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«durchgeführt am Lehrstuhl für Biochemie III der Universität Regensburg vorgelegt von: ANNA SCHRADER SENSERSTR. 8 81371 MÜNCHEN Abgabedatum: 29. ...»

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Molecular mechanisms of nucleosome

positioning and DNA methylation in chromatin

Dissertation zur Erlangung des Doktorgrades

der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen

Fakultät III - Biologie und vorklinische Medizin der Universität Regensburg

durchgeführt am Lehrstuhl für Biochemie III

der Universität Regensburg

vorgelegt von:



81371 MÜNCHEN Abgabedatum: 29. Juli 2009 Die vorliegende Arbeit wurde unter der Betreuung von Prof. Dr. Gernot Längst in der Zeit von Februar 2006 bis Juli 2009 am Institut für Biochemie III der Universität Regensburg erstellt.


Vorsitzender: Prof. Dr. Reinhard Wirth

1. Gutachter: Prof. Dr. Gernot Längst

2. Gutachter: Prof. Dr. Alexander Brehm

3. Gutachter (Prüfer): Prof. Dr. Ralf Wagner Ersatzprüfer: Prof. Dr. Michael Thomm Table of Contents Abbreviations

A. Zusammenfassung

B. Introduction

I. The Chromatin structure

1. In General

1.1. The nucleosome - basic packaging unit of chromatin

1.2. Chromatin higher order structures

1.3. Compartmentation of chromatin within the nucleus

2. Nucleosome assembly

II. Modifications in chromatin

1. Histone modifications

1.1. Posttranslational modifications of histones

1.2. Histone variants

2. Chromatin dynamics

2.1. Important chromatin remodeling subfamilies for this study

2.2. Mechanism of and influences on nucleosome mobility

3. Positioning of nucleosomes on DNA

3.1. Sequence-dependent nucleosome positioning

3.2. Nucleosome positioning by chromatin remodeling enzymes

3.3. Additional factors influencing nucleosome positioning

4. DNA methylation

4.1. DNA methylation – Enzymes and mechanism

4.2. DNA methylation in mammals

4.3. Mammalian DNA methyltransferases

III. Interplay between Chromatin remodeling and DNA methylation

1. In vitro studies on DNA methylation in chromatin

2. In vivo studies on DNA methylation in chromatin

C. Objectives

1. Nucleosome positioning by chromatin remodeling enzymes

2. Maintenance methylation in the context of chromatin

D. Material and methods

I. Material sources

1. Laboratory chemicals and biochemicals

2. Enzymes

3. Buffers and solutions

4. Kits

5. Radioactive material

6. Medias

7. Antibodies

8. Eukaryotic tissue culture cell lines

9. Bacteria

10. DNA-constructs

11. Oligonucleotides

12. Fluorescence labeled Oligonucleotides

13. Recombinant Baculoviruses for Sf9 or Sf21 cells

14. Drosophila melanogaster: maintenance, embryo collection and extracts................ 72

15. Chromatographic material

16. Blotting material

17. Instruments

18. Free software and online tools

II. Methods

1. Biochemical methods (DNA-specific methods)

1.1. Standard procedures

1.2. Determination of DNA concentration

1.3. Analysis of DNA quality and quantity

1.4. Hybridization of Oligonucleotides

1.5. Radioactive and fluorescent labeling of DNA

1.6. Precipitation and isolation of radioactive DNA fragments

1.7. Generation and analysis of hemimethylated and methylated DNA

1.8. Preparation of DNA fragments for the assembly of mononucleosomes

2. Molecularbiological methods (Protein-specific methods)

2.1. Standard procedures in protein analysis

2.2. Protein quantification

2.3. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

2.4. Coomassie blue staining of protein gels

2.5. Semi-dry Western Blot

3. Isolation of chromatin remodeling complexes and the DNA methyltransferase Dnmt1

3.1. Expression of recombinant proteins with the baculovirus system

3.2. Purification of recombinant proteins using affinity chromatography

4. Chromatin – Assembly and analysis of arrays

4.1. Chromatin reconstitution using the salt gradient dialysis technique

4.2. Chromatin assembly using the Drosophila embryo extract (DREX)

4.3. Chromatin analysis by Micrococcal Nuclease (MNase) digest

5. Chromatin – Preparation of positioned mononucleosomes

5.1. Assembly of mononucleosomes using HP-Mix

5.2. Isolation of positioned mononucleosomes

6. In vitro analysis of DNA methylation in chromatin

6.1. Methylation activity assay on free DNA

6.2 Methylation activity assay on mononucleosomes and chromatin arrays

6.3. Bisulfite genomic sequencing

7. Chromatin – functional assays

7.1. Nucleosome mobilization assay

7.2. ATPase assay

7.3. Electrophoretic mobility shift assay (EMSA)

7.4. Competition assays

7.5. Dnmt1 binding assay using small DNA fragments

7.3. DNaseI protection assays (“DNaseI footprinting”)

8. Mammalian tissue culture

E. Results

I. Nucleosome positioning by chromatin remodeling complexes

1. Chromatin remodeling factors determine specific nucleosome positions.................104

2. Specific DNA features that direct nucleosome positioning

3. Two models explaining remodeler directed nucleosome positioning

4. Nucleosome positioning on “601”-NPS DNA substrates

II. Maintenance methylation in the context of chromatin

1. DNA and nucleosome binding properties of Dnmt1

1.1. DNA binding characteristics of Dnmt1

1.2. Nucleosome binding characteristics of Dnmt1

1.3. Mapping the localization of Dnmt1 on the 77-WID-77 nucleosome

2. Methylated CpG site analysis in the mononucleosomal core

3. Generation of hemimethylated DNA as a substrate for Dnmt1

3.1. Analysis of the hemimethylated substrates

4. Dnmt1 methyltransferase activity on nucleosome arrays

4.1. Activity in the absence and presence of chromatin remodeling factors

5. Binding properties on mononucleosomes in the presence of the chromatin remodeling enzyme Snf2H

F. Discussion and Perspectives

I. Nucleosome positioning by chromatin remodeling complexes

1. Do remodelers position nucleosomes in a sequence-dependent manner?.................. 145

2. Is remodeler directed nucleosome positioning determined by the DNA?

3. How can remodeler dependent nucleosome positioning be explained?

II. Characterization of Dnmt1 in the context of chromatin

1. What are the DNA and nucleosome binding properties of Dnmt1?

2. Where does Dnmt1 bind on a nucleosome?

3. Does Dnmt1 methylate DNA within the nucleosome core region?

4. Are chromatin dynamics required for Dnmt1 activity in chromatin?

5. Do Remodelers influence the Dnmt1 nucleosome binding affinity?

G. References

H. Manuscript

I. Acknowledgements / Danksagung

Eidesstattliche Erklärung

Curriculum Vitae…………………….………………………………………………....229 Index of Figures Figure 1: The nucleosome core particle

Figure 2: “Beads-on-a-string”

Figure 3: Schematic representation of two different topologies for the 30 nm fiber........ 10 Figure 4: Miscellaneous view of chromatin fiber condensation

Figure 5: Functional compartments of the vertebrate cell nucleus

Figure 6: Posttranslational modifications of histones

Figure 7: Schematic illustration of the components that constitute chromatin................. 17 Figure 8: SNF2 family of ATPases

Figure 9: Reactions catalyzed by ATP-dependent chromatin remodeling factors........... 21 Figure 10: SNF2 family of ATPases

Figure 11: DNA movement during the nucleosome remodeling reaction

Figure 12: Sequence-dependent nucleosome positioning

Figure 13: Graphical illustrations of the mammalian DNA methyltransferase domain organization

Figure 14: Catalytic mechanism of methylgroup tranfer by DNA methyltransferases...... 40 Figure 15: Imprinting control at the Igf2/H19 differentially methylated region (DMR)..... 44 Figure 16: Most prominent interaction partners of Dnmt1

Figure 17. Generation of different 601 DNA templates

Figure 18. Expression and purification of recombinant DNA methyltransferase 1 and chromatin remodeling enzymes

Figure 19. Chromatin assembly by salt gradient dialysis

Figure 20. Recombinant Drosophila histones

Figure 21. Chromatin remodeling complexes position nucleosomes in dependence on the underlying DNA sequence.

Figure 22. A curved DNA fragment guides remodeler-dependent nucleosome positioning

Figure 23. Schematic representation of the remodeler-dependent nucleosome translocation reaction

Figure 24. Evidence for a nucleosome positioning according to the “release model”.

.....113 Figure 25. Comparative nucleosome mobilization assays on different 601 nucleosomal substrates

Figure 26. Dnmt1 requires a DNA substrates length 45 bp for efficient DNA binding 117 Figure 27.

Nucleosome assembly on modified 601 fragments

Figure 28. Characterization of the Dnmt1 binding affinity to mononucleosomal substrates differing in the length of protruding DNA

Figure 29. The binding of Dnmt1 to nucleosomes occurs on symmetrical nucleosomes harboring 30-80 bp DNA overhangs on entry and exit sites of the nucleosome

Figure 30. Labeling of the 77-WID-77 DNA substrate using fluorescently labeled oligonucleotides

Figure 31. Setting up the DnaseI protection assay using a capillary electrophoresis instrument

Figure 32. Scheme of the DNaseI protection assay (“Footprint”)

Figure 33. DNaseI protection (“Footprinting”) assay to map the localization of Dnmt1 at the preferred mononucleosomal template

Figure 34. Bisulfite sequencing of Dnmt1-methylated mononucleosomal templates.

.....132 Figure 35. Analysis of Dnmt1 activity on different mononucleosomal templates............134 Figure 36. Preparation of hemimethylated DNA

Figure 37. Effective generation of hemimethylated DNA

Figure 38. Analysis of DNA methylation efficiency in chromatin

Figure 39. Remodeling factors are required for efficient DNA methylation in chromatin 142 Figure 40.

Analysis of the DNA binding characteristics of Dnmt1 in the presence of Snf2H

Figure 41. Interactions between DNA methyltransferases and chromatin-associated proteins

Index of Tables Table 1. Dnmt1 interacting proteins

Table 2: Used enzymes and respective company

Table 3: Common buffers and solutions

Table 4: Kits with the respective company

Table 4: Utilized antibodies

Table 5: Utilized mammalian and insect cell lines

Table 6: DNA constructs with cloning strategy

Table 7: Common DNA plasmids with supplier

Table 8: Oligonucleotides with indicated name, sequence, orientation, melting temperature and respective purpose

Table 9: Fluorescence labeled oligonucleotides with indicated name, sequence, orientation, melting temperature and respective purpose

Table 10: Chromatographic material

Table 11: Material used for Western blotting

Table 12: List of instruments

Table 13: List of used software

Table 14: Used PCR protocol

Table 15: Used PCR protocol for 601 fragments

Table 16: 601 DNA templates (WID)

Table 17: Purification by affinity chromatography

Table 18: Buffers used for salt gradient dialysis

Table 19: Methylation reaction for bisulfite sequencing

Abbreviati on s

–  –  –

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