Histone lysine methylation in the context of nuclear architecture [Elektronische Ressource] / vorgelegt von Roman Zinner

Roman Zinner

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

Department Biologie II
Anthropologie und Humangenetik
Ludwig-Maximilians-Universität München
_____________________________

Histone lysine methylation
in the context of nuclear
architecture
_____________________________

Roman Zinner

Dissertation an der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
Eingereicht am 21.03.2007








Histone lysine methylation in the
context of nuclear architecture

Dissertation an der Fakultät für Biologie, Department II
Der Ludwig-Maximilians-Universität München (LMU)

Vorgelegt von
Dipl.Biol. Roman Zinner

Gutachter:
Erstgutachter: Prof. Dr. Thomas Cremer
Zweitgutachter: (PD) Dr. Angelika Böttger

Tag der mündlichen Prüfung:
25.06.2007











Stufen
Wie jede Blüte welkt und jede Jugend
dem Alter weicht, blüht jede Lebensstufe
blüht jede Weisheit auch und jede Tugend
zu ihrer Zeit und darf nicht ewig dauern
es muß das Herz bei jedem Lebensrufe
bereit zum Abschied sein und Neubeginne
um sich in Tapferkeit und ohne Trauern
in andre, neue Bindungen zu geben
und jedem Anfang wohnt ein Zauber inne
der uns beschützt und der uns hilft zu leben
wir wollen heiter Raum um Raum durchschreiten
an keinem wie an einer Heimat hängen
der Weltgeist will nicht fesseln uns und engen
er will Stuf' um Stuf' uns heben, weiten
kaum sind wir heimisch einem Lebenskreise
und traulich eingewohnt, so droht Erschlaffen
nur wer bereit zu Aufbruch ist und Reise
mag lähmender Gewöhnung sich entraffen
Es wird vielleicht auch noch die Todesstunde
uns neuen Räumen jung entgegensenden
des Lebens Ruf an uns wird niemals enden
Wohlan denn, Herz, nimm Abschied und gesunde.
Hermann Hesse, Das Glasperlenspiel

Contents
__________________________________________________________
1 Summary
2 Introduction ____________________________________________________ 1
2.1 Goals of the study _________________________________________________ 1
2.2 A higher order functional nuclear topology ____________________________ 3
2.3 Chromatin ________________________________________________________ 5
2.4 Euchromatin versus (facultative) heterochromatin ______________________ 6
2.5 Epigenetics and the histone code ____________________________________ 8
2.6 The nucleosome core particle ______________________________________ 11
2.7 Histone lysine methylation _________________________________________ 12
2.7.1 H3K4 _______________________________________________________________ 15
2.7.2 H3K9 (and interaction with HP1) __________________________________________ 16
2.7.3 H3K27 (and X-inactivation)_______________________________________________ 19
2.7.4 H4K20 ______________________________________________________________ 21
2.8 Chaetocin, inhibitor of the HMT SUV39H1 ____________________________ 22
2.9 RNA interference and heterochromatin formation ______________________ 22
2.10 DNA methylation _________________________________________________ 23
2.11 Commonly used techniques in the field ______________________________ 24
2.12 Outlook _________________________________________________________ 26
3 Methods and Protocols__________________________________________ 27
3.1 Cell culture ______________________________________________________ 27
3.1.1 Procedure for cell cultivation _____________________________________________ 27
3.1.2 Thawing and freezing of cells 28
3.1.3 Slide preparation 29
3.1.4 Seeding cells on coverslips ______________________________________________ 29
3.2 Replication labeling with BrdU ______________________________________ 30
3.3 Scratch transcription labeling with BrUTP ____________________________ 31
3.4 Polymerase chain reaction (PCR) ___________________________________ 32
3.4.1 DOP-PCR____________________________________________________________ 32
3.4.2 Label-PCR ___________________________________________________________ 35
3.5 Metaphase preparation ____________________________________________ 37
3.6 2D-FISH _________________________________________________________ 39
Contents
__________________________________________________________
3.7 Peptide competition assay _________________________________________ 42
3.8 Immunostaining __________________________________________________ 45
3.9 Sequential antibody labeling _______________________________________ 46
3.10 3D-Immuno-FISH _________________________________________________ 47
3.11 Chaetocin treatment ______________________________________________ 51
3.11.1 Calculation of the molarity out of the Chaetocin concentration__________________ 51
3.11.2 Experimental Chaetocin setup __________________________________________ 52
3.12 Microscopy______________________________________________________ 52
3.12.1 Transmitted light microscopy ___________________________________________ 52
3.12.2 Epifluorescence microscopy____________________________________________ 53
3.12.3 Confocal-laser-scanning microscopy _____________________________________ 53
3.13 Deconvolution ___________________________________________________ 54
3.13.1 Deconvolution in general ______________________________________________ 54
3.13.2 Deconvolution setup__________________________________________________ 55
3.13.3 The impact of deconvolution on image restoration___________________________ 59
3.13.4 The consistency of evaluation results at different thresholds ___________________ 61
3.14 Image processing ________________________________________________ 63
3.14.1 Documentation and shift correction ______________________________________ 63
3.14.2 Photoshop and Image J _______________________________________________ 63
3.14.3 Co-localization analysis 63
3.14.4 Amira 3D reconstruction 67
3.14.5 Radial autocorrelation (RAC) function analysis _____________________________ 68
3.14.6 Evaluation procedure in Chaetocin experiments ____________________________ 68
4 Results _______________________________________________________ 70
4.1 Nuclear patterns of distinct histone methylation sites __________________ 70
4.1.1 Methylation sites and their arrangement in regard to centromeres ________________ 70
4.1.2 Overlap assessment between centromeres and histone modifications _____________ 73
4.1.3 Relation between different histone modifications and nascent RNA _______________ 75
4.1.4 Pattern formation in cycling and quiescent nuclei _____________________________ 76
4.1.5 Interrelationship of different lysine methylation sites ___________________________ 78
4.1.6 The formation of distinct nuclear zones by lysine methylation sites ________________ 82
4.2 Changes in nuclear 3D topology after Chaetocin treatment ______________ 83
4.2.1 Test for the assessment of an appropriate Chaetocin dilution ____________________ 83
4.2.2 Test for cells in S-phase_________________________________________________ 84
4.2.3 Investigation of cell morphology in a test for Chaetocin cytotoxicity________________ 85
4.2.4 Changes in nuclear topology after Chaetocin treatment_________________________ 86
4.2.5 Investigation of HP1-alpha distribution after Chaetocin treatment _________________ 89
Contents
__________________________________________________________
4.2.6 No reorganization of chromatin occurs after Chaetocin rescue ___________________ 92
4.2.7 Evaluation of H3K9me3 pattern size in Chaetocin experiments 94
4.3 Lysine methylation sites and specific chromatin segments ______________ 97
4.3.1 Modifications correlate with gene density on the chromosomal level_______________ 98
4.3.2 “Holes” in chromosome paints are filled with heterochromatin___________________ 101
4.3.3 Distinct lysine methylations correlate with expression levels ____________________ 102
5 Discussion ___________________________________________________ 108
5.1 Lysine methylation patterns are arranged in nuclear zones _____________ 108
5.2 Changes in nuclear 3D topology after Chaetocin treatment 114
5.3 Analysis of lysine sites with regard to chromatin segments_____________ 118
5.4 The methodological approach _____________________________________ 121
6 References 125
7 Appendix ____________________________________________________ 138
7.1 Material and technical equipment __________________________________ 138
7.1.1 Cells _______________________________________________________________ 138
7.1.2 Chemicals, enzymes and reagents _______________________________________ 138
7.1.3 Media, buffers and solutions_____________________________________________ 141
7.1.4 Equipment and instrumentation __________________________________________ 142
7.1.5 BACs used in the experiments ___________________________________________ 146
8 Abbreviations_________________________________________________ 151
9 Table of Figures_______________________________________________ 152
10 Publicati