H2B monoubiquitination in the murine hematopoietic system:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Heidelberg
[2018]
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | X, 130 Seiten Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Dietlein, Nikolaus |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a H2B monoubiquitination in the murine hematopoietic system |c presented by Nikolaus Dietlein, M.Sc. |
| 264 | 1 | |a Heidelberg |c [2018] | |
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| 502 | |b Dissertation |c Ruperto-Carola University of Heidelberg |d 2018 | ||
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Record in the Search Index
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| adam_text | TABLE OF CONTENTS
1
INTRODUCTION..................................................................................................1
1.1 EPIGENETIC GENE
REGULATION..........................................................................................
1
1.1.1 DNA
METHYLATION...............................................................................................
1
1.1.2 POSTTRANSLATIONAL HISTONE MODIFICATIONS
.............................................................
3
1.1.3 ATP-DEPENDENT CHROMATIN REMODELERS, HISTONE VARIANTS AND
NON-CODING
RNAS.................................................................................................................6
1.2 H2B
MONOUBIQUITINATION.............................................................................................6
1.2.1 WRITERS AND ERASERS OF MONOUBIQUITINATED H2B (H2BUB 1)
...............................
6
1.2.2 FUNCTIONS OF
H2BUBL.........................................................................................8
1.2.3 RING FINGER PROTEIN 20
(RNF20).........................................................................
11
1.2.4 UBIQUITIN-SPECIFLC PEPTIDASE 22 (USP22)
.........................................................
12
1.2.5 THE ROLE OF H2B MONOUBIQUITINATION IN STEM CELL BIOLOGY
..............................
14
1.3 THE MURINE HEMATOPOIETIC SYSTEM
......
.
....................................................................
17
1.3.1 HEMATOPOIETIC STEM CELL (HSC) MARKERS
.........................................................
17
1.3.2 HSC
NICHE........................................................................................................
18
1.3.3 HSC SELF-RENEWAL, LINEAGE COMMITMENT AND
DIFFERENTIATION.............................19
1.3.4 EPIGENETIC REGULATION OF
HEMATOPOIESIS............................................................24
1.4 AIMS OF THIS
STUDY.......................................................................................................28
2 MATERIALS AND
METHODS.............................................................................
29
2.1
MATERIALS.....................................................................................................................29
2.1.1
KITS..................................................................................................................
29
2.1.2
ENZYMES..........................................................................................................
29
2.1.3 MOLECULAR WEIGHT
MARKERS................................................................................30
2.1.4
OLIGONUCLEOTIDES...............................................................................................30
2.1.5
ANTIBODIES.......................................................................................................
31
2.1.6
CHEMICALS........................................................................................................
33
2.1.7 BUFFERS, MEDIA AND
SOLUTIONS.............................................................................34
2.1.8
PLASMIDS..........................................................................................................
38
2.1.9
CONSUMABLES....................................................................................................
39
2.1.10
EQUIPMENT.......................................................................................................
39
2.1.11
SOFTWARE..........................................................................................................
40
2.2 MOLECULAR BIOLOGY
METHODS.......................................................................................
40
2.2.1 PCR
AMPLIFICATION............................................................................................
40
2.2.2 RESTRICTION
DIGEST.............................................................................................
41
2.2.3 AGAROSE GEL
ELECTROPHORESIS.............................................................................
41
2.2.4 GEL EXTRACTION OF DNA
FRAGMENTS....................................................................
42
2.2.5 PCR PURIFICATION OF DNA
FRAGMENTS...............................................................
42
2.2.6
LIGATION............................................................................................................
42
2.2.7
A-TAILING...........................................................................................................
42
2.2.8 TA
CLONING.......................................................................................................
43
2.2.9 TRANSFORMATION OF BACTERIAL CELLS BY ELECTROPORATION
.......................................
43
2.2.10 BACTERIAL
CULTURES.............................................................................................
43
2.2.11 PLASMID
MINI-PREPARATION..................................................................................43
2.2.12 PLASMID
MAXI-PREPARATION................................................................................
44
2.2.13 PHENOL/CHLOROFORM EXTRACTION OF GENOMIC DNA
...............................................
44
2.2.14 EXTRACTION OF GENOMIC DNA BY SODIUM ACTETATE/ETHANOL
PRECIPITATION
...........
44
2.2.15 DNA
SEQUENCING...............................................................................................45
2.2.16 IN VITRO TRANSCRIPTION (IVT) OF CAS9 MRNA
....................................................
45
2.2.17 IN VITRO TRANSCRIPTION OF
SGRNAS.......................................................................46
2.2.18 RNA EXTRACTION USING TRIZOL
REAGENT...............................................................
47
2.2.19 RNA EXTRACTION USING THE ARCTURUS PICO PURE RNA EXTRACTION K IT
.................
47
2.2.20 QUALITY CONTROL OF EXTRACTED
RNA....................................................................
48
2.2.21 CDNA SYNTHESIS BY REVERSE
TRANSCRIPTION.........................................................
48
2.2.22 QUANTITATIVE REAL-TIME PCR
(QRT-PCR).......................................................... 48
2.2.23 RNA
SEQUENCING..............................................................................................49
2.3 WESTERN
BLOT...............................................................................................................
50
2.3.1 PREPARATION OF PROTEIN
LYSATES...........................................................................
50
2.3.2 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
..................................
50
2.3.3 PROTEIN TRANSFER BY SEMI-DRY
BLOT.....................................................................
50
2.3.4 PROTEIN TRANSFER BY WET
BLOT..............................................................................
50
2.3.5 TARGET PROTEIN
DETECTION...................................................................................
51
2.4 ANIMAL
EXPERIMENTS...................................................................................................
51
2.4.1 LEGAL
REGULATIONS..............................................................................................
51
2.4.2 MOUSE
MAINTENANCE.........................................................................................
51
2.4.3 MOUSE LINES
...........
.
........................................................................................
51
2.4.4 MICROINJECTION OF MURINE ZYGOTES
....................................................................
52
2.4.5
GENOTYPING.......................................................................................................
52
2.4.6 COLLECTION OF HEMATOPOIETIC ORGANS
.................................................................
55
2.4.7 BLOOD
COLLECTION...............................................................................................
55
2.4.8 BONE MARROW
TRANSPLANTATION...........................................................................
55
2.5 CELL CULTURE
EXPERIMENTS...........................................................................................
56
2.5.1 CELL
LINES..........................................................................................................56
2.5.2 MOUSE EMBRYONIC FIBROBLAST (MEF) CULTURE
....................................................
56
2.5.3 MURINE EMBRYONIC STEM CELL (MESC) CULTURE
..................................................
56
2.5.4 TRANSFECTION OF
MESCS....................................................................................56
2.5.5 CULTIVATION OF OP9 FEEDER
CELLS........................................................................
57
2.5.6 OP9
CO-CULTURES................................................................................................57
2.6 FLOW
CYTOMETRY...........................................................................................................58
2.6.1 PREPARATION OF SINGLE CELL
SUSPENSIONS.............................................................
58
2.6.2 CELL
COUNTING....................................................................................................58
2.6.3 FLOW CYTOMETRIC ANALYSIS OF PERIPHERAL BLOOD SAMPLES
....................................
58
2.6.4 FLOW CYTOMETRIC ANALYSIS OF BONE MARROW, SPLEEN AND THYMUS SAMPLES
........
59
2.6.5 LINEAGE DEPLETION OF BONE MARROW CELLS
..........................................................
59
2.6.6 FLUORESCENCE ACTIVATED CELL
SORTING..................................................................60
2.6.7 DETECTION OF INTRACELLULAR IGM BY FLOW CYTOMETRY
...........................................
60
2.6.8 EDU INCORPORATION ASSAY BY FLOW CYTOMETRY
...................................................
60
2.6.9 DNA CONTENT MEASUREMENT USING FLOW CYTOMETRY
...........................................
61
2.6.10 APOPTOSIS DETECTION BY ANNEXIN V LABELING USING FLOW CYTOMETRY
.................
61
2.7 DATA
ANALYSIS...............................................................................................................61
2.7.1 STATISTICAL
ANALYSIS............................................................................................61
2.7.2 RNA SEQUENCING DATA
ANALYSIS.........................................................................62
3
RESULTS.......................................................................................................
63
3.1 REGULATION OF H2B MONOUBIQUITINATION DURING HEMATOPOIETIC
DIFFERENTIATION ... 63
3.2 CHARACTERIZATION OF CONDITIONAL USP22 KNOCK-OUT MICE
.........................................
64
3.2.1 VAVL-CRE EFFICIENTLY RECOMBINES THE CONDITIONAL USP22 KNOCK-OUT
ALLELE IN
HEMATOPOIETIC
CELLS...........................................................................................64
3.2.2 USP22 DEFICIENCY LEADS TO INCREASED LEVELS OF MONOUBIQUITINATED
H2B
(H2BUBL)........................................................................................................
65
3.2.3 USP22 CKO MICE SHOW DECREASED NUMBERS OF PHENOTYPIC LT-HSCS
.............
66
3.2.4 ABSENCE OF USP22 INDUCES INCREASED PROLIFERATION OF HSCS
............................
67
3.2.5 USP22 DEFICIENCY LEADS TO AN IMBALANCE BETWEEN MYELOPOIESIS AND
LYMPHOPOIESIS..................................................................................................
68
3.2.6 B-CELL DEVELOPMENT IS PARTIALLY BLOCKED AT THE TRANSITION FROM
PRO- TO
PRE-B CELLS IN THE ABSENCE OF USP22 DUE TO INCREASED APOPTOSIS RATES
.............
70
3.2.7 USP22 DEFICIENCY ALTERS THE COMPOSITION OF THE HSC COMPARTMENT
................
73
3.2.8 INCREASED MYELOPOIESIS AND DECREASED LYMPHOPOIESIS IN THE ABSENCE
OF USP22
ARE INDEPENDENT PHENOTYPES AND DO NOT RESULT FROM LINEAGE CONVERSION
..........
74
3.2.9 USP22 DEFICIENCY INCREASES THE CELLULAR OUTPUT OF MYELOID
PROGENITORS
..........
75
3.2.10 USP22-DEFICIENT BONE MARROW CELLS SHOW DIMINISHED COMPETITIVENESS
..........
77
3.2.11 USP22 DEFICIENCY DOES NOT LEAD TO PREMATURE EXHAUSTION OF HSCS
..................
78
3.2.12 USP22-DEFICIENT HSPCS SHOW A STRONG MYELOID BIAS IN VITRO
..........................
80
3.2.13 TRANSCRIPTIONAL PROFILING OF USP22 CKO HSPCS
.............................................
81
3.3 GENERATION OF RNF20 KNOCK-OUT MICE USING CRISPR/CAS9
...................................
83
3.3.1 CRISPR/CAS9 TARGETING OF RED FLUORESCENT PROTEIN (RFP) IN MURINE
ESCS
.....
83
3.3.2 RNF20 TARGETING IN MESCS USING CRISPR/CAS9
..............................................
85
3.3.3 CRISPR/CAS9-MEDIATED RNF20 TARGETING IN MURINE ZYGOTES
...........................
86
3.3.4 CRISPR/CAS9-MEDIATED RNFZO DELETION LEADS TO COMPLEX INVERSIONS IN
MURINE
ZYGOTES.................................................................................................
88
3.3.5 RNF20 DEFICIENCY LEADS TO EMBRYONIC LETHALITY
................................................
90
3.4 CHARACTERIZATION OF RNF20 KNOCK-OUT MICE
.............................................................
90
3.4.1 RNF20 HAPLO-INSUFFICIENCY HAS NO MAJOR IMPACT ON STEADY-STATE
HEMATOPOIESIS...................................................................................................
90
4
DISCUSSION....................................................................................................93
4.1 USP22 REGULATES THE BALANCE BETWEEN MYELOPOIESIS AND LYMPHOPOIESIS
................
93
4.2 USP22 IS REQUIRED FOR THE MAINTENANCE OF HSC QUIESCENCE
...................................
97
4.3 USP22 IS REQUIRED FOR EFFICIENT B-CELL DEVELOPMENT AT THE TRANSITION
FROM THE
PRO-B TO THE PRE-B CELL
STAGE......................................................................................
98
4.4 USP22 IS REQUIRED FOR PROPER H2BUBL DEUBIQUITINATION IN THE
HEMATOPOIETIC
SYSTEM........................................................................................................................
100
4.5 RNF20 HETEROZYGOSITY HAS NO MAJOR EFFECTS ON STEADY STATE
HEMATOPOIESIS..........101
4.6 GLOBAL H2BUBL LEVELS DO NOT CHANGE DURING HEMATOPOIETIC
DIFFERENTIATION
.....
102
4.7 CONCLUSIONS AND FUTURE
DIRECTIONS..........................................................................
102
5 REFERENCES
105
6
ABBREVIATIONS..........................................................................................125
7
ACKNOWLEDGEMENTS..................................................................................129
|
| any_adam_object | 1 |
| author | Dietlein, Nikolaus |
| author_facet | Dietlein, Nikolaus |
| author_role | aut |
| author_sort | Dietlein, Nikolaus |
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| bvnumber | BV045278010 |
| ctrlnum | (OCoLC)1035526825 (DE-599)GBV1022181289 |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| spelling | Dietlein, Nikolaus Verfasser aut H2B monoubiquitination in the murine hematopoietic system presented by Nikolaus Dietlein, M.Sc. Heidelberg [2018] X, 130 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto-Carola University of Heidelberg 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1160107149/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030665649&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Dietlein, Nikolaus H2B monoubiquitination in the murine hematopoietic system |
| subject_GND | (DE-588)4113937-9 |
| title | H2B monoubiquitination in the murine hematopoietic system |
| title_auth | H2B monoubiquitination in the murine hematopoietic system |
| title_exact_search | H2B monoubiquitination in the murine hematopoietic system |
| title_full | H2B monoubiquitination in the murine hematopoietic system presented by Nikolaus Dietlein, M.Sc. |
| title_fullStr | H2B monoubiquitination in the murine hematopoietic system presented by Nikolaus Dietlein, M.Sc. |
| title_full_unstemmed | H2B monoubiquitination in the murine hematopoietic system presented by Nikolaus Dietlein, M.Sc. |
| title_short | H2B monoubiquitination in the murine hematopoietic system |
| title_sort | h2b monoubiquitination in the murine hematopoietic system |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1160107149/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030665649&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT dietleinnikolaus h2bmonoubiquitinationinthemurinehematopoieticsystem |
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