The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | X, 161 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice |c von Kerstin Schmidt |
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| adam_text | TABLE OF CONTENTS
PAGE NUMBER
TABLE OF CONTENTS I
ABBREVIATIONS V
LIST OF FIGURES VII
LIST OF TABLES IX
1 INTRODUCTION
1.1 STEM CELLS AND MAMMALIAN DEVELOPMENT 1
1.2 EPIGENETICS AND CHROMATIN 4
1.2.1 CHROMATIN STRUCTURE IS KEY TO GENE REGULATION 4
1.2.2 CHROMATIN IS SUBJECT TO A WIDE VARIETY OF EPIGENETIC REGULATION 5
1.2.2.1 DNA METHYLATION 6
1.2.2.2 POSTTRANSLATIONAL HISTONE TAIL MODIFICATIONS 6
1.2.3 HISTONE LYSINE METHYLATION PLAYS A PIVOTAL ROLE FOR GENE
REGULATION 8
1.3 HISTONE LYSINE METHYLTRANSFERASES 9
1.3.1 POLYCOMB AND TRITHORAX GROUP PROTEINS FULFILL OPPOSING FUNCTIONS 9
1.3.2 THE MAMMALIAN FAMILY OF H3K4 METHYLTRANSFERASES COMPRISES SIX
DIFFERENT
ENZYMES 11
1.3.3 MAMMALIAN H3K4 METHYLTRANSFERASES PLAY DIVERSE ROLES DURING
DEVELOPMENT
AND IN THE ADULT 13
1.3.3.1 MII1 (ALL1, KMT2A) 13
1.3.3.2 MII2 (WBP7; KMT2B) 15
1.3.3.3 MII3 (KMT2C) AND MII4 (KMT2D) 16
1.3.3.4 SETDLA (KMT2F) 16
1.4 AIM OF THIS STUDY 17
2 MATERIAL AND METHODS
2.1 GENERAL MATERIALS 19
2.1.1 CHEMICALS AND REAGENTS 19
2.1.2 ENZYMES, KITS AND COMPLETED SOLUTIONS 20
2.1.3 CELL CULTURE MEDIA AND SUPPLEMENTS 21
2.1.4 CONSUMABLES 22
HTTP://D-NB.INFO/1072704129
2.1.5 BUFFERS AND SOLUTIONS 23
2.1.6 PRIMERS 29
2.1.7 ANTIBODIES 29
2.1.8 TOOLS, DEVICES AND SOFTWARE 30
2.2 METHODS 31
2.2 1 GENERATION OF THE SETDLB KNOCKOUT MOUSE 31
2.2.2 MOUSE CARE AND HOUSING 31
2.2.3 GENOTYPING 32
2.2.3.1 DNA ISOLATION 32
2.2.3.2 POLYMERASE CHAIN REACTION (PCR) 32
2.2.4 EMBRYOLOGY 33
2 2.4.1 EMBRYO DISSECTIONS 33
2.2.4.2 WHOLE MOUNT LACZ STAINING 33
2.2.4.3 IMMUNOHISTOCHEMISTRY OF BLASTOCYSTS 34
2.2.5 EMBRYONIC STEM CELLS 34
2.2.5 1 DERIVATION OF EMBRYONIC STEM CELLS (ESCS) FROM MOUSE BLASTOCYSTS
34
2.2.5.2 GENERAL CULTURE OF ESCS 36
2.2.5.3 TAMOXIFEN INDUCTION OF ESCS 36
2.2.5.4 ALKALINE PHOSPHATASE (AP) STAINING 36
2.2.5.5 CELL CYCLE ANALYSIS 37
2.2.6 WESTERN BLOT ANALYSIS 37
2.2.6.1 PROTEIN EXTRACTION 37
2.2.6.2 SDS-PAGE 38
2.2.6.3 IMMUNOBLOTTING 39
2.2.7 HISTOLOGY 39
2.2.7.1 PARAFFIN EMBEDDING AND SECTIONING 39
2.2.7.2 H&E STAINING 40
2.2.7.3 TUNEL ASSAY 40
2.2.7.4 IMMUNOHISTOCHEMISTRY 40
2.2.8 TAMOXIFEN GAVAGE 41
2.2.9 BLOOD MEASUREMENTS 41
2.2.10 FACS ANALYSIS 42
2.2.10.1 PREPARATION OF CELL SUSPENSIONS 42
2.2.10.2 STAINING OF CELL SUSPENSIONS 42
2.2.11 BONE MARROW TRANSPLANTATION 45
3 RESULTS
3.1 THE FUNCTION OF SETDL B DURING DEVELOPMENT AND IN THE ADULT WAS
EXPLORED USING A MULTIPURPOSE ALLELE STRATEGY
47
3.2 SETDLB PLAYS CRUCIAL ROLES DURING POST-IMPLANTATION DEVELOPMENT 49
3.2.1 SETDLB IS UNIFORMLY EXPRESSED DURING EMBRYONIC DEVELOPMENT 49
3.2.2 THE CONSTITUTIVE KNOCKOUT OF SETDLB IS EMBRYONIC LETHAL BEFORE
E11.5 50
3.2.3 KNOCKOUT EMBRYOS ARE SEVERELY GROWTH-RETARDED, BUT DEVELOP ALL
THREE GERM
LAYERS 51
3.2.4 THE CONSTITUTIVE EMBRYONIC PHENOTYPE IS REPRODUCED USING PGK-CRE
MEDIATED
DELETION OF SETDLB 54
3.2.5 SETDLB IS CONTINUOUSLY REQUIRED THROUGHOUT DEVELOPMENT 56
3.3 SETDLB IS DISPENSABLE FOR EARLY EMBRYONIC DEVELOPMENT AND EMBRYONIC
STEM CELLS 58
3.3.1 SETDLB-DEFICIENT ESCS CAN BE DERIVED FROM THE INNER CELL MASS OF
E3.5
BLASTOCYSTS 58
3.3.2 CONDITIONAL DELETION OF SETDLB IN ESCS DOES NOT AFFECT NORMAL
MORPHOLOGY AND
PROLIFERATION 60
3.4 THE LOSS OF SETDL B DOES NOT ALTER GLOBAL H3 METHYLATION 62
3.5 CONDITIONAL DELETION OF SETDLB IN THE ADULT MOUSE PROVOKES THE
DEVELOPMENT OF FATAL MYELOID NEOPLASIA 63
3.5.1 MICE DIE WITHIN 30 WEEKS AFTER UBIQUITOUS ROSA26-CRE MEDIATED
DELETION OF
SETDLB 63
3.5.2 KNOCKOUT MICE REVEAL MAJOR CHANGES IN HAEMATOPOIETIC ORGANS 65
3.5.3 ABNORMAL HAEMATOPOIESIS IS EVIDENT FROM QUANTITATIVE AND
DYSPLASTIC CHANGES
IN ALL LINEAGES OF PERIPHERAL BLOOD 67
3.5.3.1 KNOCKOUT MICE REVEAL SEVERE DEVIATIONS OF PERIPHERAL BLOOD
COUNTS 67
3.5.3.2 APPEARANCE OF IMMATURE FORMS IN BLOOD AND CELLULAR DYSPLASIA ARE
SIGNS OF
INEFFICIENT HAEMATOPOIESIS 72
3.5.4 HAEMATOPOIESIS IN THE BONE MARROW IS AFFECTED ON THE LEVEL OF
HAEMATOPOIETIC
STEM CELLS 75
3.5.4.1 VARIABLE DEGREES OF DENSITIES CHARACTERIZE THE BONE MARROW OF
KNOCKOUT
MICE 75
3.5.4.2 FACS-ANALYSIS OF HSCS AND HPCS REVEALS AN INCREASED FRACTION OF
ST-HSCS IN KNOCKOUT MICE 77
3.5.5 SPLENOMEGALY IN KNOCKOUT MICE IS CAUSED BY EXTENSIVE ACTIVATION OF
EXTRAMEDULLAR HAEMATOPOIESIS 86
3.5.5.1 THE KNOCKOUT SPLEEN IS DIFFUSELY INFILTRATED BY CELLS OF THE
MYELOID LINEAGE 86
3.5.5.2 FACS-ANALYSIS CONFIRMS THE PREDOMINANCE OF MOSTLY IMMATURE
MYELOID
CELLS IN KNOCKOUT SPLEENS 89
3.5.5.3 FULL-BLOWN HAEMATOPOIESIS CHARACTERIZES THE SPLEENS OF KNOCKOUT
MICE 93
3.6 BONE MARROW RECONSTITUTION EXPERIMENTS CONFIRM THE HAEMATOPOIETIC
INTRINSIC REQUIREMENT OF SETDL B 96
3.6.1 HIGH PERCENTAGE OF TRANSPLANT-DERIVED (CD45.2+) CELLS IN
PERIPHERAL BLOOD
VALIDATES SUCCESSFUL ENGRAFTMENT 99
3.6.2 CD45.2+ GRANULOCYTES EXPAND IN PERIPHERAL BLOOD UPON DELETION OF
SETDL B 103
3.6.3 ANALYSIS OF HSCS AND HPCS IN THE BONE MARROW REVEALS AN INCREASED
LSK AND
DECREASED HPC COMPARTMENT 105
3.6.4 MYELOID EXPANSION IN THE SPLEEN IS DRIVEN BY ENHANCED
EXTRAMEDULLAR
HAEMATOPOIESIS 106
3.7 THE USE OF SPECIFIC HAEMATOPOIETIC CRE LINES FURTHER SPECIFIES THE
SETDL B-DEPENDENT COMPARTMENT 109
3.7.1 VAV-CRE MEDIATED PAN-HAEMATOPOIETIC DELETION OF SETDLB LEADS TO
LETHALITY
BY 30 WEEKS 109
3.7.2 CONFINED DELETION OF SETDL B IN MATURE MYELOID CELLS DOES NOT
AFFECT NORMAL
HOMEOSTASIS 111
4 DISCUSSION
4.1 REQUIREMENT OF SETDL B DURING EMBRYONIC DEVELOPMENT 113
4.1.1 SETDLB IS CRUCIAL FOR SURVIVAL OF POST-IMPLANTATION EMBRYOS 113
4.1.2 MULTIPLE ORGAN SYSTEMS MIGHT CONTRIBUTE TO INSUFFICIENT GROWTH AND
DEATH IN
SETDLB NULL EMBRYOS 116
4.1.3 THE DEVELOPMENTAL REQUIREMENT OF SETDLB IMPLIES BOTH FUNCTIONAL
SPECIFICATION
AND REDUNDANCY BETWEEN H3K4 METHYLTRANSFERASES 120
4.2 REQUIREMENT OF SETDL B IN THE ADULT ORGANISM 123
4.2.1 THE USE OF DIFFERENT KNOCKOUT APPROACHES REVEALS AN INTRINSIC
REQUIREMENT
OF SETDLB IN THE HAEMATOPOIETIC SYSTEM 123
4.2.2 A SYNERGISTIC INTERPLAY BETWEEN HAEMATOPOIESIS IN BONE MARROW AND
SPLEEN
DEFINES THE NATURE OF THE MALIGNANT DISEASE 125
4.2.3 THE NEOPLASM UPON DELETION OF SETDL B CAN BE CLASSIFIED AS MYELOID
LEUKAEMIA
WITH FEATURES OF HUMAN CMML 131
4.2.4 THE SETDLB KNOCKOUT MOUSE MODEL ALLOWS STUDYING THE PATHOGENESIS
OF
MYELOID MALIGNANCY IN HUMANS 136
5 REFERENCES 141
6 ACKNOWLEDGEMENTS 157
STATEMENT OF ORIGINALITY 161
|
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| indexdate | 2024-07-10T07:07:23Z |
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| language | English |
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| spelling | Schmidt, Kerstin 1984- Verfasser (DE-588)1072704099 aut The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice von Kerstin Schmidt 2015 X, 161 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2015 Spiegelman, Art 1948- Maus (DE-588)4781848-7 gnd rswk-swf Histon-Methyltransferase (DE-588)4820080-3 gnd rswk-swf Tiermodell (DE-588)4140660-6 gnd rswk-swf Embryonalentwicklung (DE-588)4070792-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Tiermodell (DE-588)4140660-6 s Spiegelman, Art 1948- Maus (DE-588)4781848-7 u Histon-Methyltransferase (DE-588)4820080-3 s Embryonalentwicklung (DE-588)4070792-1 s DE-188 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028117240&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schmidt, Kerstin 1984- The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice Spiegelman, Art 1948- Maus (DE-588)4781848-7 gnd Histon-Methyltransferase (DE-588)4820080-3 gnd Tiermodell (DE-588)4140660-6 gnd Embryonalentwicklung (DE-588)4070792-1 gnd |
| subject_GND | (DE-588)4781848-7 (DE-588)4820080-3 (DE-588)4140660-6 (DE-588)4070792-1 (DE-588)4113937-9 |
| title | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice |
| title_auth | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice |
| title_exact_search | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice |
| title_full | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice von Kerstin Schmidt |
| title_fullStr | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice von Kerstin Schmidt |
| title_full_unstemmed | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice von Kerstin Schmidt |
| title_short | The role of the H3K4 histone methyltransferase Setd1b during embryogenesis and in adult mice |
| title_sort | the role of the h3k4 histone methyltransferase setd1b during embryogenesis and in adult mice |
| topic | Spiegelman, Art 1948- Maus (DE-588)4781848-7 gnd Histon-Methyltransferase (DE-588)4820080-3 gnd Tiermodell (DE-588)4140660-6 gnd Embryonalentwicklung (DE-588)4070792-1 gnd |
| topic_facet | Spiegelman, Art 1948- Maus Histon-Methyltransferase Tiermodell Embryonalentwicklung Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028117240&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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