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 MODULAR STRUCTURE OF UPSTREAM TRANSCRIPTION FACTORS Experimental approaches DNA binding domains (DBD) Transcription regulation domains (TAD, TRD) Ligand binding domains RG Clerc. March 16, 2016

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

3 MODULAR STRUCTURE OF UPSTREAM TRANSCRIPTION FACTORS Experimental approaches gel shift assay (EMSA) DNA binding domains (DBD) (modular highly ordered structure, classification of upstream TF) Transcription regulation domains (TAD, TRD): (induced structure, not ordered on their own) Hormone binding domains (structured modules, pharmacological tractable)

4 Typical gene and components involved in gene activation and inactivation: the players in transcription regulation S. Buratowski Nature 483: Assembly of the RNA Pol II preinitiation complex (PIC) and the major players in transcription regulation DBDs are responsible for localizing a TAD domain in the proximity of the basal apparatus, recruiting co-factors specificity

5 Modular structure of upstream transcription factors Transcription regulation (TAD, TRD) Structural domain DBD GENE DNA binding site (RE) N Structural domain DBD TAD C N Structural domain DBD TRD C

6 Modular structure of upstream transcription factors N Structural domain DBD 1 X X TAD 1 Structural domain DBD 2 C N TAD 2 C N domain swapping experiment Structural domain DBD 2 TAD 1 C

7 Electrophoresis mobility shift assay (EMSA): a corner stone in deciphering DNA binding specificities

8 EMSA competition experiment DNA methylation interference assay

9 DNase I footprinting assay

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11 Affinity chromatography to purify DNA:protein and protein:protein interactions

12 Staudt L. Clerc RG and Baltimore D. Science 241: Southwestern: Expression cloning of DNA binding protein eg. cloning of POU domain Oct-2

13 Bertolino E. Clerc RG. J Biol Chem 270: TRX Factor Binding Assay on Random DNA oligomers: PCR Binding Site Selection

14 Bertolino E. Clerc RG. J. Biol Chem.270: TRX Factor Binding Assay on Random DNA oligomers: PCR Binding Site Selection

15 DNA Binding Domains (DBD) DNA binding domain: the motif mindset concept: no one had anticipated that basic stuctural scaffolds would be used over and over again to recognize DNA and other proteins. Surprisingly large variety of protein familiy motifs involved in DNA sequence -specific recognition (promoter/enhancer contain matrices of short consensus sequences (RE) The role of DNA binding domains is to specifically deliver the transcription activation domain into the vincinity of the PIC

16 DNA Binding Domains (DBD) When a new protein was discovered, its gene cloned, the first questions were and to some extends still is: does it has any motifs we can understand? A helix-turn-helix? A zinc finger? An HMG domain? A β sheet DNA Motif? Uptream Trx factors are grouped with respect to their respective highly conserved DNA binding domains (DBD) In the case of DNA binding transcription factors the initial concept came from major stuctural determinations of bacterial regulators (lambda phage repressor, lambda Cro, Trp, 434 repressor, and CAP (catabolite activator protein from E. coli)

17 Number of Swissprot (SW) entries for the respective DNA binding folds helix-turn-helix zinc finger helix-loop-helix leucine zipper A proteome-wide classification of DNAbinding protein DBD families

18 Helix-Turn-Helix Motif (HTH) Domain: high degree of homology between bacterial and eukaryotes DBD

19 Typically relatively short motifs are responsible for specific binding to DNA

20 Homeodomain-DNA Complex (PDB 1hdd) 5 Computer drawing of the tridimensional structure as determined by X-Ray crystallography of Engrailed homeodomain Gln50 Ile47 Asn51 T A A Arg5 Arg3 T T 3 Vollmer JY and Clerc RG J. Neurochem. 71:1-20

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22 Homeodomain protein classes implicated in the mouse brain development Vollmer JY and Clerc RG J. Neurochem. 71:1-20

23 Body plan is constructed through interaction of the developmentally regulated homeotic genes posterior late low RA response anterior early high RA response trunk hindbrain Duboule D. editor Guidebook Homeoboxgenes. Oxford Press 1994

24 Herr W. Clerc RG and Baltimore D. Genes & Dev 12: The POU domain: a large conserved region in the mammalian pit-1, oct1,2 and C. elegans unc96 gene products

25 POU domain DNA interaction 5 ATGCAAAT 3 (Octamer) Müller-Immerglück M, Matthias P. EMBO J. 9:

26 A zinc finger domain is a DBD Originally identified in TFIIIA (5S rrna), Zinc fingers form alpha helices that insert into the major groove associated with stabilizing beta sheets

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28 DNA binding in vitro transcription TFIIIA-TFIIIC-DNA recruitment of TFIIIC TFIIIA-DNA DNA Rothfels K et al. (2007) Nucl Acid Res 35: TFIIIA contains 9 sequential CysHis zinc fingers with distinct DNA binding and TRX functions

29 Originally identified in TFIIIA (5S rrna), Zinc fingers form alpha helices that insert into the major groove associated with stabilizing beta sheets

30 Targeted genome editing using zinc finger nucleases: custom DBD for transcription factors and nucleases ( Individual zinc finger domains that recognize DNA triplets with high specificity and affinity are fused to nonspecific nuclease domain to create designer TRX F for gene targeting

31 Tumor-specific missense mutations within CTCF transcriptional repressor/activator zinc finger domains Tumor-specific mutations within CTCF Zf selectively alter CTCF DNA binding selectivity to different genome target sites Filippova GN et al Cancer Research 62:48-52

32 A distinct form of Zn finger motifs in Nuclear Receptors

33 Glucocorticoid Receptor-DNA Complex (PDB 1glu) Zn finger K461 V462 R466 5 A G A A C A NNN T G T T C T3 T C T T G T NNN A C AA G A

34 Generation of DNA binding specificity mutants: glucocorticoid receptor ZnF DBD acquires estradiol specificity (ERE)

35 bzip domain: basic leucine zipper (leucine residues spaced by 7 AA residues) bzip domain: prototypical amphipathic coiled-coil alpha helix

36 c-fos-c-jun-dna Complex (PDB 1fos) 5 T G A G T C A 3

37 NF kappa B system pathways mediate cellular responses to a wide variety of stimuli including TNFalpha and lymphotoxine beta

38 Only one of the two DNA-bound orientations of AP-1 (fos-jun) found in solution cooperates with NFATp Oakley M. and Verdine GL. Curr Biol 5:882 (1995)

39 Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp Oakley M. and Verdine GL. Curr Biol 5:882 (1995)

40 Helix-Loop-Helix Domains: Prototypical TRXF dimerizers Each amphipathic helix presents a face of hydrophobic residues on one side and charged residues on the other side bhlh proteins typically bind to a consensus sequence called an E-box, CANNTG

41 HMG High Mobility Group Motif (eg.lymphoid (T-cell) enhancer binding factor LEF-1)

42 DNA bending analysis by EMSA

43 LEF1 HMG Domain-DNA Complex (PDB 2lef) HELIX 3 HELIX 2 HELIX 1 Y76 L6 M10 M13 BASIC C-TERM E28 5 C C T T T G A A G C 3

44 LEF1 HMG Domain-DNA Complex (PDB 2lef) 5 C C T T T G A A G C 3

45 TBP : TATAA Binding protein

46 Transcription Activation Domains -TADs Acidic domains (DDD, EEE), VP16, GAL4, GCN4, GR Glutamine-rich domain (QQQXXXQQQ), SP1 Proline-rich domains (PPPXXXPPP), CTCF, AP2 Isoleucine-rich domain IIXXII), NTF Several motifs involved in transactivation protein motives commonly referred to as negative noodles, acid blobs! (Sigler P. Nature 333: )

47 Transcription Activation Domains -TADs in contrary to DBDs, few structural data on TADs available TADs are intrinsically disordered unfolded proteins (resistant to crystal packing) TADs indirectly recruit Pol II by recruiting coactivators that serve as physical bridge between TADs and PIC/RNA Pol II holoenzyme TADs were reported to become structured (eg p53 helical TAD) upon binding to co-activators Wells et al. PNAS 105:

48 Ser/Thr and Gln Transcription activation domains - SP1, a prototypical constitutive TAD (Gill G., Tjian R. PNAS 91: ) 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)- SP1 contains two glutamine-serine/threonine-rich TADs which interact with TAF110 (Gill g., Tjian R. PNAS 91: )

49 Prototypical ligand mediated transcription activation domain (eg acidic negatively charged GR TAD) mouse trap unliganded co-repressor binding liganded co-activator binding

50 HPV transactivation domain TAD forms a dimer Antson AA et al. Nature 403: Harris SF, Botchan M. Science 284:

51 HPV transactivation domain E2 binding site E2 binding site Knight JD, Botchan M. PNAS 88: Antson AA et al. Nature 403:

52 Transrepression Domain (TRD) Canonical transrepression domains in upstream transcription factors eg. Krüppel associated box (KRAB) containing zinc-finger repressor proteins are associated with strong transcriptional repression of RNA polymerase I, II and III Transcriptional repression of eukaryotic promoters by direct, (recruitment of corepressors), or indirect, eg. HDAC s recruitment to form heterochromatin, other chromatin modifications Urrutia R. (2007) Genome Biology 4:231

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