Identifizierung und funktionelle Charakterisierung des neuen MAR-bindenden Proteins, SATB2 [Elektronische Ressource] / Gergana Dobreva

Gergana Dobreva

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München




Identifizierung und funktionelle
Charakterisierung des neuen
MAR-bindenden Proteins, SATB2




Gergana Dobreva


aus

Sofia, Bulgarien



2003

Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Prof. Dr. Rudolf Grosschedl
betreut.


Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.


München, 4.12.2003



Gergana Dobreva











Dissertation eingereicht am: 4.12.2003
1. Gutachter: Prof. Dr. Rudolf Grosschedl
2. Gutachter: Priv.-Doz. Dr. Jürgen Haas
Mündliche Prüfung am: 12.01.2004










Identification and functional
characterization of the novel
MAR-binding protein, SATB2










Table of Contents


List of Figures 1 of Abbreviations 3
Sumary 5
Acknowledgements 6
I. Introduction 7
1. Nuclear matrix 7
2. Matrix attachment (association) regions (MARs) 7
2.1. MARs of the lymphocyte receptor genes 9
2.1.1. The MAR of the Igκ gene 9
2.1.2. The IgH gene locus MARs 10
2.1.3. The TCRβ gene locus MAR 11
2.1.4. The TCR α/δ gene locus MARs 12
3. MAR-binding proteins 12
3.1. Cux/CDP (CCAAT displacement protein) 13
3.2. Bright (B cell regulator of IgH transcription) 14
3.3. SATB1 (special AT-rich sequence binding protein 1) 15
4. Post-translational protein modifications. SUMO modification 19
4.1. SUMO (small ubiquitin-related modifier) 19
4.2. The SUMO conjugation pathway 21
4.3. SUMO protein ligases 23
4.4. SUMO-specific proteases (SUSPs) 24
4.5. Biological functions of SUMO modification 26
4.5.1. Nuclear pore complex shuttling 26
4.5.2. Changes in subnuclear localization and
targeting to PML bodies 27
4.5.3. Modulation of protein-protein interactions 28
4.5.4. Regulation of DNA binding 28
4.5.5. Modulation of the activity of transcription
factors 28
4.5.6. Antagonism of ubiquitination 29
II. Results 31
1. Synthetic multimerized MAR-binding sites mimic the natural
MARs in augmenting the transcription of the IgH gene 31
2. A homologue of the MAR-binding protein SATB1 is expressed
in pre-B cels 33
2.1. SATB2 has a high homology and a similar domain
organization to SATB1 33
2.2. Expression pattern of SATB2 35
2.3. SATB2 is a component of the nuclear matrix 36
2.4. SATB2 binds to the core-unwinding element of
the IgH enhancer MARs in vitro 37
2.5. SATB2 binds to the MARs of the endogenous µ locus 40
2.6. SATB2 is a B cell specific transcriptional activator 40
2.6.1. SATB2 mediates transcriptional activation
under multimerized MAR consensus sites 40
2.7.2. SATB2 mediates transcriptional activation
under the natural MARs of the immunoglobulin
enhancer 43
2.7.3. SATB2 augments immunoglobulin
transcription 43
3. SATB2 is posttranslationally modified 46
3.1. SATB2 is SUMO-modified 46
3.1.1. SATB2 is SUMO-conjugated at two
SUMO-consensus sites 49
3.1.2. SATB1 is not SUMO-modified 49
3.1.3. SATB2 is not proteolytically cleaved by
caspase 6 51
3.2. PIAS1 is the E3 ligase for SATB2 53
3.2.1. SATB2 interacts specifically with PIAS1 53
3.2.2. PIAS1 stimulates SUMO conjugation to SATB2
in vivo 53
3.2.3. PIAS1 is an E3 ligase for SATB2 55
3.3. SUMO conjugation antagonizes SATB2 mediated
gene activation 56
3.3.1. Mutations of the sumoylation sites of SATB2
augment its activation potential 56
3.3.2. Covalent attachment of SUMO1 and SUMO-3
antagonizes SATB2-dependent transcription 58
3.3.3. Wild-type and SATB2-dlmut, but not the
SUMO-SATB2 fusions, augment
immunoglobulin Cα gene transcription 58
3.4. Mutations of the sumoylation sites of SATB2 augment
the association with MAR sequences of the
endogenous immunoglobulin heavy chain locus. 60
3.5. Covalent fusion of SUMO1 and SUMO3 to
SATB2 does not affect its DNA binding in vitro 62
3.6. SUMO modification does not inhibit the
dimerization of SATB2 63
3.7. SUMO modification alters the subnuclear
localization of SATB2 64
3.8. SUMO conjugation is stimulated by stress conditions 66

III. Discussion 68
1. Synthetic multimerized MAR-binding sites mimic the natural
MARs in augmenting the transcription of the IgH gene 68
2. SATB2 is a novel MAR-binding protein 69
3. SATB2 augments immunoglobulin gene expression 69
3.1. Possible mechanisms of SATB2-mediated
transcriptional activation – comparison with other
MAR-binding proteins 70
4. Regulation of SATB2 function 72
4.1. is SUMO-modified 72
4.2. PIAS1 is the E3 ligase for SATB2 74
4.3. SUMO modification antagonizes SATB2-mediated
transcriptional activation 75
4.4. SUMO modification of SATB2 reduces its chromatin
asociaton 76
4.5. Sumoylation alters the subnuclear localization of
SATB2 77
5. Perspectives 79

IV. Materials and Methods 80
1. Reagents 80
1.1. Chemicals 80
1.2. Enzymes
2. General buffers 80
3. Cloning and related techniques 82
3.1. Cloning of SATB2 constructs 82
3.2. Cloning of the luciferase reporter constructs 83
3.3. Preparation of competent E. coli 83
3.4. Transformation and growth of transformed bacteria 83
3.5. “Mini” and “maxi” plasmid preparation 84
3.6. Restriction digests 84
3.7. Ligation of DNA 84
3.8. Agarose gel electrophoresis 84
3.9. Point mutant generation 85
4. Tissues culture and related techniques 85
4.1. Cell lines 85
4.2. Culture conditions 86
4.3. Calcium phosphate transfection 87
4.4. Transfection by electroporation 88
4.5. Stable cell line establishment 88
4.6. Nuclear matrix preparation 88
4.7. Immunofluorescensce 89
4.8. Microscopy 90
4.9. Reporter assays: luciferase and β-galactosidase assay 90
5. RNA isolation, purification and analysis 91
+ 5.1. Isolation of total and poly- ARNA 91
5.1.1. Preparation of total RNA
+5.1.2. Preparation of poly-A RNA 92
5.2. Northern blot hybridization 92
5.2.1. Agarose gel electrophoresis 92
5.2.2. Transfer of RNA to nylon membrane 93
5.2.3. Preparation of probes 93
5.2.4. Hybridization 94
5.3 RT-PCR 95
6. DNA isolation, purification and analysis 96
6.1. Isolation of genomic DNA 96
6.2. Southern blot hybridization 96
6.2.1. Restriction digest and agarose gel
electrophoresis 96
6.2.2. Transfer of the digested DNA to nylon
membrane 97
6.2.3. Preparation of probes 97
6.2.4. Hybridization 97
6.3. PCR (Polymerase chain reaction) 98
7. Protein purification and analysis 98
7.1. Expression and purification of recombinant proteins 98
7.2. Preparation of total protein extracts 100
7.2.1. Cell extracts 100
7.2.2. Organ 101
7.3. Measurement of protein concentration 101
7.4. Sodium dodecylsulphate polyacrylamide gel electro-
phoresis (SDS-PAGE) 101
7.5. Coomassie staining of polyacrylamide gels 102
7.6. Silver staining of polyacrylamide gels 102
7.7. Western blotting (immunoblotting) 102
8. Analysis of protein-protein and protein-DNA interactions 103
8.1. Protein-protein interactions 103
8.1.1. Co-immunoprecipitation 103
8.1.2. TAPtag purification 104
8.2. Protein-DNA interactions 105
8.2.1. Electromobility shift assay (EMSA) 105
8.2.2. Chromatin Immunoprecipitation 105
9. Sequence analysis 106

V. Publications 108

VI. Refrences 109

Curriculm Vitae 129
LIST OF FIGURES 1

List of Figures

Figure 1. High-resolution structure of the nuclear matrix. 8
Figure 2. Physical location of MARs in lymphoid receptor genes. 9
Figure 3. Domain organization of SATB1. 15
Figure 4. SATB1 forms a three-dimensional cage-like network,
surrounding dense regions of chromatin. 16
Figure 5. Sequence alignment of SUMO family members and ubiquitin. 21
Figure 6. The SUMO conjugation pathway. 22
Figure 7. Functional significance of SUMO-modification