Transcription factors

Transcription

1 Transcription factors R. de Martin Department of Vascular Biology and Thrombosis Research Medical University of Vienna

2 K63 A20, TTP, XIAP IκBα K48 IL-6, Il-8, MCP-1, ICAM1, SELE, ciap1/2, A20, IκBα Adapted from Beug, Cheung et al. (2012)

3 1. Transcription - general mechanisms 2. Transcription factors: structure-function 3. Methods: what can you do? 4. Regulation of TFs de Martin - Transcription

4 EUCHROMATIN Low-density (as defined by electron microscopy) regions in the nucleus that are thought to contain open chromatin structures associated with the active transcription of genes. HETEROCHROMATIN High-density regions in the nucleus that are thought to contain compacted chromatin structures associated with silent genes. SILENCERS DNA elements that repress promoter and/or enhancer activity in a position- and orientationindependent manner, possibly by influencing chromatin structure. MATRIX-ATTACHMENT REGIONS (MARs).DNA elements that are thought to tether loops of chromatin to specific sites in the nuclear matrix. Occur every kb. PROMOTER The regulatory region of DNA to which RNA polymerase binds to initiate transcription. ENHANCER A control element to which regulatory proteins bind, thereby influencing the rate of gene transcription. Enhancers function in an orientation- and positionindependent manner. BASIC CHROMATIN ELEMENTS: CIS ELEMENTS Methylated DNA Demethylated DNA Promoters Enhancers Matrix-attachment sites TRANS ELEMENTS Transcription factors RNA polymerases Chromatin-remodelling complexes Histone-modification enzymes de Martin - Transcription

5 The transcription initiation complex contains the TATA box binding protein, RNA Pol.II, general TFs, and interacts with other regulatory proteins de Martin - Transcription

6 Genes are not transcribed evenly throughout the nucleus but at dedicted sites: Factories Chromosome conformation capture (3C) Papantonis,..Cook, EMBO J 2012 de Martin - Transcription

7 min. TNF stim. SAMD4A: a TNF responsive gene, or EDN1: neg. control (green); + 7 TNF target genes (red) Papantonis,..Cook, EMBO J 2012 de Martin - Transcription

8 How to achieve tissue/celltype-specific gene expression Zhang and Glass, 2013 de Martin - Transcription

9 Zhang and Glass, 2013 de Martin - Transcription

10 Transcription factors have a modular structure: Domains: DNA binding Transactivation Dimerization Nulclear localization (NLS) Nuclear export Protein stability Other interaction domains Co-Aktivators Modular TFs de Martin - Transcription

11 DNA binding: Binding involves hydrogen bonds, ionic bonds, and hydrophobic interactions 20 contacts Tightest known in biology Specificity a helices or ß sheets de Martin - Transcription

12 Transcription factors recognize specific DNA sequences (bacteriophage lambda Cro protein): de Martin - Transcription

13 1 Superclass: Basic Domains Examples: Leucine Zipper Factors (Jun, Fos, CREB) Leucine zippers also mediate protein-protein interactions!! Phe-Ile zipper de Martin - Transcription

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15 2 Superclass: Zinc-coordinating DNA-binding domains ( Zinc Fingers ) Examples: Steroid hormon Rs, Thyroid, Retinoid, Vitamin D, PPAR ZF type TFs are usually composed of several individual ZFs cognate Zn fingers, each recognizing a specific short DNA sequence combination -> artificial TFs de Martin - Transcription

16 de Martin - Transcription GR GR

17 3 Superclass: Helix-turn-helix Examples: Homeo domain, Paired box, Forkhead Heat shock, IRFs Helix fits into major groove. Blue helix acts to position the recognition helix. Usually act as dimers Homeodomain: A HLH subtype. Side chain of helix 1 also contacts minor groove. de Martin - Transcription

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19 4 Superclass: ß-Scaffold Factors with Minor Groove Contacts Examples: NF-kB/Rel, NF-AT, p53 de Martin - Transcription

20 The bacterial met repressor: an example for DNA binding through ß-sheets de Martin - Transcription

21 The CRISPR/Cas9 system for gene editing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR associated (Cas) nuclease, Cas9 de Martin - Transcription

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24 SAM: CRISPR/Cas9 Synergistic Activation Mediator / Repression de Martin - Transcription

25 TALENs gene targeting for the masses? Engineered transcription activator-like (TAL, from Xanthomonas ssp.) effector nuclease (TALEN) cartoons. (A) Engineered TALE DNA binding domain recognizes specific bases by using a known cipher involving two key amino acid residues. These amino acids are embedded in a amino acid repeat. Nuclear access is provided by the native nuclear localization signal (NLS). (B) Total sequence specificity and formation of a fully functional nuclease is achieved from each of the two TALEN monomers after dimerization on the target sequence in the genome. (Clark, 2011) de Martin - Transcription

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27 Transactivation domains: do not show any strong amino-acid sequence homology to each other have large proportion of acidic amino acids producing a strong net negative charge (Hollenberg et al,.1988) or consists of glutamine-rich (Gerber et al.,1994) or proline-rich sequences (Mermod et al.,1989) de Martin - Transcription

28 Wiesner et al. Nucl. Acids Res de Martin - Transcription

29 Proline-rich regions: MDHTDNELQGTNSSGSLGGLDVRRRIPIKLISKQASKVKPAPRTQRTVSRMPAK APQGDEEGFDYNEEQRYDCKGGELFGNQRRFPGHLFWDFKINILGEKDDTPVHF CDKCGLPIKVYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGS LFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVHPPIAPPPTDIP DRFIMPPDKHHMSHIPPKQHIMMPPPPLQHVPHEHYNQPHEDIRAPPAELSMAP PPPRSVSQETFRISTRKHSNLITVPIQDDSSSGAREPPPPAPAPAHHHPEYQGQPV VSHPHHIMPPQQHYAPPPPPPPPISHPMPHPPQAAGTPHLVYSQAPPPPMTSAP PPITPPPGHIIAQMPPYMNHPPPGPPPPQHGGPPVTAPPPHHYNPNSLPQFTEDQ GTLSPPFTQPGGMSPGIWPAPRGPPPPPRMQGPPSQTPLPGPHHPDQTRYRPYY Q de Martin - Transcription

30 Glutamine-rich regions: MKTRQNKDSMSMRSGRKKEAPGPREELRSRGRASPGGVSTSSSDGKAEKSRQTA KKARVEEASTPKVNKQGRSEEISESESEETNAPKKTKTEQELPRPQSPSDLDSLDG RSLNDDGSSDPRDIDQDNRSTSPSIYSPGSVENDSDSSSGLSQGPARPYHPPPLFPP SPQPPDSTPRQPEASFEPHPSVTPTGYHAPMEPPTSRMFQAPPGAPPPHPQLYPGG TGGVLSGPPMGPKGGGAASSVGGPNGGKQHPPPTTPISVSSSGASGAPPTKPPTTP VGGGNLPSAPPPANFPHVTPNLPPPPALRPLNNASASPPGLGAQPLPGHLPSPHAM GQGMGGLPPGPEKGPTLAPSPHSLPPASSSAPAPPMRFPYSSSSSSSAAASSSSSSSS SSASPFPASQALPSYPHSFPPPTSLSVSNQPPKYTQPSLPSQAVWSQGPPPPPPYGR LLANSNAHPGPFPPSTGAQSTAHPPVSTHHHHHQQQQQQQQQQQQQQQQQQ QHHGNSGPPPPGAFPHPLEGGSSHHAHPYAMSPSLGSLRPYPPGPAHLPPPHSQVS YSQAGPNGPPVSSSSNSSSSTSQGSYPCSHPSPSQGPQGAPYPFPPVPTVTTSSATL STVIATVASSPAGYKTASPPGPPPYGKRAPSPGAYKTATPPGYKPGSPPSFRTGTPP GYRGTSPPAGPGTFKPGSPTVGPGPLPPAGPSGLPSLPPPPAAPASGPPLSATQIKQ EPAEEYETPESPVPPARSPSPPPKVVDVPSHASQSARFNKHLDRGFNSCARSDLYF VPLEGSKLAKKRADLVEKVRREAEQRAREEKEREREREREKEREREKERELERSV KLAQEGRAPVECPSLGPVPHRPPFEPGSAVATVPPYLGPDTPALRTLSEYARPHV MSPGNRNHPFYVPLGAVDPGLLGYNVPALYSSDPAAREREREARERDLRDRLKP GFEVKPSELEPLHGVPGPGLDPFPRHGGLALQPGPPGLHPFPFHPSLGPLERERLA LAAGPALRPDMSYAERLAAERQHAERVAALGNDPLARLQMLNVTPHHHQHSHI HSHLHLHQQDAIHAASASVHPLIDPLASGSHLTRIPYPAGTLPNPLLPHPLHENEV LRHQLFAAPYRDLPASLSAPMSAAHQLQAMHAQSAELQRLALEQQQWLHAHHP LHSVPLPAQEDYYSHLKKESDKP de Martin - Transcription

31 The 9aa TAD (Piskacek et al., 2007) de Martin - Transcription

32 Methods to analyze Transkription Factors Questions: Identification Structure-function analysis Is it active, under which conditions? Band shift (EMSA) Reporter gene analysis TAD analysis Identification of binding squence Affinity purification Yeast 1 hybrid screen ChIP de Martin - Transcription

33 Electrophoretic Mobility Shift Assay (EMSA) Protein: nuclear extract recombinant protein de Martin - Transcription

34 EMSA incl. Supershift : competitions de Martin - Transcription

35 Transcription factor ELISA: de Martin - Transcription

36 Chromatin Immunoprecipitation (ChIP): de Martin - Transcription

37 Chromatin Immunoprecipitation (ChIP): de Martin - Transcription

38 ABCD (Avidin Biotin Complex DNA) precipitation assay Hubner et al., J. Proteome Res. (2015) de Martin - Transcription

39 ChIP on chip: 1. Generation of two cell lines that are genetically matched except for Foxp3 2. IP 3. Hybridization against microarrays containing 60-mers spanning -8/+2 kb of genes Alternative: Next Gen Seq 4. Supplement by microarray data de Martin - Transcription

40 Reporter gene assay: -??? TATA +1 Promoter region Luciferase Reporter genes: Luciferase: firefly, renilla, Nluc (deep-sea shrimp, Oplophorus gracilirostris) stable LUC, secreted... ß-galactosidase Chloramphenicol acetyltransferase (CAT) Human growth hormone EGFP de Martin - Transcription

41 Minimal promoter-reporter gene assay: 5x NF-kB element Minimal (TK) promoter Luciferase de Martin - Transcription

42 Analysis of transaktivation domains: LTR Gal4DBD TAD LTR TAD GAL4 EGFP de Martin - Transcription

43 Isolation of TFs by affinity purification: de Martin - Transcription

44 Isolation of TFs by a genetic screen: yeast-1-hybrid screen Expressed as fusion proteins with a strong TAD de Martin - Transcription

45 Identification of Binding Sequence: 1. rec. Protein, Immobilisation: 5. Repeat GST I IV IIII Primer 1 gnctcncg cngagngc 4. Amplification (PCR) Primer 2 2. ds oligos w/ random sequence: nnnnnnnn nnnnnnnn I IV IIII gnctcncg cngagngc I IV IIII 3. Binding de Martin - Transcription

46 TFs: modes of regulation Transcriptional regulation Translocation from membrane (cell or ER) after proteolytic cleavage Translocation from the cytoplasm to the nucleus triggered by activation (phosphorylation, dephosphorylation) triggered by release from inhibitor Activation of a TF in the nucleus by a translocated kinase de Martin - Transcription

47 notch ß-adrenergic (7TM) EGF IFNg TNFa glutamate androgen EGF ER ATF6 de Martin - Transcription Intramembrane cleavage Relay systems Ligand passage Direct translocation

48 de Martin - Transcription