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Analysis of tissue-specific & allele-specific DNA methylation [Elektronische Ressource] / vorgelegt von Elmar Schilling

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215 pages
Publié par :
Ajouté le : 01 janvier 2009
Lecture(s) : 21
Signaler un abus

Analysis of tissue-specific & allele-
specific DNA methylation




Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen
Fakultät IV – Chemie und Pharmazie
der Universität Regensburg











vorgelegt von
Elmar Schilling
aus
Schwenningen
2009
The present work was carried out in the Department of Hematology and Oncology at the
University Hospital Regensburg from June 2005 to June 2009 and was supervised by PD.
Dr. Michael Rehli.


Die vorliegende Arbeit entstand in der Zeit von Juni 2005 bis Juni 2009 in der Abteilung
für Hämatologie und internistische Onkologie des Klinikums der Universität Regensburg
unter der Anleitung von PD. Dr. Michael Rehli.















Promotionsgesuch eingereicht am: 30. Juli 2009
Die Arbeit wurde angeleitet von PD. Dr. Michael Rehli.



Prüfungsausschuss:

Vorsitzender: Prof. Dr. Sigurd Elz
1. Gutachter: Prof. Dr. Roland Seifert
2. Gutachter: PD. Dr. Michael Rehli
3. Prüfer: Prof. Dr. Gernot Längst































Lob und Tadel bringen den Weisen
nicht aus dem Gleichgewicht.

(Budha)




TABLE OF CONTENTS

1 INTRODUCTION .............................................................................................. 1
1.1 THE CONCEPT OF EPIGENETICS ............................................................. 1
1.2 DNA METHYLATION .............................................. 2
1.2.1 DNA methyltransferases ................................ 3
1.2.2 Methyl-CpG-binding proteins.......................................................... 4
1.3 FUNCTIONS AND MOLECULAR CONSEQUENCES OF DNA METHYLATION IN HEALTHY CELLS ....... 5
1.3.1 Global methylation landscapes ...................................................... 6
1.3.2 Genomic immunity: De novo methylation of integrated foreign DNA ............................. 6
1.3.3 Development: Tissue specific DNA methylation ............................ 7
1.3.4 Imprinting ........................................................................................................................ 8
1.3.5 Resetting of imprints ....................................................................................................... 8
1.3.6 X-chromosome inactivation ............................ 9
1.3.7 Interindividual phenotypical differences and inheritance of DNA methylation ............. 11
1.4 MAPPING DNA METHYLATION ............................................................................................. 11
2 AIMS............................................... 17
3 MATERIAL ..................................................................... 18
3.1 EQUIPMENT ........................................................ 18
3.2 CONSUMABLES .................. 19
3.3 CHEMICALS ........................................................................................ 20
3.4 DNA OLIGONUCLEOTIDES ................................... 20
3.4.1 Human .......................................................... 20
3.4.1.1 Real-time primer for MCIp ................................................................. 20
3.4.1.2 Nested amplification of bisulfite-treated primer .................................. 22
3.4.2 Murine ........................................................... 23
3.4.2.1 MassARRAY Primer .......................................................................... 23
3.4.2.2 Real-time PCR primer for MCIp ......................................................................................... 26
3.4.2.3 Real-time PCR primer for validation of CNV ...................................... 26
3.4.2.4 Real-time PCR primer for RT-PCR .................... 27
3.4.2.5 Primer for amplification of genomic DNA for sequencing ................................................... 28
3.4.2.6 Additional internal sequencing primer ................................................ 29
3.5 ENZYMES, KITS AND REAGENTS ........................................................... 30
3.6 MOLECULAR WEIGHT STANDARDS ....................................................... 31
3.7 BACTERIAL STRAINS AND PLASMIDS .................... 31
3.8 DATABASES AND SOFTWARE ............................................................... 32


i
4 METHODS ...................................................................................................... 33
4.1 GENERAL MOLECULAR BIOLOGY ......................... 33
4.1.1 Bacterial culture ............................................ 33
4.1.1.1 Bacterial growth medium ................................................................................................... 33
4.1.1.2 Preparation of chemically competent E. coli ...... 34
4.1.1.3 Transformation of chemically competent E. coli. 35
4.1.1.4 Glycerol stocks .................................................................................................................. 35
4.1.1.5 Plasmid isolation from E. coli ............................. 35
4.2 MOLECULAR TECHNOLOGIES .............................................................................................. 36
4.2.1 Polymerase chain reaction (PCR) ................ 36
4.2.1.1 Primer design..................................................................................................................... 36
4.2.1.2 Standard PCR for cloning or sequencing of gDNA ............................ 37
4.2.1.3 Reverse transcription (RT-PCR) ........................ 38
4.2.1.4 Real-time quantitative PCR analysis .................................................................................. 38
4.2.2 Creation of 0% to 100% methylated DNA as a control ................ 41
4.2.3 Molecular cloning ......................................... 41
4.2.4 Restriction digest .......................................................................... 42
4.2.5 CIAP treatment ............. 42
4.2.6 Ligation reaction ........................................... 42
4.2.7 PEG-precipitation ......................................................................... 42
4.2.8 Agarose gel electrophoresis ......................................................................................... 43
4.2.9 Purification of DNA fragment by gel extraction ............................ 44
4.2.10 Sequencing of genomic DNA ....................... 44
4.3 METHYL-CPG-IMMUNOPRECIPITATION (MCIP) ..................................................................... 45
4.3.1 Preparation of MBD2-Fc Fusionprotein ........ 46
4.3.1.1 Protein production using MBD2-Fc expressing Drosophila S2 cells .. 46
4.3.1.2 MBD2-Fc protein purification ............................................................................................. 46
4.3.1.3 MBD2-Fc quality and quantity assessment ........................................ 47
4.3.2 Binding MBD2-Fc to beads .......................... 49
4.3.3 DNA fragmentation ....................................................................... 49
4.3.4 Enrichment of highly methylated DNA ......................................... 50
4.4 MICROARRAY HANDLING AND ANALYSIS .............. 51
4.4.1 Gene Expression analysis ............................................................................................ 51
4.4.1.1 Labelling reaction ............................................... 51
4.4.1.2 Microarray hybridization ..... 51
4.4.2 CGH microarrays .......................................................................... 53
4.4.2.1 Labelling reaction ............................................... 53
4.4.2.2 Microarray hybridization ..................................................................... 53
4.5 QUANTITATIVE DNA METHYLATION ANALYSIS WITH THE MASSARRAY COMPACT SYSTEM ... 55
4.5.1 General overview ......................................................................................................... 55
4.5.2 Primer Design ............... 57
4.5.3 Bisulfite treatment of genomic DNA ............. 57
ii
4.5.4 PCR-amplification ......................................................................................................... 58
4.5.5 Shrimp Alkaline Phosphatase (SAP) Treatment .......................................................... 58
4.5.6 In vitro transcription and RNaseA treatment 59
4.5.7 Desalting of Cleavage Reaction: resin treatment ......................... 60
4.5.8 Transfer on SpectroCHIP and acquisition .................................................................... 60
4.5.9 Data processing ........................................... 60
4.6 LABORATORY ANIMALS ....... 61
4.6.1 Wild-type inbred mice ................................................................... 61
4.6.2 F1 Hybrids .................................................................................................................... 61
4.7 CELL CULTURING METHODS 61
4.7.1 Cell culture.................... 61
4.7.2 Analysing cell vitality .................................................................................................... 62
4.7.3 Freezing and thawing cells ........................... 62
4.7.4 Mycoplasma assay ....... 62
4.7.5 Isolation of human monocytes...................................................................................... 62
4.7.6 Mouse bone marrow macrophage preparation ............................ 64
5 RESULTS ....................................................................... 65
5.1 GLOBAL, COMPARATIVE ANALYSIS OF TISSUE SPECIFIC PROMOTER CPG METHYLATION ........ 65
5.1.1 Adaptation of the MCIp-on-chip approach ................................................................... 65
5.1.1.1 Separation of differentially methylated DNA by MCIp ........................................................ 66
5.1.1.2 Detection of tissue specific hypomethylation by microarray readout .. 68
5.1.2 Identification of differentially methylated promoters ..................... 70
5.1.3 Correlation of promoters and nearby genetic elements (CGI and Alu repeats) ........... 71
5.1.4 Validation of MCIp microarray results by real-time PCR .............................................. 73
5.1.4.1 Y-chromosomal genes ....................................................................... 73
5.1.4.2 Autosomal Genes .............. 75
5.1.4.3 Microarray-validation using bisulfite-treatment of genomic DNA........................................ 77
5.1.4.4 Summary of validation ....................................................................... 79
5.1.5 Relationship between DNA methylation and gene expression .... 81
5.2 ALLELE-SPECIFIC DNA METHYLATION IN MOUSE STRAINS IS MAINLY DETERMINED BY CIS-
ACTING SEQUENCES ....................................................................................................................... 84
5.2.1 Inbred mice as model organisms & Preliminary work .................. 84
5.2.2 Design of custom tiling array ........................ 85
5.2.3 MCIp “mirror-image” procedure .................................................................................... 87
5.2.4 Virtual CGH to detect genetic differences .................................................................... 89
5.2.5 Detection of DMR by microarray .................. 91
5.2.6 Correlation between genetic variability and differential methylation status ................. 93
5.2.7 MALDI-TOF mass spectrometry: Validation of microarray data .................................. 95
5.2.7.1 Method characteristics ....................................................................... 95
5.2.7.2 Genomic DNA sequences in C57BL/6 and BALB/c ........................... 95
5.2.7.3 Validation of DMR by combination of bisulfite and MALDI-TOF MS .................................. 98
iii
5.2.8 Analysis of gender differences ................................................................................... 101
5.2.9 Analysis of the influence of the BMM culture system ................. 103
5.2.10 Comparison of somatic cells with germ line cells ....................... 104
5.2.11 Inheritance of DNA methylation patterns in F1-hybrid animals .................................. 105
5.2.11.1 Methylation pattern of DMRs in F1-hybrid mice ............................................................... 105
5.2.11.2 Impact of mating combination or offspring gender ........................... 108
5.2.11.3 Comparison of mean methylation levels between parents and offspring ......................... 109
5.2.11.4 Allele-specific bisulfite sequencing of DMRs ................................................................... 111
6 DISCUSSION ............................................................................................... 114
6.1 MAPPING DNA METHYLATION ........................................................... 114
6.2 CELL TYPE-SPECIFIC PROMOTER METHYLATION ................................. 116
6.3 ALLELE-SPECIFIC DNA METHYLATION IN MICE ... 121
6.4 OUTLOOK ......................................................................................................................... 128
7 SUMMARY ................................... 130
8 DEUTSCHE ZUSAMMENFASSUNG ........................................................... 132
9 REFERENCE LIST ....................................................... 134
10 ABBREVIATIONS ........................................................ 149
11 LIST OF PUBLICATIONS ............................................ 152
12 APPENDIX ................................................................... 153
12.1 APPENDIX I – TISSUE-SPECIFIC METHYLATED PROMOTERS ................. 154
12.2 APPENDIX II – DIFFERENTIALLY EXPRESSED LOCI .............................................................. 173
12.3 APPENDIX III – ANNOTATED LIST OF MOUSE STRAIN-SPECIFIC DMR.................................... 177
12.4 APPENDIX IV – SEQUENCE ALIGNMENTS ............................................ 187
13 EIDESSTATTLICHE ERKLÄRUNG ............................. 205




iv
LIST OF FIGURES

FIGURE 1.1 EPIGENETIC REGULATORY MECHANISMS: DNA METHYLATION AND CHROMATIN STRUCTURE. .. 2
FIGURE 1.2 CHARACTERISTIC DOMAINS OF THE MBD PROTEIN FAMILY.. .................................................. 5
FIGURE 1.3 IMPRINTING DURING DEVELOPMENTAL REPROGRAMMING.. .................... 9
FIGURE 1.4 CHEMICAL REACTION DURING BISULFITE TREATMENT .......................................................... 12
FIGURE 4.1 MASSARRAY WORKFLOW OVERVIEW. ............................................... 56
FIGURE 5.1 MCIP-ON-CHIP APPROACH TO DETECT PROMOTER REGIONS WITH TISSUE-SPECIFIC
DEMETHYLATION. ........................................................................................ 67
FIGURE 5.2 GLOBAL PROFILING OF TISSUE DNA METHYLATION USING HUMAN PROMOTER MICROARRAYS 70
FIGURE 5.3 ASSOCIATION OF HYPOMETHYLATED PROMOTERS WITH CPG ISLANDS AND ALU REPEATS ..... 72
FIGURE 5.4 DIFFERENT AREAS ON THE HUMAN Y CHROMOSOME. .......................................................... 73
FIGURE 5.5 TISSUE-SPECIFIC CPG METHYLATION PROFILES OF THE HUMAN Y CHROMOSOME. ................ 74
FIGURE 5.6 HEAT MAPS OF MCIP REAL-TIME PCR RESULTS. ................................ 76
FIGURE 5.7 BISULFITE SEQUENCING RESULTS OF SELECTED GENE PROMOTERS. ... 78
FIGURE 5.8 BISULFITE SEQUENCING OF ZSCAN5. ............................................................................... 79
FIGURE 5.9 COMPARISON OF METHYLATION AND EXPRESSION PROFILES 82
FIGURE 5.10 CORRELATION OF TISSUE-SPECIFIC EXPRESSION AND DEMETHYLATION ............................. 83
FIGURE 5.11 EXPRESSION PROFILING OF BMM FROM TWO INBRED MOUSE STRAINS. 86
FIGURE 5.12 SIMULTANEOUS DETECTION OF EPIGENETIC AND GENETIC DIFFERENCES USING MCIP ........ 88
FIGURE 5.13 DETECTION OF SEQUENCE VARIATION USING VIRTUAL CGH. ............................................. 90
FIGURE 5.14 SEQUENCE REGIONS WITH STRAIN-SPECIFIC CPG METHYLATION. ...... 91
FIGURE 5.15 CORRELATION OF DMR AND GENETIC VARIATION ............................................................. 94
FIGURE 5.16 DNA SEQUENCES OF 4 SELECTED DMRS IN C57BL/6 AND BALB/C. ................................ 96
FIGURE 5.17 VALIDATION OF STRAIN-SPECIFIC CPG METHYLATION BY MALDI-TOF MS OF BISULFITE
TREATED DNA ............................................................................................................................ 99
FIGURE 5.18 CORRELATION MATRIX OF MALDI-TOF MS DETECTED METHYLATION RATIOS IN BMM. ... 102
FIGURE 5.19 CORRELATION MATRIX OF MALDI-TOF MS DETECTED METHYLATED RATIOS IN BMM AND
SPLEEN. ................................................................................................................................... 103
FIGURE 5.20 REAL-TIME PCR RESULTS FOR A MATERNALLY IMPRINTED GENE ..... 105
FIGURE 5.21 INHERITANCE OF STRAIN-SPECIFIC METHYLATION PATTERNS IN F1 HYBRIDS ..................... 106
FIGURE 5.22 INHERITANCE OF A X-LINKED DMR IN F1-HYBRID MICE ................................................... 107
FIGURE 5.23 CORRELATION MATRIX OF MALDI-TOF MS DETECTED METHYLATION RATIOS IN PARENTAL
STRAINS AND F1-HYBRID MICE. .................................................................................................. 109
FIGURE 5.24 METHYLATION RATIOS OF WILDTYPE MICE VERSUS F1 HYBRIDS. ...... 110
FIGURE 5.25 STRAIN-SPECIFIC METHYLATION PATTERNS ARE MAINLY CONTROLLED IN CIS .................... 112



v
LIST OF TABLES

TABLE 1.1 STEPS AND ACTIVE COMPONENTS OF MAMMALIAN X-INACTIVATION ....................................... 10
TABLE 1.2 CURRENT METHODS FOR DNA METHYLATION ANALYSIS: SAMPLE PRETREATMENT ................. 15
TABLE 1.3 CURRENT METHODS FOR DNA METHYLATION ANALYSIS: HIGH-THROUGHPUT READOUT ......... 16
TABLE 4.1 AGAROSE CONCENTRATION FOR DIFFERENT SEPARATION RANGES ....................................... 43
TABLE 4.2 ELUTRIATION STEPS, SETTINGS AND EXPECTED CELLS ......................................................... 63
TABLE 5.1 PROFILES OF PROMOTER HYPOMETHYLATION AND MRNA EXPRESSION OF SELECTED GENES . 80
TABLE 5.2 ANNOTATED LIST OF SELECTED MOUSE STRAIN-SPECIFIC DMR ............................................ 92
TABLE 5.3 MCIP-DETECTED DMR VALIDATION SET AND GERM LINE METHYLATION ............................... 100
TABLE 12.1 MICROARRAY-DETECTED DIFFERENTIALLY METHYLATED GENE PROMOTER ........................ 154
TABLE 12.2 GENOMIC REGIONS SELECTED FOR C57BL/6-BALB/C DMR SCREENING .......................... 173
TABLE 12.3 ANNOTATED LIST OF ALL MOUSE STRAIN-SPECIFIC DMR .................................................. 177




vi
Acknowledgement

I would like to thank Prof. Dr. Reinhard Andreesen for accepting me as a PhD student in his department and
for his honest and generous support during all those years.

I would like to thank Prof. Dr. Roland Seifert for accepting to supervise and review my thesis.

I am very grateful to PD Dr. Michael Rehli for his enthusiasm, his continuous support and advice and for the
great atmosphere in our lab. He was always willing to listen to my problems. He kept patience and calmness
all the time and during his leadership I had the chance to learn many new things.

I would like to thank everybody inside our laboratory for the wonderful atmosphere, for your patience, technical
and moral support. In detail: Many thanks to: Dr Carol El-Chartouni, PhD Hang Thu Pham, Claudia Gebhard,
Maja Klug, Lucia Schwarzfischer-Pfeilschifter, Dagmar Glatz, Christian Schmidl, Katrin Dietl, Michael
Kastenberger, Julia Wegner, Prof. Dr. Marina Kreutz, Dr. Eva Gottfried, Monika Wehrstein, Alice Peuker, Gabi
Hartmannsgruber, Martina Köhler, Ireen Ritter and Sandra Vogl and all those I probably forgot to mention. In
addition, I would like to take the opportunity to thank our closely connected co-workinggroup – the Kreutz-Lab.
We not only shared room and working material, I also appreciated our combined seminars where I learned
much about tumor and monocyte metabolism. I also would like to thank the whole Forschungsbau H1 where I
could find all I needed for both: the last minute experiments and coffee breaks, climbing or grilling events.

Thanks to Carol for the help in the preparation of BMM's. Special thanks to Luci for her precious organization
skills and her technical and moral support. I will also like to thank Dagmar and Ireen for their technical support
and (not to forget) funny coffee breaks and spare time events.

I would also like to thank all former crewmembers, who already left the „ship‟ sailing to various different
harbors: Dr. Monika Lichtinger, Dr. Tobias Weil, Dr. Mike Aigner, Monika Eigner, Sabine Pape and Alex
Müller.

I am glad that I found some people having the same life of suffering each day: pangs of hunger. This group
consisted of Kaste, Chris, Tobi, Dagmar, mostly Michael and was sometimes complemented by Katrin and
Sandra, at least for the obligatory “after lunch” coffee (At this point it is indicated to thank Nespresso…)
I was also a great pleasure for me to live in our flat-sharing community consisting of my beloved brother Reini
and Martin Lachinger, who successfully substituted my good old friend Dr. Werner Müsch after three eventful
years. I‟ll also like to thank all other friends not personally mentioned yet who helped to make my stay in
Regensburg not boring at all.

Last but not least, I would like to thank my parents. They really always supported me in a very unselfish way.
Without their help I would never had the change to start and finish my study, to work in and explore the United
States for over half a year and to perform the present thesis. I really appreciate their never-ending help and
patience.

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