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Analysis of developmental epistasis by chromatin immunoprecipitation in Xenopus laevis [Elektronische Ressource] / vorgelegt von Katrin Mansperger

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Analysis of Developmental Epistasis by Chromatin Immunoprecipitation in Xenopus laevis Dissertation zur Erlangung der Doktorwürde des Dr. rer. nat. an der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Katrin Mansperger München 2007 Erklärung und ehrenwörtliche Versicherung: Ich versichere, dass ich die vorliegende Arbeit selbstständig durchgeführt habe und keine anderen als die aufgeführten Hilfsmittel und Quellen benutzt habe. Hiermit erkläre ich, dass ich mich einer Doktorprüfung anderweitig ohne Erfolg nicht unterzogen habe. Mündliche Prüfung ablegt am 25. Juli 2007 1. Gutachter: Prof. Dr. Peter Becker 2. Gutachter: Prof. Dr. Heinrich Leonhardt für meine Eltern „The final aim of all love intrigues, be they comic or tragic, is really of more importance than all other ends in human life. What it turns upon is nothing less than the composition of the next generation.“ Arthur Schopenhauer, quoted by Charles Darwin This work gave rise to the following publication: Linder, B., Mentele, E., Mansperger, K., Straub, T., Kremmer, E. and Rupp, R.A.

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Published 01 January 2007
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Analysis of Developmental Epistasis
by Chromatin Immunoprecipitation in
Xenopus laevis










Dissertation zur Erlangung der Doktorwürde des Dr. rer. nat.
an der Fakultät für Biologie der
Ludwig-Maximilians-Universität München










vorgelegt von

Katrin Mansperger

München 2007










Erklärung und ehrenwörtliche Versicherung:

Ich versichere, dass ich die vorliegende Arbeit selbstständig durchgeführt habe
und keine anderen als die aufgeführten Hilfsmittel und Quellen benutzt habe.

Hiermit erkläre ich, dass ich mich einer Doktorprüfung anderweitig ohne Erfolg
nicht unterzogen habe.

























































Mündliche Prüfung ablegt am 25. Juli 2007

1. Gutachter: Prof. Dr. Peter Becker

2. Gutachter: Prof. Dr. Heinrich Leonhardt





für meine Eltern

































„The final aim of all love intrigues, be they comic or tragic, is really of more
importance than all other ends in human life. What it turns upon is nothing less
than the composition of the next generation.“
Arthur Schopenhauer, quoted by Charles Darwin

































This work gave rise to the following publication:


Linder, B., Mentele, E., Mansperger, K., Straub, T., Kremmer, E. and Rupp, R.A.
(2007) CHD4/Mi-2beta activity is required for the positioning of the
mesoderm/neuroectoderm boundary in Xenopus. Genes Dev, 21, 973-983. Table of Contents I
Table of Contents
1 Summary..........................................................................1
2 Introduction .....................................................................3
2.1 The live cycle of the African clawed frog Xenopus laevis................... 3
2.2 Determination signals and induction events in Xenopus laevis......... 4
2.2.1 Transcriptional regulation of the muscle determination factor MyoD....................6
2.2.2 Canonical Wnt/β-catenin signaling during embryonic development.....................8
2.2.3 Distinct regulatory input of the SNF2-like chromatin remodeling
ATPase CHD4 ........................................................................................................12
2.3 Epigenetics - from genotype to phenotype ........................................ 13
2.4 Chromatin ............................................................................................. 15
2.4.1 Structural features of chromatin ............................................................................15
2.4.1.1 The nucleosome............................................................................................16
2.4.1.2 The 30nm fiber ..............................................................................................16
2.4.1.3 Higher order chromatin structure .................................................................17
2.4.2 ATP-dependent chromatin remodeling .................................................................18
2.4.2.1 SWI/SNF-containing chromatin remodeling complexes.............................19
2.4.2.2 CHD class of remodelers..............................................................................20
2.4.3 Covalent, post-translational histone modifications...............................................21
2.4.3.1 Acetylation .....................................................................................................23
2.4.3.2 Methylation.....................................................................................................24
2.4.3.3 Other modifications .......................................................................................27
2.5 Chromatin immunoprecipitation (ChIP).............................................. 27
2.6 Objectives............................................................................................. 30
3 Materials and Methods .................................................31
3.1 Reagents............................................................................................... 31
3.1.1 Fine chemicals........................................................................................................31
3.1.2 Enzymes and proteins............................................................................................31
3.2 Laboratory Equipment ......................................................................... 31
3.3 Antibodies ............................................................................................ 32
3.3.1 Primary Antibodies .................................................................................................32
3.3.1.1 Primary Antibodies, commercially available or published ..........................32
3.3.1.2 Rat monoclonal antibodies ...........................................................................32
3.3.2 Secondary Antibodies ............................................................................................34 Table of Contents II
3.3.2.1 Immunocytochemistry ...................................................................................34
3.3.2.2 Immunofluorescence.....................................................................................34
3.3.2.3 In Situ Hybridization ......................................................................................34
3.3.2.4 Western Blot analysis ...................................................................................34
3.4 Nucleic acids ........................................................................................ 34
3.4.1 Solutions..................................................................................................................34
3.4.2 Size standard..........................................................................................................34
3.4.3 Oligonucleotides .....................................................................................................35
3.4.3.1 Oligonucleotides for RT-PCR .......................................................................35
3.4.3.2 Oligonucleotides for cloning .........................................................................35
3.4.3.3 Oligonucleotides for real-time PCR..............................................................36
3.4.4 Plasmids..................................................................................................................38
3.4.4.1 Vectors for cloning.........................................................................................38
3.4.4.2 Plasmids for in vitro transcription .................................................................39
3.4.4.3 Plasmids for dig-labeled RNA in situ hybridization probes.........................40
3.4.4.4 Plasmids for recombinant GST-Fusion-Proteins.........................................40
3.4.4.5 Plasmids for real-time PCR tests .................................................................40
3.4.5 Handling of bacteria ...............................................................................................40
3.4.6 Bacteria strains .......................................................................................................40
3.5 Molecular biological methods ............................................................. 40
3.5.1 Solutions..................................................................................................................40
3.5.2 Isolation of nucleic acids ........................................................................................41
3.5.2.1 Mini-preparation with Qiagen kit...................................................................41
3.5.2.2 Isolation of RNA.............................................................................................42
3.5.3 Analysis and manipulation of nucleic acids ..........................................................42
3.5.3.1 Cloning methods ...........................................................................................42
3.5.3.2 Gel electrophoresis of nucleic acids ............................................................42
3.5.3.3 Isolation of DNA fragments from agarose gel .............................................42
3.5.4 Polymerase chain reaction (PCR).........................................................................42
3.5.4.1 PCR amplification of DNA fragments for cloning ........................................42
3.5.4.2 RT-PCR assay...............................................................................................43
3.5.4.3 Real-time PCR...............................................................................................43
3.5.5 In vitro transcription ................................................................................................43
3.5.5.1 In vitro transcription for microinjection .........................................................43
3.5.5.2 In vitro transcription of dig labeled RNA probes..........................................44
3.5.6 RNA in situ hybridization........................................................................................44
3.6 Embryological methods....................................................................... 45 Table of Contents III
3.6.1 Solutions..................................................................................................................45
3.6.2 Experimental animals.............................................................................................45
3.6.3 Superovulation of female Xenopus laevis ............................................................46
3.6.4 Preparation of testis ...............................................................................................46
3.6.5 In vitro fertilization of eggs and culture of the embryos .......................................46
3.6.6 Removal of the egg jelly coat ................................................................................46
3.6.7 Injection of embryos ...............................................................................................46
3.6.8 Preparation of animal cap explants.......................................................................47
3.7 Histological methods ........................................................................... 47
3.7.1 Solutions..................................................................................................................47
3.7.2 Immunocytochemistry ............................................................................................47
3.7.3 Immunofluorescence on Embryo Sections ...........................................................48
3.8 Protein analysis.................................................................................... 49
3.8.1 Solutions..................................................................................................................49
3.8.2 In vitro translation ...................................................................................................50
3.8.3 Protein extract for SDS-PAGE with StrataClean™ resin ......................................50
3.8.4 Protein extract for SDS-PAGE with StrataClean™ resin and
sonication ................................................................................................................50
3.8.5 SDS-PAGE and Western Blot Analysis ................................................................51
3.8.6 Immunoprecipitation (IP)........................................................................................51
3.8.7 ChIP-type Immunoprecipitation (ChIP-type IP) ....................................................51
3.8.8 Purification of GST-tagged, recombinant proteins ...............................................52
3.9 Chromatin Analysis.............................................................................. 53
3.9.1 Solutions..................................................................................................................53
3.9.2 In Situ Chromatin Immunoprecipitation (ChIP).....................................................54
3.9.3 Douncer ChIP .........................................................................................................55
3.9.4 Cesium chloride isopycnic centrifugation..............................................................57
3.9.5 Quantification of enriched DNA through ChIP with real-time-PCR.....................57
3.9.5.1 ΔΔCt Method..................................................................................................57
3.9.5.2 Quantification via standard curves...............................................................57
4 Results...........................................................................58
4.1 Generation of tools for ChIP analyses................................................ 58
4.1.1 Antibodies against MyoD .......................................................................................58
4.1.2 Antibodies against Lef1, Tcf1 and Tcf3 ................................................................61
4.1.2.1 Specificity of the antibodies ..........................................................................67
4.1.2.2 Lef/Tcf protein expression pattern ...............................................................71 Table of Contents IV
4.2 ChIP Analyses ...................................................................................... 72
4.2.1 SRF localizes to the myoD maintenance enhancer in activin-
induced animal cap explants .................................................................................72
4.2.1.1 Titration of activin containing cell culture supernatant for
mesoderm and muscle induction of animal caps .......................................73
4.2.1.2 Optimizing the chromatin shearing conditions ............................................75
4.2.1.3 Quantification of the precipitated DNA via TaqMan
technology-based real-time PCR.................................................................76
4.2.1.4 SRF is bound to the MyoD maintenance enhancer in activin-
treated animal cap explants .........................................................................80
4.2.2 αLef/Tcf ChIP at the siamois and myf5 loci was irreproducible ..........................81
4.2.3 The switch to the Douncer ChIP protocol helps to remove
excessive proteins..................................................................................................83
4.2.3.1 Titration of the chromatin lysate condition...................................................85
4.2.3.2 TaqMan amplicons and method of quantification .......................................88
4.2.3.3 Determination of the amount of antibody.....................................................90
4.2.4 Chromatin profiling of the myoD locus..................................................................91
4.2.5 CHD4 binds to the sip1 gene...............................................................................107
4.2.6 ChIP data quality assessment.............................................................................109
5 Discussion...................................................................114
5.1 Technical aspects of ChIP................................................................. 114
5.1.1 Comparison of ChIP methods .............................................................................115
5.1.1.1 Preparation of chromatin lysates................................................................115
5.1.1.2 Selection of Antibodies ...............................................................................116
5.1.1.3 ChIP controls ...............................................................................................117
5.1.1.4 Real-time PCR techniques .........................................................................118
5.1.1.5 Abundance of the investigated protein-DNA association .........................119
5.1.1.6 Quantification and representation of the ChIP data..................................119
5.1.1.7 Conclusions .................................................................................................121
5.2 Biological results of this project....................................................... 121
5.2.1 Regulation of the MyoD locus..............................................................................121
5.2.1.1 Histone ChIP................................................................................................122
5.2.1.2 αSRF ChIP ..................................................................................................125
5.2.1.3 αMyoD ChIP ................................................................................................126
5.2.2 Lef/Tcf transcription factors co-precipitate under ChIP-IP conditions ..............127
5.2.3 CHD4 binds to the sip1 locus at exon 1..............................................................128
5.2.4 Conclusions ..........................................................................................................129 Table of Contents V
5.3 Outlook ............................................................................................... 130
6 Abbreviations ..............................................................132
7 References...................................................................134
8 Appendix......................................................................151