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
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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
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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
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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
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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