Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2015
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XIII, 166 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |c von Ashish Gupta |
| 264 | 1 | |c 2015 | |
| 300 | |a XIII, 166 S. |b Ill., graph. Darst. | ||
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| 502 | |a Dresden, Techn. Univ., Diss., 2015 | ||
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| adam_text | TABLE OF CONTENTS
ABSTRACT (III)
LIST OF FIGURES (IX)
LIST OF TABLES (XI)
LIST OF ABBREVIATIONS (XII)
1 INTRODUCTION 1
1.1 EPIGENETICS 1
1.1.1 CHROMATIN STRUCTURE: HISTONES AND NUCLEOSOME MODEL 2
1.1.2 CHROMATIN MODIFICATIONS AND CHROMATIN REMODELING 4
1.1.3 POST-TRANSLATIONAL MODIFICATIONS (PTMS) 6
1.1.4 HISTONE LYSINE METHYLATION AND METHYLTRANSFERASES 8
1.1.4.1 TRITHORAX (TRXG) AND POLYCOMB (PCG) GROUP PROTEINS 9
1.2 HISTONE DEMETHYLATION 11
1.2.1 HISTONE LYSINE DEMETHYLASES AND THEIR MECHANISMS OF CATALYSIS 11
1.2.1.1 LSD DEMETHYLASES (LYSINE-SPECIFIC DEMETHYLASES) 11
1.2.1.2 JMJC DEMETHYLASES 12
1.3 HISTONE DEMETHYLASES AND THEIR ROLES IN DISEASE AND DEVELOPMENT. 13
1.3.1 KDM1 SUB-FAMILY 14
1.3.2 KDM5 SUB-FAMILY 15
1.3.2.1 KDM5A 16
1.3.2.2 KDM5B 17
1.3.2.3 KDM5C 19
1.3.2.4 KDM5D 20
1.4 EPIGENETIC REGULATION IN NEUROGENESIS 20
1.4.1 HISTONE METHYLTRANSFERASES AND DEMETHYLASES IN NEUROGENESIS 20
1.5 MOUSE EMBRYONIC STEM CELLS AS A MODEL FOR STUDYING DEVELOPMENT IN
VITRO
22
IV
HTTP://D-NB.INFO/107610164X
2 AIM OF THIS WORK 24
3 MATERIALS AND METHODS 25
3.1 GENERAL MATERIALS 25
3.1.1 CHEMICAL REAGENTS 25
3.1.2 OTHER REAGENTS (KITS, LADDERS, NUCLEOTIDES, ETC.) 26
3.1.3 CELL CULTURE MEDIA AND OTHER REAGENTS 27
3.1.4 ANTIBODIES 32
3.1.5 PLASMIDS 32
3.1.6 BACTERIAL ARTIFICIAL CHROMOSOMES (BACS) 33
3.1.7 OLIGONUCLEOTIDES/PRIMER SEQUENCES 33
3.2 METHODS - DNA 35
3.2.1 OBTAINING A BAC 35
3.2.2 DESIGN OF THE TAGGING CASSETTE 36
3.2.3 TRANSFORMATION WITH RED/ET EXPRESSION PLASMID 39
3.2.4 PREPARATION OF THE TAGGING CASSETTE BY PCR 40
3.2.5 INSERTING THE TAGGING CASSETTE INTO A BAC BY RECOMBINEERING 41
3.2.6 QUALITY CONTROL OF RECOMBINEERING 42
3.2.7 BAC/PLASMID MINI-PREP PROTOCOL AND RESTRICTION ANALYSIS OF DNA 43
3.2.8 BAC MAXI-PREP PROTOCOL 44
3.2.9 TAGGING STRATEGY 44
3.2.10 GENERATION OF GENE TARGETING CONSTRUCTS BY RECOMBINEERING 45
3.2.11 SUBCLONING OF A SPECIFIC REGION FROM A BAC BY RED/ET 47
3.2.12 VERIFICATION AND PREPARATION OF DNA FOR TARGETING 48
3.3 METHODS - CELL CULTURE 48
3.3.1 CULTURE CONDITIONS FOR MOUSE EMBRYONIC STEM CELLS (MES) 48
3.3.2 TRANSFECTION OF MOUSE ES CELLS TO GENERATE STABLE BAC TRANSGENIC
CELL
LINES 48
3.3.3 ELECTROPORATION OF MOUSE ES CELLS 49
3.3.4 PICKING COLONIES FOR CLONAL ANALYSIS 49
3.3.5 GENOMIC DNA EXTRACTION FROM 96 WELL AND GENOTYPING 50
3.3.6 LONG RANGE PCR ANALYSIS OF TARGETED CLONES 50
3.3.7 IMMUNOFLUORESCENCE ANALYSIS OF THE GFP/VENUS TAGGED PROTEINS 52
V
3.3.8 INDUCTION OF KDM5B KNOCKOUT WITH 4-OHT 52
3.3.9 CULTURE CONDITIONS AND DIFFERENTIATION OF MES CELLS TO NEURAL STEM
CELLS
(NS), NEURONS AND ASTROCYTES 53
3.3.10 SHORT HAIRPIN RNA (SHRNA) KNOCKDOWN EXPERIMENTS 54
3.3.11 CELL PROLIFERATION ASSAY 55
3.3.12 ALKALINE PHOSPHATASE (AP) STAINING OF MES CELLS 55
3.3.13 CELL CYCLE ANALYSIS BY FLOW CYTOMETRY 55
3.4 METHODS - RNA 56
3.4.1 RNA ISOLATION AND CDNA SYNTHESIS 56
3.4.2 QUANTITATIVE REAL-TIME REVERSE TRANSCRIPTION-PCR (QRT-PCR)
ANALYSIS .56
3.5 METHODS - CHROMATIN 57
3.5.1 CHROMATIN IMMUNOPRECIPITATION (CHIP) 57
3.5.2 DEEP SEQUENCING OF THE IMMUNOPRECIPITATED DNA 60
3.5.3 CHLP-SEQ, RNA-SEQ, AND GO ANALYSIS 60
3.6 METHODS - PROTEIN 63
3.6.1 WESTERN BLOT ANALYSIS 63
3.6.2 SMALL-SCALE IMMUNOPRECIPITATION (IP) OF VENUS-TAGGED PROTEINS 63
3.6.3 CO-LMMUNOPRECIPITATION (CO-IP) ASSAY 64
3.6.4 IMMUNOPRECIPITATION OF VENUS-TAGGED PROTEINS FOR MASS
SPECTROMETRY. 64
3.7 GENERATION OF TRANSGENIC MES LINES 65
3.7.1 TAGGING OF KDM5 DEMETHYLASES 65
3.7.2 GENERATION OF VENUS TAGGED BAC CONSTRUCTS FOR CANDIDATE KDM5B
PROTEIN INTERACTORS 65
3.7.3 GENERATION OF KDM5B MUTANT BAC CONSTRUCTS 66
4 RESULTS 70
4.1 CHARACTERIZATION OF KDM5B CONDITIONAL CELL LINE 70
4.2 IDENTIFICATION OF KDM5B PROTEIN INTERACTORS 73
4.2.1 EXPRESSION OF KDM5 DEMETHYLASES IN MES CELLS 73
4.2.2 CELLULAR LOCALIZATION OF KDM5 DEMETHYLASES 74
4.2.3 MASS SPECTROMETRY (MS) ANALYSIS OF KDM5 DEMETHYLASES 75
4.2.4 EXPRESSION OF VENUS TAGGED KDM5B INTERACTORS IN MES CELLS 80
VI
4.2.5 CELLULAR LOCALIZATION OF KDM5B INTERACTORS 82
4.2.6 MASS SPECTROMETRY ANALYSIS OF KDM5B INTERACTORS 84
4.2.7 VALIDATION OF PROTEIN-PROTEIN INTERACTION BY
CO-IMMUNOPRECIPITATION 91
4.3 ROLE OF KDM5B IN SELF-RENEWAL AND DIFFERENTIATION OF MES CELLS 95
4.3.1 LOSS OF KDM5B DOES NOT AFFECT PLURIPOTENCY IN MES CELLS 95
4.3.2 LOSS OF KDM5B LEADS TO REDUCED PROLIFERATION OF MES CELLS 96
4.3.3 KDM5B IS INDISPENSIBLE FOR NEURAL DIFFERENTIATION 99
4.3.4 LOSS OF KDM5B LEADS TO DOWNREGULATION OF GENES INVOLVED IN
NEUROGENESIS 106
4.4 ROLE OF KDM5B INTERACTORS IN MES CELLS PLURIPOTENCY 108
4.4.1 SHRNA MEDIATED KNOCKDOWN OF CTBP2 AND ZMYND8 IN MES CELLS 108
4.4.2 LOSS OF CTBP2 IMPEDES EXIT FROM PLURIPOTENCY AND NEURAL
DIFFERENTIATION
109
4.4.3 CTBP2 DEPLETED EARLY POPULATION OF NS CELLS CONTAIN
UNDIFFERENTIATED ES
CELLS 111
4.5 KDM5B DOMAIN DELETIONS AND CELLULAR LOCALIZATION 112
4.5.1 EXPRESSION OF MUTANT KDM5B BAC CONSTRUCTS IN MES CELLS 112
4.5.2 DELETION OF PHD2 AND PHD3 LEADS TO CYTOPLASMIC LOCALIZATION 113
4.6 GENOMIC LOCALIZATION OF KDM5B AND ITS INTERACTORS IN MES
CELLS....115
4.6.1 KDM5B CO-LOCALIZES WITH H3K4ME3 AT ACTIVE GENES IN MES CELLS 115
4.6.2 KDM5B OCCUPIES DEVELOPMENTAL REGULATORS IN MES CELLS 117
4.6.3 KDM5B AND ITS INTERACTORS CO-OCCUPY PROMOTER REGIONS 119
4.6.4 LOSS OF KDM5B LEADS TO INCREASED H3K4ME3 LEVELS, BUT DOES NOT
RESULT
IN CONCOMITANT INCREASED TRANSCRIPTION 123
5 DISCUSSION 125
5.1 IDENTIFICATION OF NOVEL KDM5B BINDING PARTNERS 125
5.1.1 COMMON INTERACTORS BETWEEN DIFFERENT KDM5 DEMETHYLASES 126
5.1.2 KDM5B AS PART OF CTBP2 CO-REPRESSOR COMPLEX; POTENTIAL ROLES IN
WNT
SIGNALING AND REDOX SENSING 127
5.1.3 KDM5B S ASSOCIATION WITH A COMPLEX OF FOUR UNCHARACTERIZED
ZINC-FINGER
PROTEINS 129
VII
5.2 KDM5B IN PLURIPOTENCY AND PROLIFERATION OF MES CELLS 130
5.2.1 KDM5B IN SELF-RENEWAL OF MES CELLS 130
5.2.2 KDM5B AFFECTS PROLIFERATION IN MES CELLS 131
5.3 REQUIREMENT OF KDM5B IN NEURAL DIFFERENTIATION OF MES CELLS 132
5.4 KDM5B BINDING PARTNER CTBP2 IN NEURAL DIFFERENTIATION 135
5.5 KDM5B PROTEIN DOMAIN DELETIONS 137
5.6 GENOME-WIDE LOCALIZATION OF KDM5B AND ITS BINDING PARTNERS IN MES
CELLS 138
5.6.1 KDM5B BINDS TO H3K4ME3-POSITIVE PROMOTERS AT ACTIVE GENES 138
5.6.2 KDM5B TARGET GENES ENCODE DEVELOPMENTAL REGULATORS 139
5.6.3 KDM5B CO-OCCUPIES DEVELOPMENTAL PROMOTERS WITH ITS BINDING PARTNER
ZNF592 140
5.6.4 LOSS OF KDM5B LEADS TO INCREASED H3K4ME3, BUT DOES NOT LEAD TO
INCREASED TRANSCRIPTION 140
6 CONCLUSION AND OUTLOOK 142
7 ACKNOWLEDGEMENTS 164
VIII
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| any_adam_object | 1 |
| author | Gupta, Ashish 1986- |
| author_GND | (DE-588)1074365615 |
| author_facet | Gupta, Ashish 1986- |
| author_role | aut |
| author_sort | Gupta, Ashish 1986- |
| author_variant | a g ag |
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| dewey-full | 572.819353 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.819353 |
| dewey-search | 572.819353 |
| dewey-sort | 3572.819353 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Gupta, Ashish 1986- Verfasser (DE-588)1074365615 aut Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation von Ashish Gupta 2015 XIII, 166 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2015 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028198821&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Gupta, Ashish 1986- Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |
| subject_GND | (DE-588)4113937-9 |
| title | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |
| title_auth | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |
| title_exact_search | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |
| title_full | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation von Ashish Gupta |
| title_fullStr | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation von Ashish Gupta |
| title_full_unstemmed | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation von Ashish Gupta |
| title_short | Role of H3K4 demethylase Kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |
| title_sort | role of h3k4 demethylase kdm5b in the maintenance of mouse embryonic stem cell pluripotency and neural differentiation |
| topic_facet | Hochschulschrift |
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