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Analysis of the Barr Body with Super-Resolution Microscopy Implications for a Structural Role of Xist RNA in Mammalian X Chromosome Inactivation

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Analysis of the Barr Body with Super-Resolution Microscopy Implications for a Structural Role of Xist RNA in Mammalian X Chromosome Inactivation Department Biologie II Humanbiologie und Bioimaging Ludwig-Maximilians-Universität München Analysis of the Barr body with super-resolution microscopy Implications for a structural role of Xist RNA in mammalian X chromosome inactivation Daniel Smeets Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München Eingereicht am 16. Juli 2013 Analysis of the Barr body with super-resolution microscopy Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von: Dipl. Biol. Daniel Smeets aus Koblenz Gutachter: Prof. Dr. Heinrich Leonhardt Prof. Dr. Thomas Cremer Eingereicht am: 16. Juli 2013 Tag der mündlichen Prüfung: 22. November 2013 II Wo kämen wir hin, wenn alle sagten, wo kämen wir hin, und niemand ginge, um einmal zu schauen, wohin man käme, wenn man ginge? Kurt Marti Schweizer Schriftsteller III ANALYSIS OF THE BARR BODY WITH SUPER-RESOLUTION MICROSCOPY Table of contents Table of contents .................................................................................................................................... IV List of figures ......................................................................................................................................... VII List of tables ........................................................................................................................................... IX 1 Summary .................................................................................................................................. 1 2 Introduction ............................................................................................................................. 3 2.1 Chromatin, nuclear architecture and gene regulation .............................................................3 2.1.1 Chromatin and its modifications ....................................................................................3 2.1.2 Nuclear architecture .......................................................................................................5 2.2 X chromosome inactivation (XCI) ..............................................................................................8 2.2.1 Brief history of the Lyon hypothesis ...............................................................................8 2.2.2 Evolution of XCI ...............................................................................................................9 2.2.3 XCI during development .............................................................................................. 11 2.2.4 Xist RNA and initiation of chromosome-wide silencing .............................................. 12 2.2.5 Xist RNA spreading, gene silencing and Barr body formation ..................................... 15 2.3 Super-resolution microscopy ................................................................................................. 20 2.3.1 Single molecule localization microscopy (PALM/STORM) ........................................... 21 2.3.2 Stimulated emission depletion (STED) ........................................................................ 22 2.3.3 Structured illumination microscopy (SIM) ................................................................... 23 2.4 Aim of this work ..................................................................................................................... 24 3 Materials and Methods .......................................................................................................... 26 3.1 Cell culture ............................................................................................................................. 26 3.1.1 Thawing cells................................................................................................................ 27 3.1.2 Passaging somatic cells ................................................................................................ 28 3.1.3 Cultivating embryonic stem cells ................................................................................. 29 3.1.4 Freezing cells ................................................................................................................ 32 3.1.5 Seeding cells on coverslips .......................................................................................... 33 3.2 Labeling cells for 3D-SIM super-resolution imaging .............................................................. 35 IV TABLE OF CONTENTS 3.2.1 Immunofluorescence ................................................................................................... 35 3.2.2 RNA-FISH ...................................................................................................................... 40 3.2.3 DNA-FISH ..................................................................................................................... 48 3.2.4 Relocating cells before and after 3D-FISH ................................................................... 57 3.2.5 Replication labeling with EdU and click-chemistry reaction ....................................... 58 3.3 3D-SIM imaging ...................................................................................................................... 61 3.3.1 Basic principles ............................................................................................................ 61 3.3.2 3D-SIM imaging and system calibration ...................................................................... 64 3.3.3 3D-SIM post-processing and data analysis .................................................................. 67 4 Results .................................................................................................................................... 76 4.1 Assessing the suitability of 3D-FISH for 3D-SIM ..................................................................... 76 4.2 X chromosome inactivation analyzed with super-resolution 3D-SIM – Implications for a novel structural role for Xist RNA in XCI ................................................... 80 4.2.1 The chromatin architecture of the Barr body.............................................................. 80 4.2.2 Xist RNA is preferentially located within decondensed chromatin sites in somatic cells................................................................................................................. 90 4.2.3 Xist RNA strongly colocalizes with the ANC-enriched nuclear matrix protein SAF-A ............................................................................................................... 93 4.2.4 Xist RNA localization in decondensed chromatin is an intrinsic property that is already present during early spreading .......................................................... 100 4.3 Xist RNA is unlikely to interact directly with PRC2 ............................................................... 105 5 Discussion............................................................................................................................. 109 5.1 3D-FISH studies can benefit from super-resolution microscopy despite inherent detrimental effects .............................................................................................................. 109 5.2 X chromosome inactivation at 3D-SIM resolution ............................................................... 111 5.2.1 The Barr body is a special case of conventional nuclear architecture ...................... 111 5.2.2 Implications of the focal localization of Xist RNA within the ANC ............................. 113 5.2.3 Implications of the spatial correlation between Xist RNA and ANC- enriched SAF-A, but not with repressed chromatin .................................................. 114 V ANALYSIS OF THE BARR BODY WITH SUPER-RESOLUTION MICROSCOPY 5.2.4 Localization of Xist RNA within the ANC is an intrinsic property already at early differentiating XX ESCs ...................................................................................... 115 5.2.5 3D-SIM reveals incomplete silencing within an autosomal Barr body ...................... 116 5.2.6 Xist RNA does not interact directly with PRC2 .......................................................... 117 5.2.7 Summary of novel insights about nuclear architecture gained by 3D-SIM and outlook on future directions ............................................................................... 117 6 Appendix .............................................................................................................................. 122 6.1 Abbreviations ....................................................................................................................... 122 6.2 Materials and equipment .................................................................................................... 124 6.2.1 Cell lines ..................................................................................................................... 124 6.2.2 Chemicals and reagents ............................................................................................. 125 6.2.3 Buffers, solutions and media ..................................................................................... 127 6.2.4 Enzymes, nucleotides and kits ................................................................................... 129 6.2.5 Antibodies .................................................................................................................. 130 6.2.6 Equipment and hardware .........................................................................................
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