Transcription Regulation And Gene Expression in Eukaryotes FS 2016 Graduate Course G2 P Matthias and RG Clerc Pharmazentrum Hörsaal 2 16h15-18h00

Transcription

1 Transcription Regulation And Gene Expression in Eukaryotes FS 2016 Graduate Course G2 P Matthias and RG Clerc Pharmazentrum Hörsaal 2 16h15-18h00 The general problem RG Clerc March 2, 2016 RNA

2 Transcription factor binding sites convey specificity to regulation RNA

3 Systematic Discovery of TFBS by Comparative genomics: key learnings Checking for promoter region only is bad! Checking for full loci (entire trx unit) is highly recommended! Check for modules of several factor binding sites Genomic sequences come in different qualities Need to mask problematic bad sequences, parts which do not make sense Do we detect transcription factor binding sites because they were conserved during evolution? Or the sequences have been conserved because they contain a functional transcription factor binding site?

4 Transcript Imaging

5 The functional complexity of the human transcriptome is not yet fully elucidated Transcribed portions of the genome are larger and more complex than expected; transcripts based on untranslated regions or noncoding RNA 65 % of the polyadenylated transcriptome maps to known genes and 35% to non annotated genomic regions Sultan et al (2008) Science, 321, 956

6 Functional classes of eukaryotic promoters 3 RNA Polymerases: Pol I rrna Pol II mrna (small RNAs) Pol III small RNAs Pol IV, V plants 3 categories of promoters Architecture of yeast RNA polymerase II (backbone models for the 10 subunits as ribbon diagram) Cramer P et al. Science 288:

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8 Definition of regulatory elements

9 Definition of regulatory elements

10 Binding Assay on Random DNA oligomers: PCR Binding Site Selection

11 Pol I promoters

12 Eukaryotic Pol II promoter: upstream and core NF-kB Fos Oct-1 CBFA-1 CREB etc Transcription activators TATA Basal machinery INR GENE RNA PolII 12 TFIIA 3 TFIIB 1 TFIID (TBP) ca. 14 (1) TFIIE 2 TFIIF 2 TFIIH ca. 9 upstream (regulatory, enhancer) core

13 The core Pol II promoter in more detail BRE TATA box G/CG/CG/CCGCC TATAAA Initiator PyPyANT/APyPy DPE GA/TCGTG TFIIB TFIID TFIID TFIID TFIIA TBP TAF II 250 TAF II 150 TAF II 60 TAF II 40

14 The TATA box Matrices are derived from known binding sites or binding assays on random DNA oligomers

15 Definition of the upstream promoter elements β Example: CAAT (4 bases) average match 1 / 4>4 (1/256 bp), 12 10>6 matches in the genome!

16 Transcriptional enhancers/silencers: First identified in viruses, then in cellular genes Activate (or repress) transcription independently of position (distance) and orientation (no polarity) Are made (and can be created) from partially redundant modules Confer cell-specific or temporal regulation No activity + Reporter Gene Low activity High activity

17 Identification of enhancer modules Enhancer activity is the result of interaction between individual modules «Super enhancers» controversial class of enhancers reported in genomic proximity with high level of mediator binding (p300, CBP, and histone marks) Pott S. (2015) Nature 47:8

18 Pol III promoters

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20 Systematic discovery of transcriptional regulatory motifs by comparative genomics Paper presentation

21 The Basics of Transcription - I Methods used to study transcription: in vitro assays Transcription proceeds in several steps DNA-dependent RNA polymerases General Transcription Factors: GTFs

22 The Players in Transcription Regulation DNA-binding transcription factors (upstream factors) Chromatin regulators Coactivators and corepressors: Mediator, etc.. Basal Machinery: RNA Pol, GTFs Chromatin regulators Mediator Coactivator Basal Machinery TFIIH TFIIE TFIID RNAPII TFIIA TFIIB TFIIF DNA-binding TFs Gene

23 In Vivo Transcription Assays

24 In Vitro Transcription Assays Purified DNA templates (plasmids) are transcribed in the test tube with nuclear extracts, highly purified or recombinant transcription factors Criteria: accuracy and quantity of transcripts The ultimate goal is the reconstitution of regulated gene expression from recombinant (i.e. completely defined) components

25 In Vitro Transcription Assays All methods require some knowledge about the DNA fragment containing the promoter of interest where is the natural start site (+1)? 5 3 mrna

26 Many transcripts will be accurately initiated on the promoter of interest, but there will be a degree of non-specific background transcription spurious promoters; gaps/nicks in template DNA Different methods are available for quantitating in vitro transcripts: primer extension assay run-off assay G-less cassettes S1 nuclease protection assay

27 Methods: primer extension, run off transcripts and G-less cassettes

28 Methods: S1 Nuclease Protection Assay Single strand-specific nuclease enzymes will not digest doublestranded nucleic acid substrates this includes RNA:RNA or RNA:DNA hybrids S1 endonuclease mrna is hybridised to an uniformly radiolabelled single-stranded DNA fragment containing the start site; the probe can also be end-labelled mrna hybridised to the DNA-fragment will protect it right up to the start sequence from degradation

29 Methods: S1 Nuclease Protection Assay 1. In vitro Transcription RNA 2. Prepare radiolabeled probe * * * * * * * * * * * * * * * * * * * * * * 3. Hybridize radiolabeled probe with RNA 4. Digest with nuclease S1 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

30 Basal vs Activated Transcription: an in vitro concept NF-kB Fos Pu.1 Oct-1 CBFA-1 etc Transcription activators TATA Basal machinery INR TFIIH TFIIE TFIID GENE RNAPII TFIIA TFIIB TFIIF RNAPII 12 TFIIA 3 TFIIB 1 TFIID (TBP) ca. 14 (1) TFIIE 2 TFIIF 2 TFIIH 10 upstream (regulatory, enhancer) core Cofactors, Mediator

31 Transcription Cycle: Multiple Steps, Multiple Opportunities for Regulation

32 Transcription Cycle: Status of Promoter- Proximal Pol II : Paused, Stalled, Poised, etc? Paused: elongation complexes halted temporarily Stalled: elongation complexes that have stopped RNA synthesis Poised: RNA polymerase detected near a gene promoter (ChIP assay terminology) Backtracked: halted elongation complexes that move backwards Arrested: complexes cannot restart transcription without additional factors

33 Transcription Initiation Steps Increasing Commitment Promoter (Start Site) Recognition Promoter Binding Promoter Melting Transcript Initiation Promoter Escape/Clearance Transcript Elongation

34 Sequential assembly of Initiation Complex Stepwise recruitement of general transcription factors or TFIIA PIC: preinitiation complex (DNA, RNAPII, TBP, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH Buratowski S and Sharp PA. Cell 56:549

35 Recruitment of preassembled RNAPII holoenzyme and general transcription factors two component pathway

36 Eukaryotic RNA polymerases: Original Discovery AMANITIN BINDING AS STABLE COMPLEX TO CALF THYMUS RNA POLYMERASE B (1970) Kédinger C. and Chambon P. FEBS Letter 9: alpha-amanitin, a toxin from the mushroom Amanita phalloides, is a potent inhibitor of DNA-dependent RNA polymerase II

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38 Eukaryotic RNA polymerases: Contacts between Alpha-amanitin and RNA Pol II as of Today Bruekner F. and Cramer P. (2008) Nature S&MB 15: alpha-amanitin chemical formula and stick model shown in orange, RNA Pol II residues and as ribbon model in grey

39 Engel Nature 2013

40 Cramer P et al. (2008) Ann Rev Biophys 37: Cramer P et al. (2000, 2001) Science 288: 642

41 RNA Polymerase I Initiation Engel Nature 2013

42 RNA Polymerase I : Purification and Crystalization litres S. cerevisiae Engel C. and Cramer P. (2013) Nature 502:

43 RNA Polymerase I Engel C. and Cramer P. (2013) Nature 502:

44 RNA Polymerase II All eukaryotic RNAPIIs characterized so far contain 12 different subunits (involved in transcription initiation, elongation and termination) RPB1 to RPB12 RNAPIIs are highly conserved across eukaryotic species 53% overall identity between yeast and human RNAPII The two largest subunits, RPB1 and RPB2, contain the catalytic center Four subunits (RPB3, RPB10, RPB11 and RPB12) serve as an assembly scaffold for RPB1/RPB2 Post-transcriptional mechanisms also depend on RNAPII capping splicing 3 end formation

45 Cramer, Bushnell and Kornberg (2001). Structural basis of transcription: RNA polymerase II at 2.8 Angstrom resolution. Science 292,

46 RNA Polymerase II: Initiation-elongation transition Kostrewa D, Cramer P. (2009). Nature 462:

47 Structural Conservation: 53% overall Identity between Yeast and Human all across distributed Residues Identical Between Yeast and Human RNAPII Residues Identical Between Yeast and E. coli

48 Structural Conservation: overall Homology Sequence Homology Between Yeast RNAPII and Bacterial RNAPs Structural Homology Between Yeast RNAPII and Bacterial RNAPs

49 Differences between Pol I and Pol II

50 The Carboxy Terminal Domain: CTD repeating heptapeptide sequences: 52 repeats human, 26 in yeast Tyr-Ser-Pro-Thr-Ser-Pro-Ser YSPTSPS n target for phosphorylation RNA Pol II O = phosphorylated RNA Pol II A = unphosphorylated form RNA Pol II is phosphorylated at Ser5 (Ser7)to initiate elongation RNA Pol II is phosphorylated at Ser2 after bp +50 during elongation Phosphorylation is removed for termination

51 The Carboxy Terminal Domain: CTD

52 The Carboxy Terminal Domain: CTD RNA Kinase chip-chip occupancy. Mayer A and Cramer P (2010) Nature 17: 1272

53 RNA Polymerase III : Rapid Repression by Maf1 ensures cell survival during stress Pol III specific subunits on Pol II structure (C53, C34, C82) ribbon diagram of Maf1 crystal Vannini A. and Cramer P. (2010) Cell 143:59-70

54 Prokaryotic vs eukaryotic RNA Polymerases In the presence of σ factors, bacterial RNA polymerases (RNAPs) can recognize promoters without the help of any other transcription factors Although eukaryotic RNAPs are structurally much more complex, they do not have any obvious sequence-specific binding activities by themselves. Promoter recognition requires large number of additional factors (GTFs).

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56 Cramer et al. (2008) Ann Rev Biophys 37:

57 E. coli RNA polymerase Hochschild A and Dove SL. Cell 92: 597 (1998)

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59 σ 54 is dedicated for long-range activation: response to nitrogen deprivation in prokaryotes Su et al. DNA looping and enhancer activity at glutamine synthetase gene. (1990) PNAS 87: 5504

60 Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less Marie Curie Sklodowska