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 Regulation of 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 3.2.1 Co-enrichment for Gad67 expression in eGFP+ mTEChigh 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 Chromatic shifts between Oregon Green-Alexa Fluor 647 are small when directly measured in parallel with probes....................................................................................................................................................... 71 3.3.4 Chromatin in the Casein locus is more compact