Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | IV, 135 S. Ill., graph. Darst. |
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| adam_text | MAX-PLANCK-INSTITUT FUR IMMUNBIOLOGIE UND GPIGENETIK
ABTEILUNG FUR ZELLULARE UND MOLEKULARE IMMUNOLOGIC
FREIBURG IM BREISGAU
MOLECULAR
AND FUNCTIONAL
CHARACTERIZATION OF THE ARGININE
METHYLTRANSFERASE CARM1 IN NOTCH
SIGNALING
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTAT FUR BIOLOGIE
DER ALBERT-LUDWIGS-UNIVERSITAT
FREIBURG IM BREISGAU
VORGELEGT MARZ 2013
VON KERSTIN HEIN
GEBOREN IN SCHWEINFURT
HTTP://D-NB.INFO/1065829450
CONTENTS I
CONTENTS
CONTENTS I
ZUSAMMENFASSUNG 1
SUMMARY 2
1. INTRODUCTION 3
1.1 THE NOTCH
PATHWAY 3
1.1.1 DISCOVERY OF THE NOTCH PATHWAY 3
1.1.2 NOTCH RECEPTORS AND LIGANDS 4
1.1.3 THE NOTCH SIGNALING CASCADE 6
1.1.4 NOTCH TARGET GENES 9
1.1.5 FUNCTIONAL ROLE OF NOTCH SIGNALING 11
1.1.5.1 NOTCH SIGNALING IN HEMATOPOIESIS 11
1.1.5.2 N OTCH SIGNALING IN CANCER 13
1.1.5.3 NOTCH SIGNALING IN.OTHER DEVELOPMENTAL PROCESSES 15
1.2 POSTTRANSLATIONAL MODIFICATIONS IN TRANSCRIPTIONAL REGULATION OF THE
NOTCH
PATHWAY 16
1.2.1 PHOSPHORYLATION 17
1.2.2 UBIQUITINATION 18
1.2.3 ACETYLATION 19
1.2.4 METHYLATION 20
1.3 ARGININE METHYLATION 22
1.3.1 THE PROTEIN ARGININE METHYLTRANSFERASE FAMILY 22
1.3.2 FUNCTIONAL ROLE OF ARGININE METHYLATION 24
1.3.3 CARM1 26
2. AIM OF STUDIES 27
3. MATERIALS AND METHODS 28
3.1 MATERIALS 28
3.1.1 CHEMICALS 28
3.1.2 ENZYMES AND STANDARDS 29
3.1.3 BUFFERS AND SOLUTIONS 29
3.1.4 TECHNICAL EQUIPMENT 32
CONTENTS II
3.1.5 CELL CULTURE - CELL LINES, MEDIA AND REAGENTS 33
3.1.6 ANTIBODIES, KITS, RESINS AND OTHER MATERIALS 34
3.1.7 PLASMIDS 35
3.1.8 BACTERIAL STRAINS 37
3.1.9 PRIMER, SHRNA AND PEPTIDE SEQUENCES 37
3.1.10 DATABASES AND SOFTWARES 39
3.2 METHODS 40
3.2.1 BACTERIAL CELL CULTURE METHODS 40
3.2.1.1 CULTIVATION OF E.
COLI 40
3.2.1.2 PREPARATION OF ELECTROCOMPETENT CELLS 40
3.2.1.3 TRANSFECTION OF BACTERIAL CELLS BY ELECTROPORATION 40
3.2.1.4 PROTEIN EXPRESSION IN BL21 41
3.2.2 EUKARYOTIC CELL CULTURE METHODS 41
3.2.2.1 CELL CULTURE CONDITIONS AND CRYOPRESERVATION 41
3.2.2.2 TRANSFECTION OF ADHERENT CELLS USING THE CALCIUM-PHOSPHATE
METHOD 42
3.2.2.3 TRANSFECTION OF ADHERENT CELLS USING LIPOFECTAMINE 42
3.2.2.4 TRANSFECTION AND ANALYSIS OF MGF CELLS
FOR FACS BASED STABILITY ASSAY 43
3.2.2.5 RETROVIRAL INFECTION OF SUSPENSION CELLS 43
3.2.2.6 LENTIVIRAL INFECTION OF SUSPENSION CELLS 44
3.2.2.7 LUCIFERASE ASSAYS 44
3.2.2.8 PHARMACOLOGICAL TREATMENT OF CELLS 44
3.2.3 MOLECULAR BIOLOGICAL METHODS 45
3.2.3.1 POLYMERASE CHAIN REACTION 45
3.2.3.2 RESTRICTION DIGEST 45
3.2.3.3 SITE DIRECTED MUTAGENESIS 46
3.2.3.4 LIGATION 46
3.2.3.5 PURIFICATION OF PLASMID DNA FROM BACTERIAL CELLS 46
3.2.3.6 ISOLATION OF RNA AND REVERSE TRANSCRIPTION 47
3.2.3.7 QUANTITATIVE REAL-TIME PCR 48
3.2.4 PROTEIN BIOCHEMICAL METHODS 48
3.2.4.1 PURIFICATION OF GST FUSION PROTEINS 48
3.2.4.2 IN VITRO TRANSCRIPTION/TRANSLATION OF PROTEINS 49
3.2.4.3 GST-PULLDOWN 50
3.2.4.4 PREPARATION OF NUCLEAR EXTRACTS 50
3.2.4.5 PREPARATION OF WHOLE CELL EXTRACTS 50
3.2.4.6 CELL FRACTIONATION 51
3.2.4.7 QUANTIFICATION OF PROTEIN CONCENTRATION USING BRADFORD ASSAY 51
3.2.4.8 IN VITRO METHYLATION ASSAY 51
3.2.4.9 HEAVY-METHYL SILAC 52
CONTENTS III
3.2.4.10 IN VIVO UBIQUITINATION ASSAY 53
3.2.4.11 CO-IMMUNOPRECIPITATION 54
3.2.4.12 IMMUNOFLUORESCENCE 54
3.2.4.13 PURIFICATION OF RABBIT POLYCLONAL ANTIBODIES 55
3.2.4.14 PEPTIDE PULLDOWN 57
3.2.4.15 SDS-PAGE 58
3.2.4.16 COOMASSIE AND SILVER STAINING 59
3.2.4.17 WESTERN BLOT 59
3.2.4.18 CHROMATIN-IMMUNOPRECIPITATION 60 .
3.2.5 QUANTIFICATION AND STATISTICAL ANALYSIS 61
4. RESULTS 62
4.1 ARGININE METHYLATION OF THE NICD BY CARM1 62
4.1.1 ANTIBODY AGAINST ADMA RECOGNIZES PURIFIED NICD 62
4.1.2 CARM1 METHYLATES THE C-TERMINAL PART OF THE NICD IN VITRO 63
4.1.3 IDENTIFICATION OF METHYLATION SITES BY MASS SPECTROMETRY IN VIVO
64
4.1.4 VALIDATION OF METHYLATION SITES BY IN
VITRO
METHYLATION 70
4.1.5 CHARACTERIZATION OF AN ANTIBODY SPECIFIC FOR METHYLATED NICD 71
4.1.6 ANALYSIS OF SINGLE ARGININE MUTANTS 73
4.2 CARM1 IN THE
NOTCH PATHWAY 75
4.2.1 CARM1 INTERACTS DIRECTLY WITH RBP-J IN GST-PULLDOWN ASSAYS 75
4.2.2 RBP-J AND NICD CO-IMMUNOPRECIPITATE WITH CARM1 76
4.2.3 ENDOGENOUS NOTCHL AND CARM1 INTERACT ONLY IN NOTCH-ACTIVE CELLS 77
4.2.4 CARM1 IS METHYLATING THE NICD IN THE NUCLEUS 79
4.2.5 CARM1 FUNCTIONS AS CO-ACTIVATOR AT NOTCH TARGET GENES 80
4.2.6 CHIP ANALYSIS OF NOTCH TARGET GENES 83
4.2.6.1 ANALYSIS OF NOTCH TARGET GENES FOR NOTCHL/RBP-J BINDING SITES 84
4.2.6.2 CARM1 BINDING
TO NOTCHL OCCUPIED SITES 84
4.2.6.3 HISTONE MODIFICATION H3R17ME2A AT NOTCHL BINDING SITES 86
4.3 FUNCTIONAL ROLE OF ARGININE METHYLATION OF THE NICD 87
4.3.1 INFLUENCE OF METHYLATION ON STABILITY 87
4.3.1.1 CYCLOHEXIMIDE-CHASE 87
4.3.1.2 FACS BASED STABILITY ASSAY USING A BICISTRONIC VECTOR 89
4.3.2 CROSSTALK OF METHYLATION WITH OTHER PTMS 90
4.3.2.1 ACETYLATION 91
4.3.2.2 PHOSPHORYLATION 91
4.3.2.3 UBIQUITINATION 93
4.3.3 SPECIFIC INTERACTION PARTNERS OF METHYLATED NICD 94
CONTENTS IV
4.3.3.1 SILAC APPROACH TO IDENTIFY METHYL-NOTCHL SPECIFIC INTERACTION
PARTNERS 94
4.3.3.2 INTERACTION OF TDRD3 AND NOTCHL 96
4.3.3.3 FUNCTIONAL ROLE OF TDRD3 98
4.3.4 INFLUENCE OF ARGININE METHYLATION ON NICD DEPENDENT TRANSCRIPTION
98
4.4 IN VIVO
SIGNIFICANCE OF CARM1 IN NOTCH
SIGNALING 101
5. DISCUSSION 102
5.1 CARM1 METHYLATES
THE NICD 102
5.2 CARM1 HAS
A FUNCTIONAL ROLE AS CO-ACTIVATOR OF NOTCH SIGNALING 105
5.3 ARGININE METHYLATION REGULATES STABILITY OF THE NICD 107
5.4 ARGININE METHYLATION INFLUENCES BINDING PARTNERS OF THE NICD 108
5.5 ARGININE METHYLATION OF THE NICD PLAYS
A ROLE IN TRANSCRIPTION 109
5.6 IN VIVO
SIGNIFICANCE OF ARGININE METHYLATION OF THE NICD 113
6. OUTLOOK 115
ACKNOWLEDGEMENTS 117
ABBREVIATIONS 118
REFERENCES 121
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| spelling | Hein, Kerstin Verfasser (DE-588)1065825315 aut Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling vorgelegt von Kerstin Hein 2013 IV, 135 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2013 Methyltransferasen (DE-588)4276195-5 gnd rswk-swf Gen notch (DE-588)4762370-6 gnd rswk-swf Arginin (DE-588)4142987-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Gen notch (DE-588)4762370-6 s Arginin (DE-588)4142987-4 s Methyltransferasen (DE-588)4276195-5 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027809721&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hein, Kerstin Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling Methyltransferasen (DE-588)4276195-5 gnd Gen notch (DE-588)4762370-6 gnd Arginin (DE-588)4142987-4 gnd |
| subject_GND | (DE-588)4276195-5 (DE-588)4762370-6 (DE-588)4142987-4 (DE-588)4113937-9 |
| title | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling |
| title_auth | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling |
| title_exact_search | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling |
| title_full | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling vorgelegt von Kerstin Hein |
| title_fullStr | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling vorgelegt von Kerstin Hein |
| title_full_unstemmed | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling vorgelegt von Kerstin Hein |
| title_short | Molecular and functional characterization of the arginine methyltransferase CARM1 in Notch signaling |
| title_sort | molecular and functional characterization of the arginine methyltransferase carm1 in notch signaling |
| topic | Methyltransferasen (DE-588)4276195-5 gnd Gen notch (DE-588)4762370-6 gnd Arginin (DE-588)4142987-4 gnd |
| topic_facet | Methyltransferasen Gen notch Arginin Hochschulschrift |
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