Promiscuous gene expression in the thymic medulla [Elektronische Ressource] : on regulation at the epigenetic and single cell level / vorgelegt von Anna Sinemus

Promiscuous gene expression in the thymic
medulla – on regulation at the epigenetic and
single cell level










INAUGURAL – DISSERTATION




zur Erlangung der Doktorwürde
der
Naturwissenschaftlich - Mathematischen Gesamtfakultät
der
Ruprecht – Karls – Universität
Heidelberg











vorgelegt von
Diplom-Biochemikerin Anna Sinemus
aus Göttingen




Gutachter: Prof. Dr. Günter Hämmerling
Prof. Dr. Bruno Kyewski


Die vorliegende Arbeit wurde angefertigt in der
Abteilung Entwicklungsimmunologie,
Leitung Prof. Dr. Bruno Kyewski,
im Deutschen Krebsforschungszentrum Heidelberg.



























Hiermit erkläre ich, dass ich die vorgelegte Dissertation selbst verfasst und mich dabei keiner
anderen, als der von mir ausdrücklich bezeichneten Quellen bedient habe.

Heidelberg,


Anna Sinemus _______________________________________________________________________CONTENTS
Contents
ZUSAMMENFASSUNG ............................................................................................. 5
SUMMARY................................................................................................................. 6
LIST OF ABBREVIATIONS ....................................................................................... 7
1. INTRODUCTION .................................................................................................... 9
1.1 Thymocyte maturation and central T cell tolerance.................................................................................... 9
1.1.1 T cell maturation and selection.................................................................................................................. 9
1.1.2 Dominant Tolerance................................................................................................................................ 12
1.2 Promiscuous gene expression ....................................................................................................................... 12
1.2.1 Regulation of pGE at the cellular level ................................................................................................... 13
1.2.2 Reof pGE at the molecular level ............................................................................................... 15
1.3 Mechanisms of epigenetic regulation........................................................................................................... 18
1.3.1 DNA methylation .................................................................................................................................... 18
1.3.2 The histone code...................................................................................................................................... 19
1.3.3 The role of chromatin structure and nuclear organization....................................................................... 20
1.4 Objective of this study .................................................................................................................................. 22
2. MATERIALS AND METHODS ............................................................................. 24
2.1 Materials ........................................................................................................................................................ 24
2.1.1 Chemicals................................................................................................................................................ 24
2.1.2 Buffers and commercial solutions........................................................................................................... 25
2.1.3 Enzymes, Proteins ................................................................................................................................... 26
2.1.3 Antibodies, dyes ...................................................................................................................................... 27
2.1.4 Primers and (Oligo-) Nucleotides................... 27
2.1.5 Commercial Kits ..................................................................................................................................... 30
2.1.6 Mice, cell lines, bacteria............................................................................................... 30
2.1.7 Consumables ........................................................................................................................................... 31
2.1.8 Equipment............................... 31
2.1.9 Software .................................................................................................................................................. 32
2.2 Methods.......................................................................................................................................................... 33
2.2.1 Distance measurements using fluorescence – in situ – hybridization (FISH) ......................................... 33
2.2.2. Agarose gel electrophoresis.................................................................................................................... 39
2.2.3 RNA isolation.......................................................................................................................................... 40
2.2.4 RT-PCR................................................................................................................................................... 40
2.2.5 Isolation of genomic DNA from murine tails.......................................................................................... 40
2.2.6 “Conventional” and Quantitatve PCR (qPCR)........................................................................................ 41
2.2.7 Single-cell PCR (SC PCR) ...................................................................................................................... 42
2.2.8 Chromatin-Immunoprecipitation............................................................................................................. 45 _______________________________________________________________________CONTENTS
2.2.9 Counting of live cells .............................................................................................................................. 47
2.2.10 Antibody labeling....................................................................................................... 47
2.2.11 Isolation of specific cell populations from mice.................................................................................... 47
2.2.12 Immunohistochemistry.......................................................................................................................... 50
3. RESULTS............................................................................................................. 52
3.1 Chromatin structure is modified at the level of individual genes, not the whole locus ........................... 52
3.1.1 The casein gene locus as a model locus for promiscuous gene expression ............................................. 52
3.1.2 Optimization of the ChIP protocol for low numbers of ex vivo sorted cells............................................ 53
3.1.3 Active chromatin marks are present only at the Casein beta promoter ................................................... 54
3.1.4 Detection threshold of ChIP is above the frequency of most promiscuously expressed genes in the casein
locus ................................................................................................................................................................. 57
3.1.5 Enrichment of mTEC expressing a single antigen: Chromatin marks in Gad67/eGFP mice .................. 58
3.2 Expression patterns of a single promiscuously expressed antigen – lessons from the Gad67 locus using
Gad67-eGFP knock-in mice ............................................................................................................................... 61
+ high3.2.1 Co-enrichment for Gad67 expression in eGFP mTEC on the population level................................. 61
3.2.2 SC PCR analysis of the Gad67 locus shows decoupling between eGFP protein and mRNA expression 62
3.2.3 Strong preference for biallelic expression at the Gad67 locus ................................................................ 63
+3.2.4 Gad67/eGFP co-expressors are enriched in Aire cells........................................................................... 65
3.2.5 Gad67 and eGPF are co-expressed at the protein level in the brain ........................................................ 65
3.3 Nuclear positioning and DNA compaction of the casein locus during mTEC maturation ..................... 67
3.3.1 Two probes are localized upstream and downstream of Csna/Csnb to measure chromatin decompaction
.......................................................................................................................................................................... 68
3.3.2 Both decompaction of the locus and nuclear localization can be measured with the same probes ......... 68
3.3.3