ATP-dependent remodelling of linker histone containing nucleosomal fibres [Elektronische Ressource] / Verena K. Maier

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









ATP-dependent Remodelling
of Linker Histone-Containing
Nucleosomal Fibres













Verena K. Maier

München
22. Januar 2009




















Eingereicht am 22. Januar 2009
Mündliche Prüfung am 5. März 2009


1. Gutachter: Prof. Peter Becker
2. Gutachter: Prof. Charles David
3. Gutachter: Prof. Dirk Eick
4. Gutachter: Prof. Kirsten Jung
5. Gutachter: Prof. Heinrich Leonhardt
6. Gutachter: Dr. Angelika Böttger










Ehrenwörtliche Versicherung


Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir
selbständig und ohne unerlaubte Hilfe angefertigt ist.



München, den .................................... ............................................................
(Unterschrift)

Acknowledgements



First of all, I would like to thank Prof. Peter Becker for giving me the opportunity to
join his group, for encouraging and supporting me, for always finding time for
discussions even in busy times and for providing a stimulating scientific environment
and an exceptionally nice lab atmosphere.

I am especially grateful to Cristina Chioda for constant support, valuable advice,
useful discussions, teaching me her technical expertise, careful reading of this
manuscript and for being a great friend both inside and outside of the lab.

I also want to express my gratitude to the other members of my thesis advisory
committee, Prof. Gernot Längst, Andreas Hochheimer and Prof. Ulrich Hartl for
their time and helpful comments.

Moreover, I want to thank Daniela Rhodes for sharing her expertise on chromatin
reconstitution.

Additional thanks go to Anton Eberharter for fruitful discussions, to Ragnhild
Eskeland, Natascha Kunert and Angie Mitterweger for technical advice and to
Catherine Regnard, Andreas Hochheimer, Cristina Chioda and Andrew Routh for
providing reagents.

I also would like to thank all the people working at the molecular biology department
creating a great working atmosphere, especially my bench neighbours Charlotte,
Roxane and Raffaella, the ‘lunch group’ Torsten, Christian, Sara, Clemens, Ana and
Henrike and the members of the ‘ABI orchestra’, Sandra, Antonia, Florian, Sonja,
Franziska and Alexandra. You made it fun to work here!

Philipp, thank you for being who you are and for being there for me.

Last but not least, I want to thank my parents Anton and Anna Maria Maier for
supporting me throughout my education. Table of Contents I
Table of contents

Summary...........................................................................................................................1

Zusammenfassung ...........................................................................................................2

1. Introduction..................................................................................................................4
1.1 Levels of chromatin condensation ..........................................................................4
1.2 Linker histones........................................................................................................7
1.2.1 The somatic linker histone H1............................................................................7
1.2.2 Linker histone variants........................................................................................9
1.3 Principles of regulating chromatin structure.........................................................10
1.4 ATP-dependent chromatin remodelling factors....................................................12
1.4.1 Subfamilies of ATPases....................................................................................12
1.4.2 ISWI-containing chromatin remodellling complexes.......................................16
1.4.3 Mechanism of ISWI-dependent nucleosome repositioning..............................16
1.4.4 Regulation of ISWI and its complexes .............................................................18
1.5 Interplay between linker histones and ATP-dependent chromatin remodelling...20
1.5.1 ATP-dependent remodelling in the presence of linker histones in vitro ..........20
1.5.2 Role of ISWI complexes in linker histone incorporation .................................21
1.6 Goals .....................................................................................................................22

2. Materials and Methods..............................................................................................24
2.1 Material sources....................................................................................................24
2.1.1 Laboratory chemicals and biochemicals...........................................................24
2.1.2 Enzymes............................................................................................................ 25
2.1.3 Antibodies.........................................................................................................25
2.1.4 Organisms, cells and bacteria ...........................................................................26
2.1.5 Oligonucleotides, plasmids, and baculoviruses ................................................26
2.1.6 Other materials..................................................................................................27
2.2 Buffers and solutions ............................................................................................28
2.3 Methods for the preparation and analysis of DNA ...............................................32
2.3.1 General methods for working with DNA .........................................................32
2.3.2 Cloning of 4x601 in pUC19 vector...................................................................33
2.3.3 Preparation of DNA fragments for the assembly of nucleosomal arrays .........34
2.3.4 Radioactive DNA end-labelling........................................................................35
2.4 Methods for protein analysis and purification of proteins....................................36
2.4.1 Protein quantification........................................................................................36 Table of Contents II
2.4.2 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ...................................36
2.4.3 Western blotting................................................................................................36
2.4.4 Purification of endogenous histone octamers from Drosophila embryos ........37
2.4.5 Expression and purification of recombinant core histones in E. coli and
reconstitution of histone octamers ....................................................................38
2.4.6 Purification of linker histone H1 from Drosophila embryos............................40
2.4.7 Purification of HMG-D from Drosophila embryos..........................................41
2.4.8 Expression and purification of recombinant HMG-D in E. coli.......................42
2.4.9 Expression and purification of ACF, ISWI, CHD1 and BRG1 in Sf9 cells .....43
2.4.10 Sources of other proteins/extracts.....................................................................44
2.4.11 Small scale preparation of nuclei from Drosophila embryos...........................44
2.5 Methods for the reconstitution and analysis of nucleosomal arrays.....................45
2.5.1 Chromatin salt assembly...................................................................................45
2.5.2 Electrophoretic mobility shift assays (EMSA) .................................................46
2.5.3 MgCl -precipitation of nucleosomal arrays......................................................47 2
2.5.4 Determination of histone stoichiometry ...........................................................47
2.5.5 ATPase assays...................................................................................................47
2.5.6 Chromatin remodelling reactions......................................................................48
2.6 Maintenance and analysis of Drosophila stocks...................................................49
2.6.1 Fly strains..........................................................................................................49
2.6.2 Embryo collection and staining ........................................................................49

3. Results.........................................................................................................................51
3.1