Histone H3 lysine 9 methylation [Elektronische Ressource] : a signature for chromatin function / Ragnhild Eskeland

Ragnhild Eskeland

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München






Histone H3 lysine 9 methylation:
A signature for chromatin function



















Ragnhild Eskeland
aus
Stord, Norwegen

Dezember 2006
Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, 21.12.2006



Ragnhild Eskeland













Dissertation eingereicht am: 21.12.2006

1. Gutachter: Prof. Dr. Peter B. Becker
2. Gutachter: Prof. Dr. med. Thomas Cremer


Mündliche Prüfung am: ___19.04.2007____




1

Til mor og far



















































"The mind is not a vessel to be filled but a fire to be kindled." Mestrius Plutarchus
2
The results presented in this PhD thesis have been published in following
publications:

Eskeland, R.*, Czermin, B.*, Boeke, J., Bonaldi, T., Regula, J. T., and Imhof, A.
(2004). The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization
and regulates its methyltransferase activity. Biochemistry 43, 3740-9.

Eskeland, R., Eberharter, A., and Imhof, A. (2007). HP1 binding to chromatin
methylated at H3K9 is enhanced by auxiliary factors. Mol Cell Biol 27, 453-
465.

Greiner, D., Bonaldi, T., Eskeland, R., Roemer, E., and Imhof, A. (2005).
Identification of a specific inhibitor of the histone methyltransferase
SU(VAR)3-9. Nat Chem Biol 1, 143-5.

Stabell, M., Eskeland, R., Bjorkmo, M., Larsson, J., Aalen, R. B., Imhof, A., and
Lambertsson, A. (2006). The Drosophila G9a gene encodes a multi-catalytic
histone methyltransferase required for normal development. Nucleic Acids Res
34, 4609-21.


*Co-first author.























These publications can be found in the Appendix.


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Acknowledgements

I would like to thank my supervisor Axel Imhof for giving me the opportunity to work
in his lab. You have taught me a lot the last four years and given me the freedom to
develop my own ideas.

My gratitude is also to Peter Becker for being my official supervisor and providing an
excellent scientific environment in the lab. Thank you for following up on my work, I
have learned a lot from your critical comments.

Prof. Dr.Thomas Cremer I would like to thank for being my “Zweitgutachter”.

Many thanks to Cristina for corrections on my thesis. Thank you for always
encouraging me:-) I am also grateful to Jan Postberg for corrections.

I would also like to thank the Imhof lab for the nice every day life in the lab.
Thank you all for the wonderful leaving present! It will be hanging in my living room
and always remind me of my time here. Birgit for sharing your knowledge and
reagents. Annette for everyday chats, I had a great time with you in DomRep. Tizi for
help with the mass spec and support in the lab. Also thanks to: Jörn, Irene, Lars,
Pierre, Ana, Tilman and Doro.

Thanks goes to Anton Eberharter for scientific discussions and for providing me with
active ACF and ACF1 plus viral stocks and constructs. I am grateful to Alex Brehm
and Gernot Längst for always being available for questions and comments on my
work. Andreas Hocheimer for many inspiring discussions and showing me the
excellent NE protocol.

I also wish to thank the rest of the Becker lab, both for the nice environment you have
created and help and advices throughout my time here.

I am grateful to the International graduate program of Elitenetzwerk Bayern for
supporting me to visit a Chromatin Structure and Function Conference in December
2006.

A special thanks goes to Even. Thank you for always being understanding, supportive
and for all the late dinners you have had ready for me. I will never forget that you
came here to be with me. My life in Munich would not have been the same without
you!

Mange takk til familien heime. Mor og far for at dåke forstod kor viktig dette var for
meg og for støtte heile vegen. Det har vore langt heim. og eg har savna dåke mykje.
Heldigvis blir turen heim mykje kortare etter jul.

I am also very grateful to all my friends here in Munich. Simone and Silvia for a
wonderful holiday in Italy and many, many cinema memories. Isabel and Armin for
all the times at Mozart and for introducing us to good German wine. Juliane and Ingo
for lending me the laptop and for all the nice dinners. Birgit for borrowing me the
bike. It was nice to have you around in the lab and I am happy we managed to stay in
touch after you left. Many thanks go also to Mattia and Anna, Marica and Thomas,
Javier and Robert for all the great nights out in Munich. Warm thanks goes to Jo, Ina
and Dan who was here the first years of my time in Munich. Lastly, many thanx goes
to friends at home that I did not get to see very much these years.
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1. Summary………………………………………………............................. 10
2. Introduction............................................................................................... 11
2.1 Chromatin 11
2.2 Position-Effect Variegation 14
2.3 ATP dependent chromatin remodeling 16
2.4 Histone modifications 19
2.4.1 Acetylation 20
2.4.2 Phosphorylation 21
2.4.3 Other histone post-translational modifications 21
2.5 Methylation 22
2.5.1 Arginine methylation 23
2.5.2 Lysine me 24
2.6 The SET domain 27
2.6.1 SU(VAR)3-9 31
2.6.2 G9a 34
2.7 Heterochromatin protein1 37
2.7.1 The chromo domain 39
2.7.2 chromo shadow domain 43
2.7.3 HP1 targeting to chromatin 48
2.7.4 Methyl/Phospho Switch and a subcode within the histone code 50
2.7.5 HP1 in euchromatin 51
2.7.6 Other functions of HP1 51
2.8 The Aim 54
3. Material and Methods…………………………………..………….......... 55
3.1 Material 55
3.1.1 Chemicals, material and radioactive isotopes 55
3.1.2 Enzymes and Kits 55
3.1.3 Chromatographic material 56
3.1.4 Vectors 56
3.1.5 Oligonucleotides 56
3.1.6 Plasmids 58
3.1.7 E.coli strains 59
3.1.8 Insect cell lines 59
3.1.9 Fly lines 59
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3.1.10 Antibodies 60
3.1.11 DNA and Protein markers 60
3.2 Methods 61
3.2.1 General molecular biology methods 61
3.2.1.1 Standard PCR setup 61
3.2.1.2 Spectophotometric concentration measurements of DNA
and RNA 61
3.2.1.3 Agarose gel electrophoresis
3.2.1.4 Isolation of DNA fragments from agarose gels 62
3.2.1.5 Preparation of competent cells 62
3.2.1.6 Transformation of competent
3.2.1.7 Isolation of Plasmid DNA from E. coli 62
3.2.1.8 Site-directed mutagenesis 63
3.2.2 General protein-biochemistry methods 63
3.2.2.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 63
3.2.2.2 Coomassie Blue staining of protein gels 64
3.2.2.3 Silver staining of protein gels 64
3.2.2.4 Western Blotting 65
3.2.2.5 Li-Cor 65
3.2.2.6 Trichloroacetic acid (TCA) precipitation of proteins 66
3.2.2.7 Determination of protein concentration 66
3.2.2.8 In vitro translation 66
3.2.3 Tissue culture methods 66
3.2.3.1 Cultivation of Drosophila cell lines 66
3.2.3.2 Generation SL2 stable cell-lines 67
3.2.3.3 of Baculoviruses and protein expression 67
3.2.4 Recombinant protein expression and purification 68
3.2.4.1 Expression and purification of recombinant Drosophila
His-SU(VAR)3-9 68
3.2.4.2 Drosophila
HP1 69
3.2.4.3 Purification of Flag-dG9a 69
3.2.4.4 Expression and purification of GST-tagged proteins 70
3.2.5 Histone purification and octamer preparation 70
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3.2.5.1 Expression and purification of Drosophila histones 70
3.2.5.2 Purification of Native Drosophila histones 71
3.2.5.3 Native H1 purification 72
3.2.5.4 Octamer preparation 72
3.2.5.5 Generation of H3K9 methylated octamer 72
3.2.6 Nucleosome assembly 73
3.2.6.1 Nucleosome assembly by salt dialysis 73
3.2.7 Extract preparations 74
3.2.7.1