Transcription factor Sp3 as target for SUMOylation in vivo [Elektronische Ressource] / vorgelegt von Grigore Rişchitor

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 34
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Transcription factor Sp3 as target for
SUMOylation in vivo


Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem Fachbereich Biologie
der Philipps-Universität, Marburg/Lahn
vorgelegt von

Grigore Ri şchitor
aus
Negre şti / Rumänien
Marburg, Februar 2005 Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation
angenommen am:

Prufungvorsitzender Prof. Dr. Renate Renkawitz-Pohl
Betreuer der Arbeit Prof. Dr. G. Suske

Tag der mündlichen Prüfung:
2Die Untersuchungen zur vorliegenden Arbeit wurden von August 2001 bis
Dezember 2004 im IMT (Institut für Molekularbiologie und Tumorforschung) in
Marburg unter der Leitung von Herrn Prof. Dr.Guntram Suske durchgeführt.
Ich versichere, dass ich meine Dissertation mit dem Titel “Transcription factor
Sp3 as target for SUMOylation in vivo“ selbstständig, ohne unerlaubte Hilfe
angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten
Quellen und Hilfen bedient habe.




























3
























Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner
anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken
gedient.



Marburg, 25 Februar 2005 Grigore Rischitor


4Im Zusammenhang mit der Thematik der vorliegenden Dissertation wurden folgende
Publikationen erstellt:




Sapetschnig A., Rischitor G., Braun H., Doll A., Schergaut M., Melchior F., Suske
G. (2002). Transcription factor Sp3 is silenced through SUMO modification by PIAS1.
EMBO Journal 21, 5206-5215.

Sapetschnig A., Koch F., Rischitor G., Mennenga T., Suske G. (2004). Complexity of
translationally controlled transcription factor Sp3 isoform expression. The Journal of
Biological Chemistry 279, 42095-42105.
.



5Contents
Contents

Contents ...........................................................................................................................6
Abbreviations .................................................................................................................10
Summary........................................................................................................................12
Zusammenfassung.........................................................................................................15
1. Introduction ...............................................................................................................18
1.1. Properties of Sequence-Specific DNA Binding Transcription Factors ................ 18
1.1.1. Sequence-Specific Transcription Factors Are Modular ................................19
1.1.2. Sequence-Specific Factors Regulate Transcription via Recruitment of
Coactivators and Corepressors...............................................................................20
1.1.3. Sequence-Specific Factors Can Be Regulated by Posttranslational
Modifications...........................................................................................................21
1.1.4. Sequence-Specific Factors Are Members of Multiprotein Families..............22
1.1.5. Chromatin Is an Integral Component in the Function of Sequence-Specific
Factors ....................................................................................................................22
1.1.6. Recognition Sites for Sequence-Specific Factors Tend to Be Located in
Clusters23
1.1.7. Other Properties of Sequence-Specific Transcription Factors .....................23
1.2. Basic Classification of Sp Family Transcription Factors ..................................... 24
1.2.1. The Sp/KLF Zinc Finger Transcription Factors ...........................................24
1.2.2. Sp3: Activator versus Repressor ...................................................................29
1.3. SUMO Small Ubiquitin-like MOdifier ................................................................ 31
1.3.1. What is SUMO?.............................................................................................32
1.3.2. The Eukaryotic Family of PIAS Proteins ......................................................38
1.4. Aim of the Project ................................................................................................ 41
2. Materials and Methods..............................................................................................44
2.1. Materials............................................................................................................... 44
2.1.1. Chemicals and Equipment................................................................................. 44
2.1.2. Mammalian and Insect Cell Lines.................................................................45
2.1.3. Buffers and Solutions.....................................................................................49
2.1.4. Enzymes and Antibodies................................................................................49
6Contents
2.1.5. Oligonucleotides............................................................................................50
2.1.6. Plasmids ........................................................................................................54
2.1.6.1. Expression Plasmids for Mammalian Cells............................................54
2.1.6.2. Expression Plasmids for Generation of Stable Cell Lines:.....................60
2.1.6.3. Expression plasmids for insect cells:......................................................62
2.2. Methods................................................................................................................ 62
2.2.1. Molecular Biological Methods......................................................................62
2.2.1.1. PCR.........................................................................................................62
2.2.1.2. Purification of Nucleic Acids..................................................................63
2.2.1.3. Enzymatic Manipulation of DNA............................................................64
2.2.2. Microbiological Methods ..............................................................................66
2.2.2.1. Cultivation and Growing of Microorganisms .........................................66
2.2.2.2. Plasmid DNA Preparation from E. coli ..................................................68
2.2.3. Working with Eukaryotic Cell Lines..............................................................68
2.2.3.1. Trypsinizing and Passaging Cells...........................................................69
2.2.3.2. Cell Freezing...........................................................................................69
2.2.3.3. Cell Thawing70
2.2.4. Transient Transfection and Transfection Methods ........................................70
2.2.4.1. Calcium Phosphate.................................................................................71
2.2.4.2. DEAE-Dextran........................................................................................71
2.2.4.3. PEI ..........................................................................................................72
2.2.4.4. FuGENE6................................................................................................73
2.2.4.5. Effectene..................................................................................................73
2.2.4.6. Lipofectin ................................................................................................74
2.2.5. Stable Transfection75
2.2.5.1. Establishing a Kill Curve (Dose-Response Curve).................................75
2.2.5.2. Stably Transfection and Selection of Double-Stable Cell Lines..............77
2.2.6. Luciferase Assay............................................................................................78
2.2.7. Biochemical Methods ....................................................................................78
2.2.7.1. Isolation of Proteins from Eukaryotic Cells ...........................................78
2.2.7.2. Immunoprecipitation...............................................................................82
2.2.7.3. Two-Step Affinity Purification.................................................................83
2.2.7.4. TCA (Trichloroacetic Acid) Protein Precipitation..................................85
2.2.7.5. Determination of Protein Concentration................................................86
2.2.8. Western Blotting86
7Contents
2.2.8.1. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........................86
2.2.8.2. Blotting....................................................................................................87
2.2.8.3. Blocking and Incubation With Antibodies...............................................87
2.2.8.4. ECL-Detection ....