Transcription factors R. de Martin Department of Vascular Biology and Thrombosis Research Medical University of Vienna
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)
1. Transcription - general mechanisms 2. Transcription factors: structure-function 3. Methods: what can you do? 4. Regulation of TFs de Martin - Transcription 2016 3
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 50-100 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 2016 4
The transcription initiation complex contains the TATA box binding protein, RNA Pol.II, general TFs, and interacts with other regulatory proteins de Martin - Transcription 2016 5
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 2016 6
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 2016 7
How to achieve tissue/celltype-specific gene expression 1 4 2 5 3 Zhang and Glass, 2013 de Martin - Transcription 2016 8
Zhang and Glass, 2013 de Martin - Transcription 2016 9
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 2016 10
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 2016 11
Transcription factors recognize specific DNA sequences (bacteriophage lambda Cro protein): de Martin - Transcription 2016 12
1 Superclass: Basic Domains Examples: Leucine Zipper Factors (Jun, Fos, CREB) Leucine zippers also mediate protein-protein interactions!! Phe-Ile zipper de Martin - Transcription 2016 13
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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 30-40 cognate Zn fingers, each recognizing a specific short DNA sequence combination -> artificial TFs de Martin - Transcription 2016 15
de Martin - Transcription 2016 16 GR GR www.ncbi.nlm.nih.gov/structure/
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 2016 17
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4 Superclass: ß-Scaffold Factors with Minor Groove Contacts Examples: NF-kB/Rel, NF-AT, p53 de Martin - Transcription 2016 19
The bacterial met repressor: an example for DNA binding through ß-sheets de Martin - Transcription 2016 20
The CRISPR/Cas9 system for gene editing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR associated (Cas) nuclease, Cas9 www.horizondiscovery.com de Martin - Transcription 2016 21
https://www.addgene.org/crispr/guide/#pam de Martin - Transcription 2016 22
https://www.addgene.org/crispr/guide/#pam de Martin - Transcription 2016 23
SAM: CRISPR/Cas9 Synergistic Activation Mediator / Repression de Martin - Transcription 2016 24
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 33 35 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 2016 25
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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 2016 27
Wiesner et al. Nucl. Acids Res. 2002 de Martin - Transcription 2016 28
Proline-rich regions: MDHTDNELQGTNSSGSLGGLDVRRRIPIKLISKQASKVKPAPRTQRTVSRMPAK APQGDEEGFDYNEEQRYDCKGGELFGNQRRFPGHLFWDFKINILGEKDDTPVHF CDKCGLPIKVYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGS LFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVHPPIAPPPTDIP DRFIMPPDKHHMSHIPPKQHIMMPPPPLQHVPHEHYNQPHEDIRAPPAELSMAP PPPRSVSQETFRISTRKHSNLITVPIQDDSSSGAREPPPPAPAPAHHHPEYQGQPV VSHPHHIMPPQQHYAPPPPPPPPISHPMPHPPQAAGTPHLVYSQAPPPPMTSAP PPITPPPGHIIAQMPPYMNHPPPGPPPPQHGGPPVTAPPPHHYNPNSLPQFTEDQ GTLSPPFTQPGGMSPGIWPAPRGPPPPPRMQGPPSQTPLPGPHHPDQTRYRPYY Q de Martin - Transcription 2016 29
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 2016 30
The 9aa TAD (Piskacek et al., 2007) de Martin - Transcription 2016 31
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 2016 32
Electrophoretic Mobility Shift Assay (EMSA) Protein: nuclear extract recombinant protein de Martin - Transcription 2016 33
EMSA incl. Supershift : competitions de Martin - Transcription 2016 34
Transcription factor ELISA: de Martin - Transcription 2016 35
Chromatin Immunoprecipitation (ChIP): de Martin - Transcription 2016 36
Chromatin Immunoprecipitation (ChIP): de Martin - Transcription 2016 37
ABCD (Avidin Biotin Complex DNA) precipitation assay Hubner et al., J. Proteome Res. (2015) de Martin - Transcription 2016 38
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 16.000 genes Alternative: Next Gen Seq 4. Supplement by microarray data de Martin - Transcription 2016 39
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 2016 40
Minimal promoter-reporter gene assay: 5x NF-kB element Minimal (TK) promoter Luciferase de Martin - Transcription 2016 41
Analysis of transaktivation domains: LTR Gal4DBD TAD LTR TAD GAL4 EGFP de Martin - Transcription 2016 42
Isolation of TFs by affinity purification: de Martin - Transcription 2016 43
Isolation of TFs by a genetic screen: yeast-1-hybrid screen Expressed as fusion proteins with a strong TAD de Martin - Transcription 2016 44
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 2016 45
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 2016 46
notch ß-adrenergic (7TM) EGF IFNg TNFa glutamate androgen EGF ER ATF6 de Martin - Transcription 2016 47 Intramembrane cleavage Relay systems Ligand passage Direct translocation
rainer.demartin@meduniwien.ac.at de Martin - Transcription 2016 48