Locus specific analysis of PcG-TrxG proteins using bio-tagging technology [Elektronische Ressource] / vorgelegt von Ana Laura Monqaut

Ana Laura Monqaut

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 16 Mo

Extrait

INAGURAL-DISSERTATION




zur
Erlangung der Doktorwürde
der
Ruprecht-Karls-Universität
Heidelberg





vorgelegt von
Mikrobiologin Ana Laura Monqaut
aus Argentinien

Tag der mündlichen Prüfung:









Locus specific analysis of
PcG/TrxG proteins using
Bio-tagging technology










Gutachter: Prof. Dr. Renato Paro
Prof. Dr. Christof Niehrs




Acknowledgements

First I would like to express my gratitude to Professor Renato Paro for his guidance and
advice throughout this study.

I am also grateful to Professor Christof Niehrs for being my second advisor.

My most sincere thanks to Dr. Leonie Ringrose, for co-supervising this work at the
initial stages, for encouraging me at all times and for the helpful discussions about this
manuscript.

I would also like to thank Dr. Christian Beisel, for his continuous guidance throughout
the course of this thesis, and Dr. Gunter Merdes for his advice and friendship; to both
thanks for your time and patience.

My deepest gratitude to all members of the Paro lab, everybody contributed to a great
atmosphere. Working with you has been a pleasure, both scientifically and personally.
First, I do not want to forget the people who were there at the beginning, who welcomed
me and made me feel at home: Stephan who thought me all the German I needed to
know for survival, “Nach dem Spiel ist vor dem Spiel”; Nara and Tariq and their
fundamental discussions about evolution; Michaela and Sabine, who gave me all their
support and friendship; Britta, Alex, and Ian “always think positively”, also Andrea,
Sylvia (thanks for the embryo injections) and Melanie, and finally Heidi, for all her help
in and outside the lab and for being a friend. To the present members of the lab, I would
like to thank for their friendship and support, for all the discussions and also for putting
up with me on my bad days (quite many on the last year). Thanks to Gero for useful
protocols exchanges, Daniel for the translations, Yujie (and the Chinese connections,
always knows what to do and how to do it), Abil (an open book on fly genetics), Denise
for the help with the move, Tomo (the biggest football fan ever), Tariq for the good
advice, Tobias (ironman) thanks for the motivation and bringing the Unisports world to
us, Ute for helping us to get organized, and also Gunter, Christian, Makiko and Cem. To
all thanks again, for making this lab a great one to be part of.
I also want to extend my gratitude to the people at the ZMBH, and D-BSSE who also
contributed with their work. Thanks to Ursi, Rosita and Esther for so much help with the
fly plates; special thanks to Heidi for all her help with the paper work and Linda for the
connections. To Rolf, the Its and the administration also many thanks.

Thanks, many thanks to my good friends Fernanda, Marina, Elsa and Carmen for being
always there for me and together with Claudio, Pablo and Rodrigo for all the great
moments, so many good memories I will never forget. Muchas gracias por todo!!!!

I would like to say thanks to my family, for always believing in me and for their love and
support. No estaría aquí si no fuera por ustedes. A los que ya no están gracias porque
siempre creyeron en mi y siguieron mis pasos de cerca.

Finally, I want to thank Claudio, for his love and great support; for always staying
positive and teaching me to seek for the bright side of each experience; gracias por tu
apoyo y paciencia, hacemos un gran equipo.






Table of contents

Zusammenfassung
Summary
1. Introduction 1
1.1 Early Drosophila development: the specification of embryonic
cells 3
1.2 Maintaining cell identity: the cellular memory components 7
1.2.1 PcG, TrxG and their Protein Complexes 7
A. Polycomb group repressive complexes 7
B. Trithorax group complexes 13
1.2.2 PREs: DNA sequences with memory 15
1.3 Chromatin marks linked to silence and activation 17
1.4 Molecular mechanisms of the cellular memory 18
1.5 Genome-Wide distribution and biological functions of
PcG/TrxG proteins 23
1.6 Gene regulation by PcG/TrxG, other players come to action 24
1.7 Aims of the thesis 26
2. Results 29
2.1 A transgenic reporter system to study the PcG and TrxG
composition at different PREs 31
2.2 Establishing the transgenic system 34
2.2.1 DNA Constructs 34
A. Fab-7 and vestigial PRE constructs 34
B. TetR constructs 35
C. BirA construct 36
2.2.2 TetR expression and in vivo biotinylation 36
A. TetR is expressed and in vivo biotinylated in Drosophila SL2
cells 36
B. TetR is expressed and in vivo biotinylated in flies 38
2.2.3 Mapping of the PRE transgenes 39

2.2.4 Generating the correct controls for the 4 elements system 41
2.3 Testing the system’s performance 47
2.3.1 Biotinylated TetR is located to the nucleus of fly embryos 47
2.3.2 Efficient Chromatin IP using TetR as bait 47
2.3.3 Protein pull-down from protein-chromatin nuclear extract 50
2.4 Testing the efficiency of each step 51
2.4.1 Formaldehyde cross-link, is it really reversible? Can the
efficiency be improved? 51
2.4.2 In vivo biotinylation of TetR: How efficient is it in
Drosophila? 54
2.5 How to improve the efficiency of the system? 56
2.5.1 Addition of DSP and DTBP, protein-protein cross-linkers 56
2.5.2 Shearing the chromatin at the right size 58
2.5.3 In vitro-biotinylation using recombinant BirA ligase 60
2.6 Chromatin purification, separating the DNA bound proteins
from the unbound 62
2.6.1 Separation of nuclear extract and nuclear pellet with salt
extraction 62
2.6.2 Vivaspin ultrafiltration spin columns for chromatin
separation 64
2.6.3 High molecular weight cutoff dialysis 65
2.6.4 TetO DNA beads to deplete extracts from soluble TetR 67
2.6.5 Cesium Chloride density gradients partially solve the
chromatin separation problem 70
2.7 Protein pull-down with optimized conditions 71
3. Discussion 75
3.1 Single locus analysis: any other options? 78
3.2 Pulling from the bait: the proof of principle 81
3.3 Ineffective protein pull-down: a matter of bait abundance or
low efficiencies? 81
3.4 Problems associated to working with lines carrying many
transgenes 84
3.5 What could and could not be done to increase efficiency 84


3.5.1 Cross-link and reversal of cross-link 85
3.5.2 Chromatin shearing 86
3.5.3 Biotinylation 86
3.5.4 Soluble TetR and biotinylated proteins 88
3.6 The future of locus-specific factors identification 90
3.6.1 What is to be done: other possible applications of the
system 91
3.6.2 What is to be modified: re-design of the system 91
3.7 Conclusion and outlook 94
4. Materials 95
4.1 Antibodies 97
4.2 Molecular Weight markers 97
4.3 Enzymes 97
4.4 Oligonucleotides 97
4.4.1 Oligos and Primers for PRE constructs 98
4.4.2 Oligos and Primers for TetR constructs 98
4.4.3 Oligos for sequencing of plasmids and cloned fragments 98
4.4.4 Primers used to test Cre/loxP and Flp/FRT recombination 99
4.4.5 Primers used to make probes for Southern Blot 99
4.4.6 Additional primers used to check fly stocks 99
4.4.7 Primers used in ChIP 99
4.4.8 TetO oligos for beads coupling 100
4.5 Plasmids 100
4.6 Bacterial cell lines 102
4.7 Drosophila culture cells 103
4.8 Fly Lines 103
4.8.1 General Fly stocks 103
4.8.2 Gal4 driver lines 103
4.8.3 Transgenic lines expressing site-specific recombinases 103
4.8.4 Biotin Ligase (BirA) expressing flies 103
4.8.5 Generated fly lines 104
4.9 Technical Devices 107

4.9.1 Microscopes 107
4.9.2 Microinjection 107
4.9.3 Agarose Gel electrophoresis 107
4.9.4 SDS-PAGE gel electrophoresis and Western Blotting 107
4.9.5 Data processing 107
4.10 Further Materials 108
4.11 Chemicals 109
5. Methods 111
5.1 Molecular methods 113
5.1.1 Phenol-chloroform extraction of DNA 113
5.1.2 Ethanol precipitation of DNA 113
5.1.3 Analysis of DNA fragments by agarose gel electrophoresis 113
5.1.4 Gel Extraction/ PCR purification 114
5.1.5 Restriction Endonuclease digestion of DNA 114
5.1.6 Phosphatase treatment of DNA fragments 114
5.1.7 Ligation of DNA fragments 114
5.1.8 End-filling of DNA protruding ends 115
5.1.9 Annealing of DNA oligos 115
5.1.10 Preparation of agar plates 115
5.1.11 Freezing of bacteria stocks 115
5.1.12 Production of E. coli chemo-competent cells 116
5.1.13 Transformation of chemo-competent E. coli cells 116
5.1.14 Isolation of Plasmid DNA from bacteria by alkaline lysis
method 116
A. Small scale DNA preparation (Mini Prep) 116
B. Large scale DNA preparation (Midi Prep) 117
5.1.15 Polymerase Chain Reaction (PCR) 117
5.1.16 Colony PCR 118
5.1.17 Isolation of genomic DNA (Quick Fly Genomic DNA Prep) 118
5.1.18 Single fly PCR 119
5.1.