Unravelling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers, in particular, have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA-organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to a magnetic bead inside a flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleoprotein filaments can be extracted from the data.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm.

Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead's position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 µm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

Rigid Basepair Monte Carlo Simulations of One-Start and Two-Start Chromatin Fiber Unfolding by Force.

The organization of chromatin in 30 nm fibers remains a topic of debate. Here, we quantify the mechanical properties of the linker DNA and evaluate the impact of these properties on chromatin fiber folding. We extended a rigid basepair DNA model to include (un)wrapping of nucleosomal DNA and (un)stacking of nucleosomes in one-start and two-start chromatin fibers. Monte Carlo simulations that mimic single-molecule force spectroscopy experiments of folded nucleosomal arrays reveal different stages of unfolding as a function of force and are largely consistent with a two-start folding for 167 and one-start folding for 197 nucleosome repeat length fibers. The major insight is that nucleosome unstacking and subsequent unwrapping is not necessary to obtain quantitative agreement with experimental force extension curves up to the overstretching plateau of folded chromatin fibers at 3-5 pN. Nucleosome stacking appears better accommodated in one-start than in two-start conformations, and we suggest that this difference can compensate the increased energy for bending the linker DNA. Overall, these simulations capture the dynamic structure of chromatin fibers while maintaining realistic physical properties of the linker DNA.

Unraveling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers in particular have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to magnetics bead inside flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleo-protein filaments can be extracted from the data.

Probing Chromatin Structure with Magnetic Tweezers.

Magnetic tweezers form a unique tool to study the topology and mechanical properties of chromatin fibers. Chromatin is a complex of DNA and proteins that folds the DNA in such a way that meter-long stretches of DNA fit into the micron-sized cell nucleus. Moreover, it regulates accessibility of the genome to the cellular replication, transcription, and repair machinery. However, the structure and mechanisms that govern chromatin folding remain poorly understood, despite recent spectacular improvements in high-resolution imaging techniques. Single-molecule force spectroscopy techniques can directly measure both the extension of individual chromatin fragments with nanometer accuracy and the forces involved in the (un)folding of single chromatin fibers. Here, we report detailed methods that allow one to successfully prepare in vitro reconstituted chromatin fibers for use in magnetic tweezers-based force spectroscopy. The higher-order structure of different chromatin fibers can be inferred from fitting a statistical mechanics model to the force-extension data. These methods for quantifying chromatin folding can be extended to study many other processes involving chromatin, such as the epigenetic regulation of transcription.

Single-molecule force spectroscopy on histone H4 tail-cross-linked chromatin reveals fiber folding.

The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.