ausblenden:
Sprache(n):
eng - English
Datum:
2009-12-09
Publikationsstatus:
Angenommen
Seiten:
162
Ort, Verlag, Ausgabe:
Berlin : Freie Universität Berlin
Inhaltsverzeichnis:
ABBREVIATIONS IV
ABSTRACT VII
ABSTRACT (GERMAN) 1
1 INTRODUCTION 3
1.1 OCT4 regulated networks in pluripotent cells 3
1.1.1 Early Development – how a single cell develops to a complex organism 3
1.1.2 The POU family transcription factor OCT4 10
1.1.3 The diversity of binding site recognition motifs of OCT4 13
1.1.4 Transcriptional network for pluripotency 16
1.2 Analysis of transcription factor binding sites 19
1.2.1 RNA interference 19
1.2.2 Chromatin Immunoprecipitation 21
1.2.3 Chromatin Immunoprecipitation followed by microarray
hybridization (ChIP-chip) 22
1.2.4 ChIP followed by sequencing (ChIP-seq) 24
1.2.5 Identification of enriched sequences in ChIP-chip experiments as a way to access potential binding sites 26
1.2.6 Motif analysis and modules 28
1.3 Aim of this work 29
2 MATERIAL AND METHODS 30
2.1 Molecular biology 30
2.1.1 Polymerase Chain Reaction 30
2.1.2 Isolation of plasmid DNA 31
2.1.3 Gel extraction and PCR purification 31
2.1.4 Cloning and Sequencing of PCR-Products 32
2.1.5 Ligation 32
2.2 RNA analyses 32
2.2.1 Total RNA isolation using RNeasy® Mini Kit 32
2.2.2 RNA and cDNA quantification 32
2.2.3 Agarose gel electrophoresis 33
2.2.4 Reverse transcription 33
2.2.5 Real-time polymerase chain reaction (Real-Time PCR) 34
2.2.6 Illumina bead chip hybridisation 34
Contents
II
2.3 Protein analyses 35
2.3.1 Protein isolation 35
2.3.2 Protein quantification (Bradford) 35
2.3.3 SDS-PAGE gel electrophoresis 35
2.3.4 Western blotting 36
2.3.5 Chromatin Immunoprecipitation (ChIP) 37
2.3.6 Amplification of ChIP and Input DNA 38
2.3.7 Band shift assays (EMSA) 38
2.3.8 Pulldown assays using biotinylated DNA 38
2.3.9 Chromatin Immunoprecipitation followed by sequencing (ChIPseq) 39
2.4 Cell culture 39
2.4.1 Embryonal carcinoma cells, NCCIT cells 39
2.4.2 Transient Transfections 39
2.5 Data analysis 41
2.5.1 OCT4 ChIP array analysis 41
2.5.2 Microarray expression analysis 42
2.5.3 ChIP-seq in silico methods 43
3 RESULTS 46
3.1 ChIP-Chip data analysis 46
3.1.1 Specifity of the OCT4 antibody and the impact amplification bias has on site specific enrichment 46
3.1.2 Real time validation of known and putative OCT4 targets 48
3.1.3 ChIP-chip raw data normalization and quality control49
3.1.4 Impact of different peak detection strategies on peal quality 54
3.1.5 Comparison of different peak algorithms and rank based peak detection 57
3.1.6 Functional analysis and comparison with literature 59
3.1.7 Six distinct OCT4 binding modules 64
3.1.8 Validation of selected conserved OCT4 binding sites 71
3.1.9 Validation of an octamer site in the 5 prime proximal promoter of GADD45G 76
3.2 Induction of GADD45G expression in NCCIT cells by Genistein 78
3.3 Transient overexpression of GADD45G in NCCIT cells 79
3.4 Differences in the transcriptional levels between human
embryonal stem cells and human embryonal cancer cells 82
3.4.1 Comparison with other published datasets 84
3.4.2 Functional annotation analysis of differential expressed genes 84
3.5 ChIP-seq data analysis of OCT4 targets in NCCIT cells 86
3.5.1 Data quality control 86
3.5.2 Saturation analysis 88
3.5.3 Genome wide distribution of sequencing reads 90
3.5.4 Genome wide distribution of binding regions 91
Contents
III
3.5.5 Comparative analysis of gene associated binding regions 92
3.5.6 Motif mapping to binding regions 94
3.5.7 Functional regulation of OCT4 target genes 99
3.5.8 Functional enrichment analysis 99
3.5.9 Summary 102
4 DISCUSSION 105
4.1 General considerations regarding the ChIP technique 105
4.2 ChIP-chip results compared to literature 105
4.3 Data integration in the form of an Embryonic Stem Cell
database 107
4.4 Different modules of OCT4 binding 109
4.5 USP44 is a potential cell cycle regulator, controlled by OCT4 117
4.6 Genistein induces the upregulation of GADD45G and has an
effect on the expression of key pluripotency markers 117
4.7 GADD45G induces the upregulation of differentiation related genes 118
4.8 Differences between human EC and human ES cells 120
4.9 ChIP-seq discovered OCT4 binding sites 121
5 CONCLUSION 123
REFERENCES A
PUBLICATIONS L
APPENDIX M
5.1.1 Solutions, Buffers and Media M
5.1.2 Buffers for SDS-PAGE gel electrophoresis N
5.1.3 Buffers for western blotting O
5.1.4 Cells, Vectors and antibodies P
5.1.5 Equipment and Reagents P
5.1.6 Supplemental material T
Art der Begutachtung:
-
Identifikatoren:
eDoc: 451721
Art des Abschluß:
Doktorarbeit
