The role of DNA modifications in pluripotency and differentiation:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext http://d-nb.info/103138068X/34 https://nbn-resolving.org/urn:nbn:de:bvb:19-153089 Inhaltsverzeichnis |
| Beschreibung: | IV, 234 S. Ill., graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Schmidt, Christine Silvia |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a The role of DNA modifications in pluripotency and differentiation |c Christine Silvia Schmidt |
| 264 | 1 | |c 2012 | |
| 300 | |a IV, 234 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Univ., Diss., 2012 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 776 | 0 | 8 | |i Erscheint auch als |n Online-Ausgabe |o urn:nbn:de:bvb:19-153089 |
| 856 | 4 | 1 | |u http://edoc.ub.uni-muenchen.de/15308/ |x Verlag |z kostenfrei |3 Volltext |
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Datensatz im Suchindex
| _version_ | 1804150224397008896 |
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| adam_text | IMAGE 1
CONTENT
CONTENT
SUMMARY
1. INTRODUCTION 1
1.1 EPIGENETIC GENE REGULATION 1
1.1.1 DNA METHYLATION 2
1.1.2 HISTONE MODIFICATIONS 3
1.2 REGULATION OF DNA METHYLATION IN MAMMALIAN CELLS 6
1.2.1 WRITERS OF DNA METHYLATION MARKS - THE FAMILY OF DNA
METHYLTRANSFERASES 6
1.2.1.1 DNMTL 7
1.2.1.2 DNMT2 ; 9
. 1.2.1.3 THE DNMT3 FAMILY 10
1.2.1.4 COOPERATIVE FUNCTIONS OF MAMMALIAN DNMTS 11
1.2.2 READERS OF DNA METHYLATION MARKS - METHYLCYTOSINE BINDING PROTEINS
12
1.2.2.1 THE MBD PROTEIN FAMILY 12
1.2.2.2 THE KAISO PROTEIN FAMILY 14
1.2.2.3 THE UHRF PROTEIN FAMILY * 15
1.2.3 MODIFIERS OF DNA METHYLATION MARKS - THE FAMILY OF TET PROTEINS 16
1 1.2.3.1 TET1 17
1.2.3.2 TET2 . 19
1.2.3.3 TET3 19
1.2.3.4 DNA HYDROXYMETHYLATION - 5HMC 20
1.2.3.5 POSSIBLE MECHANISMS OF DNA DEMETHYLATION 21
1.3 EMBRYONIC STEM CELLS AS A MODEL SYSTEM FOR DIFFERENTIATION PROCESSES
IN VITRO 23
1.3.1 THE PLURIPOTENCY NETWORK 23
1.3.2 THE EPIGENETIC LANDSCAPE IN EMBRYONIC STEM CELLS 27
1.3.3 DIFFERENTIATION OF EMBRYONIC STEM CELLS IN VITRO RECAPITULATES
EARLY DEVELOPMENTAL
PROCESSES 29
1.3.4 THE EPIGENETIC LANDSCAPE CHANGES DYNAMICALLY DURING
DIFFERENTIATION 30
1.3.4.1 ROLE OF DNA METHYLATION DURING DEVELOPMENT 31
1.4 AIMS OF THE WORK 33
I
HTTP://D-NB.INFO/103348654X
IMAGE 2
CONTENT
2. MATERIALS AND METHODS 35
2.1 MATERIALS 35
2.1.1 TECHNICAL DEVICES 35
2.1.2 CONSUMABLES 36
2.1.3 REAGENTS AND CONSUMABLES 36
2.1.4 CELL LINES 39
2.1.5 PRIMER SEQUENCES 39
2.1.5.1 TAQMAN ASSAY ID NUMBERS FOR RELATIVE QUANTIFICATION USING QPCR
39
2.1.5.2 SYBR GREEN PRIMER SEQUENCES FOR RELATIVE QUANTIFICATION USING
QPCR 40
2.1.5.3 SYBR GREEN PRIMER SEQUENCES FOR QUANTIFICATION OF PVURTSI I
DIGESTED PRODUCTS
USING QPCR 40
2.1.5.4 PRIMER SEQUENCES FOR BISULFITE SEQUENCING 40
2.1.5.5 PRIMER SEQUENCES FOR PYROSEQUENCING 41
2.2 METHODS 42
2.2.1 METHODS OF CELL BIOLOGY 42
2.2.1.1 CULTIVATION OF ESCS AND SOMATIC CELLS 42
2.2.1.2 GENERATION OF TRANSGENIC CELL LINES 42
2.2.1.3 DIFFERENTIATION OF ESCS TO NEURAL STEM CELLS (NSCS) 42
* 2.2.1.4 DIFFERENTIATION OF ESCS TO EBS 42
2.2.1.5 REPLATING OF EBS * 43
2.2.1.6 TRANSFECTION OF PLASMIDS 43
2.2.1.7 TRANSIENT KNOCK- DOWN USING SMALL INTERFERING RNAS (SIRNAS) 43
2.2.1.8 TREATMENT OF OGNSCS WITH EPIGENETIC INHIBITORS 43
2.2.1.9 INTRACELLULAR PROTEIN STAINING USING FACS 44
2.2.1.10 SERUM STARVATION OF FIBROBLASTS 44
2.2.1.11 ANALYSIS OF CELL CYCLE PROFILE USING FACS 44
2.2.1.12 IMMUNOFLUORESCENCE STAINING 44
2.2.2 METHODS OF MOLECULAR BIOLOGY 45
2.2.2.1 RNA EXTRACTION 45
2.2.2.2 CDNA SYNTHESIS 45
2.2.2.3 QUANTIFICATION OF MRNA LEVELS USING REAL- TIME PCR 45
II
IMAGE 3
CONTENT
2.2.2.4 GENOME- WIDE EXPRESSION PROFILING USING MICROARRAYS 46
2.2.2.5 DNA EXTRACTION 46
2.2.2.6 PREPARATION OF REFERENCE DNA FRAGMENTS FOR 5HMC GLUCOSYLATION
ASSAY 46
2.2.2.7 QUANTITATIVE 5HMC GLUCOSYLATION ASSAY 47
2.2.2.8 PREPARATION OF REFERENCE DNA FRAGMENTS FOR TESTING PVURTSI I
SPECIFICITY 47
2.2.2.9 PREPARATION OF DNA SUBSTRATES FOR LINKER - PVURTSI I - ANALYSIS
48
2.2.2.10 PREPARATION OF LINKER 48
2.2.2.11 DIGESTION WITH PVURTSI I 48
2.2.2.12 DETECTION OF PVURTSI I DIGESTED FRAGMENTS USING QPCR 48
2.2.2.12 DETECTION OF PVURTSI I DIGESTED FRAGMENTS USING LINKER - PCR
STRATEGY 49
2.2.2.13 DNA METHYLATION ANALYSIS 49
2.2.2.14 CLONING OF OCT4 DTALES 49
2.2.2.15 CLONING OF OCT4 REPORTER CONTRUCT 50
2.2.3 METHODS OF BIOCHEMISTRY 50
2.2.3.1 PROTEIN EXPRESSION AND PURIFICATION 50
2.2.3.2 REPORTER GENE ASSAY FOR DTALE ACTIVITY 51
3. RESULTS 53
3.1 REVERSION OF DIFFERENTIATION PROGRAMS IN GLOBALLY HYPOMETHYLATED
EMBRYONIC STEM CELLS 53 3.1 11NCOMPLETE SILENCING OF PLURIPOTENCY
GENES DURING DIFFERENTIATION IN GLOBALLY
HYPOMETHYLATED CELLS * 53
3.1.2 COMPLETE AND UNIFORM DOWNREGULATION OF OCT4 PROTEIN LEVEL IN
HYPOMETHYLATED EBS 54
3.1.3 DNA METHYLATION IS DISPENSABLE FOR THE INITIATION OF
DIFFERENTIATION PROGRAMS 55
3.1.4 CELLS LACKING DNMTL POSSESS A GREATER DIFFERENTIATION POTENTIAL
THAN TKO CELLS 61
3.1.5 MOST BIVALENT GENES ARE SILENCED INDEPENDENTLY OF DE NOVO
METHYLATION OR DNMT PROTEINS
DURING EARLY EB DIFFERENTIATION 66
3.1.6 DNMTS ARE REQUIRED FOR SILENCING SELECTED BIVALENT GENES DURING
DIFFERENTIATION 67
3.1.7 HYPOMETHYLATED CELLS FROM LATE EBS REVERT TO THE UNDIFFERENTIATED
STATE 70
3.2 DISTINCT FUNCTIONS OF THE TWO MEMBERS OF THE UHRF PROTEIN FAMILY,
UHRFL AND UHRF2 73
3.2.1 UHRFL AND UHRF2 ARE DIFFERENTIALLY EXPRESSED IN ESCS, VARIOUS
ADULT TISSUES, DURING
DIFFERENTIATION TO EBS AND QUIESCENCE (SERUM STARVATION) 73
3.2.2 UHRF2 DOES NOT PLAY A ROLE IN MAINTENANCE DNA METHYLATION IN
PROLIFERATING CELLS 75
3.3 NOVEL METHODS TO QUANTIFY AND MAP 5 HMC IN GENOMIC DNA 81
III
IMAGE 4
CONTENT
3.3.1 SENSITIVE ENZYMATIC QUANTIFICATION OF GLOBAL HMC LEVELS 81
3.3.2 THE 5HMC SPECIFIC ENDONUCLEASE PVURTSLL AS A TOOL TO PROFILE
GENOMIC 5HMC PATTERNS 83
3.4 TARGETED TRANSCRIPTIONAL ACTIVATION OF SILENT OCT4 PLURIPOTENCY
GENES BY COMBINING
DESIGNER TALES AND INHIBITION OF EPIGENETIC MODIFIERS 89
4. DISCUSSION 95
4.1 GLOBAL DNA HYPOMETHYLATION PREVENTS CONSOLIDATION OF DIFFERENTIATION
PROGRAMS AND
ALLOWS REVERSION TO THE ESC STATE 95
4.1.1 DNA METHYLATION IS NOT REQUIRED FOR THE INITIAL DOWN REGULATION OF
PLURIPOTENCY GENES 95
4.1.2 DNMT1 V AND TKO ESCS SHOW DIFFERENCES IN THEIR DEVELOPMENTAL
POTENTIAL 97
4.1.3 PARALLELS AND CROSSTALK BETWEEN THE TWO MAJOR REPRESSIVE PATHWAYS-
DNA METHYLATION
AND POLYCOMB REPRESSIVE SYSTEM 100
4.1. 4 IMPROVED REPROGRAMMING BY TRANSIENT, SIMULTANEOUS INACTIVATION OF
DNMTL AND P53? 102
4.2 UHRF PROTEINS LINK THE TWO MAJOR REPRESSIVE EPIGENETIC PATHWAYS 105
4.2.1 UHRFL AND UHRF2 SHOW NO FUNCTIONAL REDUNDANCY 105
4.2.2 WHAT IS THE FUNCTION O F UHRF2? 106
4.3 ROLE OF 5HMC AND TETS DURING DEVELOPMENT 109
4.3.1 NOVEL METHODS TO QUANTIFY AND MAP 5HMC 109
4.3.2 5HMC- AN INTERMEDIATE OF DEMETHYLATION OR A STABLE EPIGENETIC
MODIFICATION? 112 % L
4.4 DESIGNER TALES- NOVEL TOOLS FOR GENOME EDITING 115
5. ANNEX 117
5.1 REFERENCES 117
5.2 ABBREVIATIONS 143
5.3 DECLARATION 147
5.4 ACKNOWLEDGEMENTS 149
6. APPENDIX 151
6.1 DIFFERENTIALLY EXPRESSED GENES IN THE PLURIPOTENT STATE 151
6.2 DIFFERENTIALLY REGULATED GENES DURING THE FIRST DIFFERENTIATION
PERIOD (DAY 0 -4) 155
6.3 DIFFERENTIALLY REGULATED GENES DURING THE SECOND DIFFERENTIATION
PERIOD (DAY 4 - 1 6 ) 192
IV
|
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| spelling | Schmidt, Christine Silvia Verfasser aut The role of DNA modifications in pluripotency and differentiation Christine Silvia Schmidt 2012 IV, 234 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-153089 http://edoc.ub.uni-muenchen.de/15308/ Verlag kostenfrei Volltext http://d-nb.info/103138068X/34 Langzeitarchivierung Nationalbibliothek https://nbn-resolving.org/urn:nbn:de:bvb:19-153089 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025906083&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schmidt, Christine Silvia The role of DNA modifications in pluripotency and differentiation |
| subject_GND | (DE-588)4113937-9 |
| title | The role of DNA modifications in pluripotency and differentiation |
| title_auth | The role of DNA modifications in pluripotency and differentiation |
| title_exact_search | The role of DNA modifications in pluripotency and differentiation |
| title_full | The role of DNA modifications in pluripotency and differentiation Christine Silvia Schmidt |
| title_fullStr | The role of DNA modifications in pluripotency and differentiation Christine Silvia Schmidt |
| title_full_unstemmed | The role of DNA modifications in pluripotency and differentiation Christine Silvia Schmidt |
| title_short | The role of DNA modifications in pluripotency and differentiation |
| title_sort | the role of dna modifications in pluripotency and differentiation |
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