstream element

upstream elements BRE TATA INR DCEI DCEII DCEIIIDPE

G/C G/C G/C CGCCC

TATA A/T A A/T Py Py AN T/A Py Py CTTC RG A/T CGTG

AGC

-37 to -32

-31 to -26

-2 to +4

+6 to +11

+16 to +21

CTTC

+28 to +30

+32 to +34

SP1

TFIID TFIID TFIID

TFIIE, IIH, RNAPolII

TFIIA

TFIIB IIB

Core promoterProximal Promoter

5ʼ 3ʼ

INRDCE

DCE

DPE

DCE IIIII

I

TATABRE

-37 to-32

-31 to -26

-2 to +4

+6 to -11

+16 to +21

+28 to +30

+32 to +34

Upstream

<-- upstream downstream -->

core promoter

+1

proximal promoter elements

TFIID TFIID TFIID

TFIIB TFIIE, IH, RNApolI

TFIIASP1

TFIID: What is TFIID?

TFIID=TBP + 8-10 TAFIIS

TBP

TAF250

TAF110

TAF150

40

30!

TAF60

30"

80

TFIID

• Crystal structureof TBP suggesteda saddle.

• TBP DNA co-crystalindicated that notlike a saddle on ahorse.

TFIID: TBP can bend DNA

TFIID: Who recognizes what?

• TATA-less promotersare not recognized by TBP

• TAFII250 & TAFII150impart ability torecognize 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

Kinase activity of TFIIH activates polymerase for elongation.

ATP-dep DNA helicase activity of TFIIH is required for promoter clearance.

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 complexeg. 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 visibleChanges to histones can stabilize the preinitiation complexChanges 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 silencersThese 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.