List of figures 9. Abbreviations Introduction 14

Table of Contents List o f figures 9 Abbreviations 11 1. Introduction 14 1.1 The discovery o f mobile genetic elements 14 1.2 Classification o...
4 downloads 0 Views 251KB Size
Table of Contents List o f figures

9

Abbreviations

11

1. Introduction

14

1.1 The discovery o f mobile genetic elements

14

1.2 Classification o f Eukaryotic Transposable elements

15

1.2.1 Retroelements

16

1.2.2 DNA Transposons

21

1.2.3 Mechanisms of DNA elements transposition

23

1.3 T e l / m a r i n e r superfamily and the Sleeping Beauty transposon

28

1.3.1 The structure of t h e Sleeping Beauty t r a n s p o s o n

29

1.3.2 Functional domains of the SB transposase

31

1.4 Interactions between Transposons and their hosts

32

1.4.1 t h e life cycle o f transposable elements

32

1.4.2 The impact o f Transposable elements on h o s t genomes

32

1.4.3. Regulation o f transposition

34

2. Aims and s c o p e s o f this study

38

3. Materials and m e t h o d s

40

3.1 Chemicals a n d kits

40

3.2 Consumables

41

3.3 Technical equipments

41

3.4 Software

42

3.5 Bacteria strains

42

3.6 Eukaryotic cells

43

3.7 Plasmids used in these studies

43

3.8 Oligonucleotides

51

3.9 Enzymes

52

3.10 Antibodies a n d fluoroconjugates

53

3.11 DNA manipulation and general molecular biology techniques

53

3.11.1 Transformation a n d plasmid isolation from E. coli cells

53

3.11.2 Restriction enzyme digests

54

3.11.3 Dephosphorylation of DNA fragments

54

4

http://d-nb.info/1024781887

Table of contents

3.11.4 DNA electrophoresis a n d isolation from agarose gel

55

3.11.5 Ligation

55

3.11.6 Standard PCR

55

3.11.7 High fidelity PCR

56

3.11.9 PCR-based mutagenesis

56

3.11.10 DNA sequencing

56

3.12 Transcription analysis and RNA methods

57

3.12.1 Luciferase r e p o r t e r assays

57

3.12.2 RNA isolation from eukaryotic cells

58

3.12.3 Reverse transcription and cDNA synthesis

59

3.13 Protein methods

59

3.13.1 Protein concentration m e a s u r e m e n t s

59

3.13.1 Protein expression a n d isolation from E.coli cells

60

3.13.2 Total protein extraction from eukaryotic cells

61

3.13.3 SDS-PAGE separation of protein samples

61

3.13.4 Coomassie Blue Staining

62

3.13.5 W e s t e r n blot analysis

62

3.13.6 Protein immunoprecipitation 3.13.7 CK2 phosphorylation assay

3.14 Cell culture methods

:

63 64

65

3.14.1 General cells handling

65

3.14.2 Transient transfections

66

3.14.3 Generation o f stable cell lines

66

3.14.4 Chemical a n d stress t r e a t m e n t

67

3.15 Microscopy and immunochemical techniques

67

3.16 Transposase activity assays

71

3.16.1 PCR-based transposon excision assay

71

3.16.2 Colony forming transposition assay

72

3.17 Statistical analysis 4. Results

73 74

4.1 Molecular mechanisms regulating transcription o f the Sleeping Beauty transposase

74

4.1.1 The endogenous p r o m o t e r o f the Sleeping Beauty t r a n s p o s o n responds to certain stress conditions

75

5

Table of contents

4.1.2 The activity o f the SB 5'UTR p r o m o t e r is elevated in germ cells

77

4.1.3 Sleeping Beauty transposon contains multiple regulatory sites located in its 5'UTR

78

4.1.4 Members o f SRY-box containing transcription factors a n d Heat Shock Factors induce transcription from t h e transposons' 5'UTR

81

4.1.5 Mutagenesis of the transposons 5'UTR confirms t h e specificity o f Sox/SRY a n d HSFs-mediated regulation o f the SB p r o m o t e r activity

83

4.2 Subcellular localization o f the Sleeping Beauty transposase. Influence o f the heat stress,

Heat Shock Proteins and

post-translational modifications on

transposase distribution and activity

87

4.2.1 Sleeping Beauty transposase translocate t o t h e nucleoli of h e a t stressed cells

87

4.2.2 The cell cycle block in G l / S a n d G2/M stages h a s no a p p a r e n t effect on t h e transposase localization

91

4.2.3 The SB transposase associates with centrosomes o f dividing heat-shocked cells

93

4.2.4 The nucleolar accumulation o f the Sleeping Beauty transposase after h e a t shock is a conserved process i n d e p e n d e n t on t h e tissue a n d species origin

....97

4.2.5 Nucleolar accumulation o f t h e protein is t h e c o m m o n p r o p e r t y of T c l related transposases

101

4.2.6 Sleeping Beauty transposase distribution after h e a t shock and the recovery from t h e r m a l stress is a dynamic process

104

4.2.7 Sleeping Beauty transposase physically interacts with certain Heat Shock Proteins following thermal stress

109

4.2.8 Hsp40 a n d Hsp70 chaperon proteins localize in nucleoli of heat-shocked cells

112

4.2.9 Members of Hsp70 family a n d t h e h e a t shock enhance the enzymatic activity o f t h e Sleeping Beauty transposase

117

4.2.10 The Sleeping Beauty transposase contains a putative CK2 kinase recognition sites a n d undergoes in vitro phosphorylation

120

4.2.11 The S152E mutation o f CK2 motif o f t h e SB t r a n s p o s a s e causes protein nucleolar sequestration

122

4.2.12 Inhibition o f the CK2 kinase activity decreases efficiency o f t h e Sleeping Beauty transposition and results in nucleolar accumulation of t h e transposase enzyme

126

6

Table of contents

5. Discussion

131

5.1 Transcriptional regulation o f the Sleeping Beauty transposition

132

5.1.1 Stress-mediated a n d tissue-specific regulation o f t h e transposon p r o m o t e r 132 5.1.2 The presence o f c/s-acting regulatory elements in transposon p r o m o t e r regions

133

5.1.3 Host-encoded factors acting on t h e t r a n s p o s a s e expression level

134

5.2 The influence o f stress and stress-induced host factors on the Sleeping Beauty transposase's localisation and activity 5.2.1

The

subcellular

localization

135 and

compartmentalization

of the

SB

t r a n s p o s a s e in interphase cells

135

5.2.2 The association of the transposase with centrosomes o f dividing cells

137

5.2.3 The species a n d cell-type dependence o f t h e Sleeping Beauty transposase localization a n d cellular distribution o f o t h e r transposases

138

5.2.4 The intracellular compartmentalisation o f t h e transposase during stress a n d t h e recovery from the h e a t shock

139

5.2.5 The physical interactors of the SB transposase in stress-exposed cells .... 140 5.2.6 The role o f t h e h e a t shock a n d stress-induced proteins in regulation o f the t r a n s p o s a s e localization a n d activity

141

5.3 The role o f transposase modifications and CK2 signalling pathway in the regulation o f transposition

142

5.3.1 Identification of the CK2 putative recognition motifs and in vitro phosphorylation o f t h e transposase protein

142

5.3.2 The subcellular localization o f the SB t r a n s p o s a s e variants with mutated CK2 phosphorylation sites

143

5.3.3 The effect o f t h e CK2 inhibition on t h e transposase's activity and subcellular distribution of t h e protein

143

6. Bibliography

145

7. Supplementary materials

171

Appendix 1. Transcriptional response o f t h e Sleeping Beauty transposon to stress treatments

171

Appendix 2. The activity of the SB p r o m o t e r in germ-line a n d somatic cells

174

Appendix 3. Prediction of c/s-acting regulatory elements located in the 5'UTR of t h e Sleeping Beauty transposon

175

7

Table of contents

Appendix 4. Induction o f t h e Sleeping Beauty t r a n s p o s o n cotransfected Sox/SRY a n d HSF transcription factors

p r o m o t e r by 178

Appendix 5. Confirmation o f Sox/SRY a n d HSFs binding specificity to the SB t r a n s p o s o n 5'UTR

180

Appendix 6. Interactions b e t w e e n t h e Sleeping Beauty t r a n s p o s a s e enzyme and various nucleolar proteins

181

Appendix 7. The effect of Heat Shock Proteins overexpression on the SB transposition activity

183

Appendix 8. Summary of post-translational modifications identified by ELM and PROSITE screens

184

Appendix 9. The effect of CK2 kinase inhibition on t h e SB transposition activity 185 Summary

186

Zusammenfassung

188

Acknowledgements

190

Curriculum Vitae

191