Centromere nucleosome sequence and centromere architecture

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Table of contents

1. ACKNOWLEDGEMENTS
2. RÉSUMÉ
3. ABSTRACT
4. INTRODUCTION
I. The Nucleus
I.1. Histones, Nucleosomes and Chromatin
I.3 Chromatin and Epigenetics
I.4 Chromosome Segregation
I.5 Chromosome Segregation Error
II The Centromere
II.1 The “Meshwork” and its “Blueprint” – Organization and Function of the Centromere Protein Network
a) CCAN organization and assembly
b) Inner kinetochore (CCAN) to outer kinetochore connections
II.2 A region like no other? – Centromeric Chromatin/Epigenetic Environment
a) The Histone H3 variant CENP-A
b) The CENP-A Nucleosome
c) Post Translational Modifications of CENP-A nucleosomes at the centromere
d) Centrochromatin beyond CENP-A
e) Pericentromeric chromatin
f) Centromere nucleosome sequence and centromere architecture
II.3 The Never-ending Story of Centromere Identity – CENP-A’s Epigenetic Self-Assembly Loop26
a) HJURP – CENP-A’s chaperone
b) Mis18 complex – the licensing factor
c) The CCAN – the reader of the epigenetic mark
d) CENP-A – the epigenetic mark
II.4 The Curious Case of Centromeric DNA (CenDNA)
a) Centromere Evolution
b) Centromere DNA sequence organization
c) Contribution of DNA sequence to the centromere architecture
II.5 CENP-B
a) CENP-B’s functions
b) Regulation of CENP-B binding at the centromere
II.6 De Novo Centromere Formation
a) Human Neocentromeres and inactivated centromeres
b) Artificial Generation of Neocentromeres
III.1 Summary and aims of this thesis
III.2 Résumé et objectifs de cette thèse
Previously deposited CENP-A is not essential for new CENP-A deposition at endogenous centromeres
De novo CENP-A localization remains unaltered in absence of old centromeric CENP-A
De novo CENP-A deposition is impaired in CENP-B deficient cells
Neocentromere formation occurs on the Y chromosome under selective pressure.
CENP-B bound to an ectopic location promotes CENP-C recruitment independently of CENP-A
CENP-B marks native centromere position to promote de novo CENP-A/C reloading
CENP-B initiates the CENP-A self-assembly epigenetic loop by recruiting CENP-C at native human centromeres
CENP-A-negative resting CD4+ T cells re-assemble CENP-A de novo upon cell cycle entry
6. RESULTATS (Résumé en français)
7. DISCUSSION
8. DISCUSSION (Résumé en français)
9. METHODS
Cell culture
Gene Targeting
Generation of Stable Cell Lines
siRNA, transient transfections, EdU staining and Colony Formation Assay
Immunoblotting
Immunofluorescence, Chromosome Spreads, IF-FISH and Live-Cell Microscopy
Single molecule microscopy
CUT&RUN-sequencing and -qPCR
Bioinformatic analysis
IF-FISH chromatin fiber
Intensity quantifications in microscopy experiments
Cloning, Expression, and protein Purification
GST pull down assay
CD4+ T cell staining and sorting
Data availability
10. CONTRIBUTIONS
11. REFERENCES
12. ABBREVIATION REGISTER
13. CURRICULUM VITAE
14. PUBLICATIONS