Cover Page
The handle
http://hdl.handle.net/1887/74050
holds various files of this Leiden University
dissertation.
Author: Kaczmarczyk, A.
Title: Nucleosome stacking in chromatin fibers probed with single-molecule force- and
torque-spectroscopy
Nucleosome stacking in chromatin
fibers probed with single-molecule
Nucleosome stacking in chromatin
fibers probed with single-molecule
force- and torque-spectroscopy
PROEFSCHRIFT
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van Rector Magnificus prof. mr. C.J.J.M. Stolker,
volgens besluit van het College voor Promoties
te verdedigen op woensdag 19 juni 2019
klokke 11:15 uur
door
Artur Kaczmarczyk
geboren te Ruda Śląska, Polen
Promotores:
Prof. dr. ir. S. J. T. van Noort
Prof. dr. N. H. Dekker (Delft University of Technology)
Promotiecommissie: Prof. dr. J. Lipfert
(Ludwig-Maximilian-University, Munich, Germany)Dr. F. Mattiroli (Hubrecht Institute, Utrecht)
Prof. dr. E. R. Eliel
Prof. dr. H. Schiessel
Prof. dr. T. Schmidt
©2019 Artur Kaczmarczyk. All rights reserved.
Cover: Little silhouettes pulling and twisting chromatin inside a cellular matrix.
(image of silhouettes adapted from: Sergey Nivens/Shutterstock.com)
Casimir PhD Series, Delft-Leiden, 2019-22
ISBN 978-90-8593-398-4
C o n t e n t s
1 Introduction 1
1.1 From DNA through nucleosomes to chromatin . . . 2
1.2 Regulation of the structure of chromatin fibers . . . 5
1.3 Single-molecule magnetic tweezers for studying the dynamics of chromatin . . 7
1.4 Mechanical properties of a stretched and supercoiled DNA . . . 10
1.5 Statistical mechanics . . . 12
1.6 Outline of this thesis . . . 13
2 Methods for probing chromatin structure with magnetic tweezers 21 2.1 Introduction . . . 22
2.2 List of materials . . . 24
2.3 DNA cloning . . . 27
2.4 DNA digestion and labelling . . . 27
2.5 Chromatin reconstitution . . . 28
2.6 Electrophoretic band shift assay . . . 29
2.7 Assembly of the flow cell chamber . . . 30
2.8 Flow cell functionalization . . . 33
2.9 Dynamic force spectroscopy on chromatin fibers . . . 35
2.10 Data analysis . . . 39
2.11 Additional notes . . . 44
3 Single-molecule force spectroscopy on histone H4 tail-cross-linked chro-matin reveals fiber folding 53 3.1 Introduction . . . 54
3.2 Results . . . 56
3.2.1 Chromatin with short NRL folds into a stiffer structure than chromatin with long NRL . . . 56
3.2.2 The H4 tail mediates the nucleosome-nucleosome interactions in folded chromatin fibers . . . 58
3.2.3 H4-V21C/H2A-E64C mutations affect chromatin stacking . . . 60
3.3 Discussion and conclusions . . . 63
3.4 Materials and methods . . . 67
3.4.1 Chromatin reconstitution and cross-linking . . . 67
3.4.2 Sample preparation . . . 67
3.4.3 Magnetic tweezers . . . 68
3.4.4 Data analysis . . . 68
viii
CONTENTS
4 Chromatin fibers stabilize nucleosomes under torsional stress 77
4.1 Introduction . . . 78
4.2 Results . . . 81
4.2.1 Chromatin fibers fold into left-handed superhelices that absorb positive twist . . . 81
4.2.2 Nucleosome unstacking is influenced by torque . . . 84
4.2.3 Positive twist can stabilize and destabilize chromatin fibers . . . 86
4.2.4 The torsional modulus of chromatin fibers can be determined experi-mentally and computationally . . . 90
4.2.5 The response of chromatin fibers to torsion does not vary with NRL . 92 4.3 Discussion and conclusions . . . 94
4.4 Materials and methods . . . 99
4.4.1 DNA constructs and chromatin assembly . . . 99
4.4.2 Flow cell preparation . . . 99
4.4.3 Magnetic tweezers . . . 100
4.4.4 Magnetic torque tweezers . . . 100
4.5 Supplementary Information . . . 101
4.5.1 Model of elastic response of DNA to torsion . . . 101
4.5.2 Model of elastic response of chromatin to torsion . . . 103
4.5.3 Distribution of the linking number between the chromatin fiber and DNA handles . . . 104
4.5.4 Computational determination of twist-induced chromatin unstacking . 105 4.6 Supplementary Figures . . . 107
5 Linker histone H1 stabilizes nucleosome stacking in chromatin fibers 127 5.1 Introduction . . . 128
5.2 Results . . . 130
5.2.1 Chromatin higher-order structure is stabilized by the linker histone . . 130
5.2.2 Chromatin unstacking enhances H1 dissociation . . . 133
5.2.3 Quantification of discrete unstacking steps reveals asymmetric unwrap-ping of chromatosomes . . . 135
5.3 Discussion and conclusions . . . 138
5.4 Materials and methods . . . 141
5.4.1 DNA and chromatin preparation . . . 141
5.4.2 Chromatin with linker histone . . . 142
5.4.3 Flow cells . . . 142
5.4.4 Magnetic tweezers . . . 143
5.5 Supplementary Information . . . 143
5.5.1 Non-equilibrium statistical mechanics model . . . 143
5.5.2 Quantification of discrete unfolding steps . . . 147
5.6 Supplementary Figures . . . 151
6 Summary 161
