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

Abstract

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.

DNA sequence is a major determinant of tetrasome dynamics

“…Also in agreement with previous observations (35)(36)(37) is the lack of observable interactions between NAP1 chaperones and DNA molecules in the absence of histones (Supplementary Figure S3). To probe the effects of ambient conditions on tetrasome structure and dynamics, we performed the experiments in three different buffers employed in prior studies (35)(36)(37)(51)(52)(53) that mainly differed in the concentrations of mono-and divalent salts ( Table I) and obtained highly similar results ( Supplementary Table SIII). We also flushed in (H3.1-H4) 2 tetramers in the absence of NAP1 (n exp,subset =11).…”

“…Individual tetrasomes were assembled and monitored in real time using magnetic tweezers (see below) in three buffers with varying compositions of core components employed in previous studies (35)(36)(37)(51)(52)(53) (Table I). To allow for direct comparison to our previous study (35), the protein samples were prepared similarly, by incubating 51 nanomolar (nM) of an equimolar solution of H3.1-H4 histones -either without or with 192 nM NAP1 -on ice for 30 min.…”

“…Using freely-orbiting magnetic tweezers (FOMT) (49), we systematically investigated the structure and handedness dynamics of single (H3.1-H4) 2 tetrasomes assembled onto the well-characterized high-affinity Widom 601 nucleosome-binding sequence (50). In addition, we have examined the contribution of different buffers commonly employed in chromatin studies (51)(52)(53) to the dynamics of high-affinity tetrasomes. Our results revealed intriguing effects of DNA sequence on handedness flipping dynamics that can be counteracted by mono-and divalent salts.…”

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail [2][3][4][5]. In addition, the interactions with proteins, like the DNA compaction by histones in eukaryotic chromatin [6][7][8][9][10][11] and prokaryotic architectural proteins [12][13][14][15][16], supercoiling [17][18][19][20], and repair processes [21][22][23] were extensively studied with magnetic tweezers (MT) or optical tweezers (OT), Acoustic Force Spectroscopy (AFS) [24][25][26] or tethered particle motion (TPM) [13,27,28]. These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures, such as extracellular protein collagen [29][30][31], or even entire cells [32].…”

“…Previously, we implemented the LUT bead tracking algorithm in our MT and used it to study the various transitions of chromatin fiber unfolding [6,7,52], as schematically depicted in Fig 1a. In these applications, we noticed that the dynamic range of the LUT method was sometimes insufficient, yielding an accuracy that depended on the bead height. As the composition of chromatin fibers may vary due to the quality of reconstitution [11], disassembly [6] or reflecting naturally occurring variations [53], we found it imperative to improve both the computation speed, allowing for tracking a large number of independent tethers, and the accuracy over a dynamic range that spans up to 10 micrometer. In our hands, the empirical LUT algorithm frequently flawed due to non-perfect imaging conditions, which led to discarding a large fraction of the beads, limiting the throughput.…”