Chromatin architecture : advances from high-resolution single molecule DNA imaging : doctoral thesis accepted by Institute of Molecular Biology, Mainz and Heidelberg University, Germany

This book sheds new light on the current state of knowledge concerning chromatin organization. Particular emphasis is given to the new imaging potential offered by super-resolution microscopy, which allows DNA imaging with a very high labeling density. From the early work on chromosomes by Walther Flemming in the nineteenth century to recent advances in genomics, the history of chromatin research now spans more than a century. The various milestones, such as the discovery of the double helix structure, the sequencing of the human genome, and the recent description of the genome in 3D space, show that understanding chromatin and chromosome function requires a clear understanding of its structure. Presenting cutting-edge data from super-resolution single molecule microscopy, the book demonstrates that chromatin manifests several levels of folding, from nucleosomes to chromosomes. Chromatin domains emerge as a new fundamental building block of chromatin architecture, with functions possibly related to gene regulation. A detailed description of chromatin folding in the pachytene stage of meiosis serves as a model for exploring this functionality, showing the apparent interplay between structure, function, and epigenetic regulation. Lastly, the book discusses possible new avenues of innovation to describe chromatinℓ́ℓs organization and functions. Gathering essential insights on chromatin architecture, the book offers students an introduction to microscopy and its application to chromatin organization, while also providing advanced readers with new ideas for future research Supervisor’s Foreword 7 Preface 10 Parts of this thesis have been published in the following journal articles 13 In Peer-Reviewed Journals 13 In Conferences 17 Acknowledgements 21 Contents 23 Abbreviations 27 List of Figures 30 1 A Condensed History of Chromatin Research 51 1.1 The Early Research on the Nucleus and Chromatin 51 1.2 Chromatin Bares Information: The Chromosomes ƒ 54 1.3 Chromatin as a Decision Center of the Cellular Factory: The Golden ƒ 57 1.4 Chromatin as a Highly Structured System: Genomic Data, Localisation ƒ 61 1.5 The Substratum of Chromatin Memory: Epigenetic Regulation 65 1.6 Fine-Scale Chromatin Architecture: A New Modelling Area 68 1.7 Conclusion 69 References 70 2 Investigating Chromatin Organisation Using Single Molecule Localisation Microscopy 75 2.1 Introduction 75 2.2 Single-Molecule Localization Microscopy: State-of-the-Art 77 2.2.1 Principle of SMLM 77 2.2.2 The Different SMLM Methods: A Historical Perspective 78 2.3 Application of SMLM to Image Chromatin 79 2.3.1 The Tao of SMLM 80 2.3.2 Importance of a Good Localization Precision in Order to Improve Resolution 80 2.3.3 Importance of High Signal Density to Improve Signal-to-Noise Ratio 81 2.3.4 Limitations of Previous Approaches to Study Chromatin Organisation 83 2.4 A Method to Reach High Labelling Density of Chromatin with SMLM 84 2.4.1 Theory of DNA Dye Fluorescence 86 2.4.2 Adapting Study of DNA Dyes Fluorescence to SMLM 86 2.4.3 Optimization of the Photoconversion Process 87 2.4.4 Optimization of the Buffer Conditions 88 2.4.5 Multicolor Imaging with DNA 88 2.4.6 A Summary of Various Approaches Used to Study DNA with SMLM 88 2.5 SMLM Microscope Design and Imaging Pipeline 91 2.5.1 Sample Preparation for SMLM 92 2.5.2 Imaging Medium 92 2.6 Data Acquisition for SMLM 93 2.7 Data Reconstruction for SMLM 94 2.7.1 Spot Finding for SMLM 95 2.7.2 Drift Correction Algorithms for SMLM 97 2.7.3 Data Visualisation for SMLM 100 2.7.4 Data Analysis for SMLM 101 2.8 Some Further Considerations for Localisation Microscopy 103 2.8.1 Artefacts in Localisation Microscopy 103 2.8.2 Difference Between Localisation Precision and Accuracy 105 2.9 Summary 106 References 107 3 Structure, Function and Dynamics of Chromatin 112 3.1 Introduction 112 3.2 The Hierarchical Organisation of Chromatin 114 3.2.1 Chromosome Territories (Scale: 1000--2000 nm) 115 3.2.2 Sub-chromosomal Domains (Scale: 500--1000 nm) 118 3.2.3 Chromatin Domains (Scale: 100--400 nm) 119 3.2.4 Chromatin Fibres (Scale: 30--100 nm) 123 3.2.5 A Cluster-on-a-String Model to Describe the Fibre/Domain Transition 126 3.2.6 Nucleosome Domains (Scale: 10--30 nm) 126 3.2.7 Inference of Further Intermediate Chromatin Structures Using Local Chromatin Density Maps 131 3.2.8 Hierarchical Organisation of Chromatin Structure 132 3.3 The Dynamics of Chromatin 133 3.3.1 Contrasting Arrangement of eu- and Hetero-Chromatin Inside the Cell Nucleus 133 3.3.2 Classifier Identifies Intermediate States Between eu- and Heterochromatin Regions in Differentiated Cells 134 3.3.3 Chromatin Dynamics During Differentiation of Mesenchymal Stem Cells 135 3.3.4 Dynamics of Chromatin upon Stress 136 3.3.5 Reversible Compaction of Chromatin Under Stress 138 3.3.6 Conclusion 141 3.4 The Function of Chromatin 143 3.4.1 Periphery of Chromatin Domains is Associated with High DNA Synthesis 143 3.4.2 Stress-Dependent Transcription at the Periphery of Chromatin Domains 145 3.4.3 Histone Modifications Allow to Further Dissect Chromatin into Active and Inactive Domains 145 3.4.4 SMLM Identifies Potential Sites of Transcription Machineries in the Mammalian Nucleus 148 3.5 Summary and Discussion 148 References 149 4 Periodic and Symmetric Organisation of Meiotic Chromosomes 153 4.1 Introduction 154 4.2 Organisation of the Synaptonemal Complex (SC) 157 4.2.1 Superresolution Imaging of the SC Substructures 157 4.2.2 Quantification of SC Substructures 157 4.2.3 A Model for Organisation of SC 160 4.3 Periodic Organisation of Pachytene Chromosomes 161 4.3.1 Superresolution Imaging of Pachytene Chromosomes Reveals Periodic Clusters of Chromatin 162 4.3.2 Quantification of Periodic Chromatin Clusters 162 4.4 Functional Organisation of Pachytene Chromosomes 164 4.4.1 Rational 164 4.4.2 Clustering Method Sorts Chromatin into Functional Epigenetic Compartments 166 4.4.3 Centromeric Histone Mark (H3K9me3) Labels One End of the SC 167 4.4.4 Repressive Histone Mark (H3K27me3) Shows Characteristic Periodic Clusters Along the SC 169 4.4.5 Histone Mark (H3K4me3) Associated with Active Transcription Emanates Radially from the Axis of the SC 171 4.5 Structure and Dynamics of Meiotic Chromosomes 171 4.5.1 Lampbrush-Like Structures in Mammalian Meiotic Chromosomes 171 4.5.2 A Model for SC Spiralisation During the Zygotene/Pachytene Transition 173 4.6 A Model of Spatial Distribution of Chromatin Around the SC 176 4.6.1 A `Cluster-on-a-String' Model for Spatial Distribution of Pachytene Chromosomes 177 4.7 Summary and Conclusion 177 References 179 5 Conclusions 182 5.1 Originality of the Work Presented Here 182 5.2 A General Methodology to Study Chromatin Architecture 183 5.3 Limitations of the Method and Possible Improvements 184 5.4 New Avenues for the Study of Chromatin Patterns During Meiosis 185 5.5 Enlarging the Spectrum of Questions: Chromatin Organisation as a Fundamental Principle of Nucleus Formation 185 Appendices Appendix A 186 Appendix Appendix B 192 This book sheds new light on the current state of knowledge concerning chromatin organization. Particular emphasis is given to the new imaging potential offered by super-resolution microscopy, which allows DNA imaging with a very high labeling density. From the early work on chromosomes by Walther Flemming in the nineteenth century to recent advances in genomics, the history of chromatin research now spans more than a century. The various milestones, such as the discovery of the double helix structure, the sequencing of the human genome, and the recent description of the genome in 3D space, show that understanding chromatin and chromosome function requires a clear understanding of its structure. Presenting cutting-edge data from super-resolution single molecule microscopy, the book demonstrates that chromatin manifests several levels of folding, from nucleosomes to chromosomes. Chromatin domains emerge as a new fundamental building block of chromatin architecture, with functions possibly related to gene regulation. A detailed description of chromatin folding in the pachytene stage of meiosis serves as a model for exploring this functionality, showing the apparent interplay between structure, function, and epigenetic regulation. Lastly, the book discusses possible new avenues of innovation to describe chromatinĺls organization and functions. Gathering essential insights on chromatin architecture, the book offers students an introduction to microscopy and its application to chromatin organization, while also providing advanced readers with new ideas for future research "This book sheds new light on the current state of knowledge concerning chromatin organization. Particular emphasis is given to the new imaging potential offered by super-resolution microscopy, which allows DNA imaging with a very high labeling density. From the early work on chromosomes by Walther Flemming in the nineteenth century to recent advances in genomics, the history of chromatin research now spans more than a century. The various milestones, such as the discovery of the double helix structure, the sequencing of the human genome, and the recent description of the genome in 3D space, show that understanding chromatin and chromosome function requires a clear understanding of its structure. Presenting cutting-edge data from super-resolution single molecule microscopy, the book demonstrates that chromatin manifests several levels of folding, from nucleosomes to chromosomes. Chromatin domains emerge as a new fundamental building block of chromatin architecture, with functions possibly related to gene regulation. A detailed description of chromatin folding in the pachytene stage of meiosis serves as a model for exploring this functionality, showing the apparent interplay between structure, function, and epigenetic regulation. Lastly, the book discusses possible new avenues of innovation to describe chromatin's organization and functions"--Page 4 of cover Front Matter....Pages i-liii A Condensed History of Chromatin Research....Pages 1-24 Investigating Chromatin Organisation Using Single Molecule Localisation Microscopy....Pages 25-61 Structure, Function and Dynamics of Chromatin....Pages 63-103 Periodic and Symmetric Organisation of Meiotic Chromosomes....Pages 105-133 Conclusions....Pages 135-138 Back Matter....Pages 139-152