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 • 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 TATA Inr TFIID=TBP + 8-10 • A TAFIIS A B TFIID • TFIID binds minor groove BTFIID A of the TATA box. DAB complex Pol II • TFIIF - ATP Pol II B F dependent F Pol II Pol II E helicase activity A B F H BTFIIDF mediator mediator 2 proteins, Holoenzyme also reduces E affinity of Pol II E H A polymerase for BTFIIDF H mediator non- Complete promoter DNA TFIID TATA Inr A A B TFIID BTFIID A Upstream TFIIDDABTFIID complexTFIID TFIIB TFIIE, IH, RNApolI element TFIID TFIID TFIID SP1 TFIIA +1 TFIIB IIB Pol II Not <-- upstream downstream --> TFIIE, IIH, RNAPolII shown Pol II 5ʼ TFIIA 3ʼ B F SP1 Upstream TATA INR DCE F II DCE - CpG III BRE DCE upstream elements BRE TATAI INRDPE DCE DCE DPE DCE -31 to -26 I II III islands -2 to +4 +16 to +21 +6 to +32+11 to +34 +32 to +34 -31 to -26 Pol II E -37 to-32 +6 to -11Pol II+28 to +30 -37 to -32 -2 to +4 +16 to +21 +28 to +30 G/C G/C G/C CGCCC ACTTC AGC B F H TATA A/T A A/Tcore promoterBPyTFIID Py ANF T/A Py Py CTTC RG A/T CGTG mediator mediator proximal promoter elements Proximal Promoter Core promoter Holoenzyme E • BRE: TFIIB binding element E • TATA: TATA box Pol II H A BTFIIDF • INR: Initiator element H mediator • DCE: Downstream core element Complete • DPE: downstream promoter element TFIID TFIID TFIID TFIIB TFIIE, IH, RNApolI SP1 TFIIA +1 <-- upstream downstream --> 5ʼ 3ʼ Upstream TATA INR DCE II DCE III BRE DCE I DPE -31 to -26 -2 to +4 +16 to +21 +32 to +34 -37 to-32 +6 to -11 +28 to +30 core promoter proximal promoter elements A few common combinations Not shown: TATA or a DPE usually not both MTE stands for "motif ten element" TATA with DCE Found at +18 to +29 TATA INR Found in Drosophila INR DPE 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 390 N. D. Heintzman and B. Ren Eukaryotic transcriptional promoters 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 downstreamTFIID of the promoter,TFIID while methylation of histoneTFIID 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 isTFIIB a composite TFIIE,of features IH, determined RNApolI in yeast, fly and mammalian systems; it is 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- gene regulation, revealing a collection of short regula<-- upstream- mined downstream by comparison --> of 5a flanking regions in several tory DNA sequence elements conserved across species. organisms5ʼ [31]. The 3TATA-boxʼ is located approximately 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 up- been discovered recently, emphasizing the importance of stream [32]. It is typically recognized by the TATA bind- continued research of these Upstreamregulatory sequences. TATAMost ingINR protein (TBP)DCE subunit of the general transcription II DCE III of the canonical core promoter elements haveBRE been thor- factor TFIIDDCE [33], though additional related but distinct oughly reviewed elsewhere [2], but it is useful to describe proteins can alsoI recognize DPEthis 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 are no ‘universal’ core promoter elements; the-37 sequences to-32 promoters+6 containing to -11 or lacking+28 to +30 a TATA-box. While the described below are found in only a subset of promoters, Inr can stimulate transcription independently of a TATA- and 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 consensusproximal promoter sequence elements (TATAWAAR; degener- The downstream promoter element (DPE; RGWYV) [38] 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 TATA- box [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 tran- ments. scription [40]. Like the TATA-box and Inr, this element is Core Position Consensus Frequency in pro- recognized by TFIID, likely the TAF6 and TAF9 subunits, element 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 interac- Flies Vertebrates tions with enhancers [42] and transcriptional activation or TATA approx. TATAWAAR 33–43% 10–16% repression [43], suggesting multiple regulatory mecha- –31 to –26 nisms acting at the core promoter. Inr –2 to +4 YYANWYY 69% 55% The TFIIB recognition element (BRE; SSRCGCC) con- sists of the 7 bp immediately upstream of the TATA-box, DPE +28 to RGWYV 40% 48% +32 and as its name suggests, it is bound by transcription fac- BRE approx. SSRCGCC – 12–62% tor IIB [44]. The BRE has been shown to both stimulate –37 to and repress transcriptional activity [45]. –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 * The TSS is assigned to position +1. and overlapping slightly with the 5a-end of the DPE. The ** Degenerate nucleotides represented using IUPAC codes. MTE requires Inr and functions synergistically with the TFIID TFIID TFIID TFIIB TFIIE, IH, RNApolI SP1 TFIIA +1 <-- upstream downstream --> 5ʼ 3ʼ Upstream TATA INR DCE II DCE III BRE DCE I DPE -31 to -26 -2 to +4 +16 to +21 +32 to +34 -37 to-32 +6 to -11 +28 to +30 core promoter proximal promoter elements Why so many elements? 228 T. Juven-Gershon, J.T. Kadonaga / Developmental Biology 339 (2010) 225–229 TRF1 does not exist in yeast and humans but is present in Droso- phila.Inmanyeukaryotes,includingyeastandhumans,TBP participates in transcription by RNA polymerases I, II, and III.