ERKLARUNG ....................................................... 2
ZUSAMMENFASSUNG ................................................. 3
SUMMERY ......................................................... 5
Contributions ................................................ 7
Acknowledgements ............................................. 7
Annotations .................................................. 8
Abbreviations ................................................ 9
TABLE OF CONTENTS .............................................. 10
1 INTRODUCTION ................................................ 14
1.1 Dictyostelium .......................................... 14
1.1.1 Genome Features ................................. 15
1.2 Regulation of gene expression .......................... 16
Histone Acetylation .................................... 19
Histone Methylation .................................... 20
Arginine Methylation ................................... 21
H3K4me ................................................. 21
H3K9me ................................................. 21
H3K27me ................................................ 21
НЗКЗбmе ................................................ 22
H3K79me ................................................ 22
H4K20me ................................................ 22
Histone Phosphorylation ................................ 22
Histone Ubiquitination ................................. 23
Histone Sumoylation .................................... 24
Histone Citrullination ................................. 24
Histone ADP-Ribosylation ............................... 24
1.3 Chromatin Associated Proteins .......................... 26
1.4 The role of RNAi in gene expression and nuclear
organisation ........................................... 27
1.5 Nuclear Organisation ................................... 28
Aims of this Thesis .................................... 41
2 MATERIALS AND METHODS ....................................... 42
2.1 Materials .............................................. 42
2.2 Methods ................................................ 47
2.4 Western Blotting ....................................... 50
2.5 Tap-tag Purification ................................... 51
2.6 Identification of Homologs. Alignments ................. 51
2.7 Immunofluorescence ..................................... 52
2.8 Florescent Labelling of DNA Probes by Nick
Translation ............................................ 52
2.9 Fluorescence In Situ Hybridisation (FISH) .............. 52
2.10 IF-FISH ................................................ 53
2.11 Microscopy ............................................. 53
2.12 Chromatin Immunoprecipitation .......................... 53
2.13 Analysis СЫР Samples ................................... 54
2.14 Developmental Assay .................................... 54
2.15 GFP Reporter Constructs ................................ 55
2.16 LacZ Reporter Constructs ............................... 55
2.17 Anti-EB4 staining of developing Dictyostelium .......... 55
3 RESULTS ..................................................... 57
3.1 Cloning vectors for Dictyostelium ....................... 57
3.2 Nuclear organisation in Dictyostelium ................... 66
3.3 Transgenes .............................................. 96
3.4 Development ............................................ 103
4 DISCUSSION ................................................. 120
4.1 Cloning Vectors ....................................... 120
4.2 Nuclear Organisation and Histone Modifications ........ 120
4.3 Transgenes ............................................ 123
4.4 Epigenetic regulation of Development .................. 124
5 FUTURE DIRECTIONS .......................................... 129
6 REFERENCES ................................................. 130
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