Single-molecule force spectroscopy probes chromatin mechanics by resolving force-induced conformational transitions across multiple length scales. Recent studies extend these measurements beyond canonical nucleosomes to chromatin assembled on native genomic DNA, incorporating histone variants, post-translational modifications, and specialized chromatin architectures. These approaches further scale to chromatosomes, folded fibers, and intact chromosomes, demonstrating that chromatin composition and organization modulate mechanical properties across multiple length scales. Single-molecule measurements further demonstrate that regulatory proteins, including transcription factors, architectural proteins, and chromatin-associated cofactors, actively sense and reshape this mechanical landscape. Together, these observations support a unified view of chromatin as a mechanically encoded medium that can be both written and read.

Eukaryotic transcription factors recognize short motifs, creating abundant binding sites within gene bodies and potential collisions with elongating RNA polymerases, yet how such encounters are resolved remains unclear. Here, we use optical tweezers to monitor RNA polymerase transcription through DNA-bound Egr-1, a zinc-finger transcription factor. Using DNA-fluctuation suppression as a readout of polymerase arrival, we show that Egr-1 delays elongation in an orientation- and rNTP-dependent manner, whereas force measurements indicate that RNAP does not bypass the TF by mechanical eviction. Instead, RNAP destabilizes the Egr-1-DNA complex over a short, structured interaction zone, increasing TF dissociation non-monotonically with distance. Monte Carlo simulations incorporating these kinetic changes recapitulate passage-time distributions. CpG methylation shortens Egr-1 residence time and largely eliminates the TF-dependent delay, suggesting a role for gene-body methylation in reducing kinetic barriers to elongation. These results reveal DNA-bound TFs as tunable barriers that locally shape transcription elongation.

Transcription factors regulate gene expression by binding specific DNA motifs, yet only a fraction of putative sites is occupied in vivo. Intrinsically disordered regions have emerged as key contributors to promoter selectivity, but the underlying mechanisms remain incompletely understood. Here, we use single-molecule optical tweezers to dissect how disordered regions influence DNA binding by Msn2, a yeast stress-response regulator. We show that these regions power a search mechanism, facilitating initial non-specific association with DNA and promoting one-dimensional scanning toward target motifs, supported by charge-mediated interactions. Remarkably, this mechanism displays sequence sensitivity, with promoter-derived sequences enhancing both initial binding and scanning rates, demonstrating that Msn2-DNA interactions alone are sufficient to confer promoter selectivity in the absence of chromatin or cofactors. Our findings provide direct mechanistic evidence for how intrinsically disordered regions tune transcription factor search dynamics for Msn2 and expand sequence recognition beyond canonical motifs, supporting promoter selectivity in complex genomic contexts.

DNA origami is a mainstay of DNA nanotechnology and several efforts have been devoted to understanding how various factors of the self-assembly reaction affect the final yield of the target origami structure. This study analyses how base sequence affects origami yield through the generation of off-target side reactions during self-assembly. Off-target bindings are an under-explored phenomenon and can potentially introduce unwanted assembly barriers and kinetic traps in the origami folding pathway. We developed a multi-objective computational approach that takes a given origami design and scores different scaffold sequences (and their complementary staples) for the prevalence of four different types of off-target binding events. Using our method on DNA origami, we can select bad regions of biological sequences (like lambda DNA phage) that, when used as origami scaffold sequences, have an excessive number of off-target side reactions for each shape. We show, using high-resolution atomic force microscopy (AFM), that these scaffold sequences largely fail to fold into the target triangle or rectangle structure in vitro, despite the scaffold sequence having a fully complementary staple set present. Conversely, using our method we can also select good regions of biological sequences. These sequences are deficient in off-target reactions and when used as origami scaffolds, fold more successfully into their target structures as characterised by AFM. These results have been validated in blind folding experiments at two different laboratories in which the experimenters did not know which scaffolds were good or bad folders. To further investigate assembly behaviour, optical tweezers experiments revealed distinct mechanical response profiles, correlating with scaffold-specific off-target interactions. While variants with higher GC content show a high mean unfolding force, variants with lower off-target binding demonstrated more uniform force-extension curves. Our analysis confirmed that high off-target binding leads to increased structural heterogeneity, as seen in the clustering behaviour of unfolding traces of OT experiments. Overall, our work demonstrates how the off-target reactions implicit in base sequences can derail the origami self-assembly process if sufficiently prevalent, and we provide a software tool to select scaffold sequences that minimise off-target reactions for any DNA origami design. ### Competing Interest Statement The authors have declared no competing interest.

Transcription factors (TFs) regulate gene expression by binding specific DNA motifs, yet only a fraction of putative sites is occupied in vivo . Intrinsically disordered regions (IDRs) have emerged as key contributors to promoter selectivity, but the underlying mechanisms remain incompletely understood. Here, we use single-molecule optical tweezers to dissect how IDRs influence DNA binding by Msn2, a yeast stress-response regulator. We show that IDRs facilitate initial non-specific association with DNA and promote one-dimensional diffusion toward target motifs, supported by charge-mediated interactions. Remarkably, the IDR-dependent search mechanism displays sequence sensitivity, with promoter-derived sequences enhancing both initial binding and sliding rates, demonstrating that Msn2–DNA interactions alone are sufficient to confer promoter selectivity in the absence of chromatin or cofactors. These findings provide direct mechanistic evidence for how IDRs tune transcription factor search dynamics and expand sequence recognition beyond canonical motifs, supporting a mechanism for promoter selectivity in complex genomic contexts. ### Competing Interest Statement The authors have declared no competing interest. Israel Science Foundation, 937/20

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping, essential for break repair. The energetics of RecBCD during translocation along the DNA track are quantitatively not defined. Specifically, it's essential to understand the mechanism by which RecBCD switches between its binding states to enable its translocation. Here, we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. In the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to 'sense' the nucleotide state. On the contrary, AMPpNp shows weak coupling as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of the RecBCD reaction mechanism suggests an 'energetic compensation' between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a plausible stepping mechanism' during the processive translocation of RecBCD.

Genome-wide studies have demonstrated regulatory roles for diverse non-coding elements, but their precise and interrelated functions have often remained enigmatic. Addressing the need for mechanistic insight, we studied their roles in expression of Lhb which encodes the pituitary gonadotropic hormone that controls reproduction. We identified a bi-directional enhancer in gonadotrope-specific open chromatin, whose functional eRNA (eRNA2) supports permissive chromatin at the Lhb locus. The central untranscribed region of the enhancer contains an iMotif (iM), and is bound by Hmgb2 which stabilizes the iM and directs transcription specifically towards the functional eRNA2. A distinct downstream lncRNA, associated with an inducible G-quadruplex (G4) and iM, also facilitates Lhb expression, following its splicing in situ. GnRH activates Lhb transcription and increased levels of all three RNAs, eRNA2 showing the highest response, while estradiol, which inhibits Lhb, repressed levels of eRNA2 and the lncRNA. The levels of these regulatory RNAs and Lhb mRNA correlate highly in female mice, though strikingly not in males, suggesting a female-specific function. Our findings, which shed new light on the workings of non-coding elements and non-canonical DNA structures, reveal novel mechanisms regulating transcription which have implications not only in the central control of reproduction but also for other inducible genes.

Proper regulation of gene expression is critical for the development and homeostasis of all organisms, and the regulation of RNA transcription is a central layer in it. In contrast to the vast knowledge obtained on the initiation phase regulation, how transcription is regulated at the elongation phase is only beginning to be understood. In particular, as RNAP advances on the DNA and transcribes an RNA molecule, it encounters a multitude of DNA-bound proteins and complexes. As a result, RNAP may pause, backtrack, or even completely arrest.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the damage of dopaminergic neurons as seen in Parkinson's disease. Oxidative stress has been as one of several pathogenic hypotheses for Parkinson's disease. Here we investigated whether arundic acid, an astrocyte-modulating agent, can protect against alterations of nitric oxide synthase (NOS) and superoxide dismutase (SOD) expression on MPTP neurotoxicity in mice, utilizing an immunohistochemistry. For this purpose, anti-tyrosine hydroxylase (TH) antibody, anti-dopamine transporter (DAT) antibody, anti-Cu/Zn-SOD antibody, anti-Mn-SOD antibody, anti-nNOS antibody, anti-eNOS antibody and anti-iNOS antibody were used. The present study showed that the arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and substantia nigra of mice. The protective effect may be, at least in part, caused by the reductions of the levels of reactive nitrogen (RNS) and oxygen species (ROS) against MPTP neurotoxicity. These results suggest that the pharmacological modulation of astrocyte may offer a novel therapeutic strategy for the treatment of Parkinson's disease. Furthermore, our results provide further evidence that a combination of nNOS inhibitors, iNOS inhibitors and free radical scavengers may be effective in the treatment of neurodegenerative diseases. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as approximately ±140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosome's entry and exit. These interactions are dynamic, exhibit rapid binding and dissociation, are sensitive to phosphorylation of a specific residue, and are crucial to determining the symmetry of the chromatosome's core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin, to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein’s binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

RecBCD is a DNA helicase-nuclease, powering the initiation of ds break repair. It is a highly processive fast helicase, with unwinding rates approaching 1,600 bp/s. By employing a combination of equilibrium and time-resolved unwinding and binding experiments(both ensemble and single-molecule) to unravel RecBCD's molecular mechanism, we had demonstrated the existence of auxiliary binding sites in RecBCD, specifically in the RecC subunit, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. According to our model, RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites. The number of nucleotide-binding sites determined by equilibrium dialysis has been estimated to be at least four. While RecB and RecD, each have one known structurally and chemically well-defined nucleotide-binding site, the additional two (or more) auxiliary nucleotide-binding sites have not been located. Our current study strongly supports that they are likely to be located in RecC. In this work, we have attempted to abolish the nucleotide-binding sites in RecC by using a multi-step approach. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking, mass spectrometry and molecular docking studies. Based on this, several mutations in RecC were carefully designed and successfully cloned and purified to produce RecBCmutD. RecBCmutD has an activated DNA ATPase activity, with a higher KM,ATP. Furthermore, equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same biphasic binding isotherm as the WT RecBCD. Based on this, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.

Reproductive function adjusts in response to environmental conditions in order to optimize success. In humans, this plasticity includes age of pubertal onset, hormone levels and age at menopause. These reproductive characteristics vary across populations with distinct lifestyles and following specific childhood events, and point to a role for the early-life environment in shaping adult reproductive trajectories. Epigenetic mechanisms respond to external signals, exert long-term effects on gene expression and have been shown in animal and cellular studies to regulate normal reproductive function, strongly implicating their role in these adaptations. Moreover, human cohort data have revealed differential DNA methylation signatures in proxy tissues that are associated with reproductive phenotypic variation, although the cause–effect relationships are difficult to discern, calling for additional complementary approaches to establish functionality. In this Review, we summarize how adult reproductive function can be shaped by childhood events. We discuss why the influence of the childhood environment on adult reproductive function is an important consideration in understanding how reproduction is regulated and necessitates consideration by clinicians treating women with diverse life histories. The resolution of the molecular mechanisms responsible for human reproductive plasticity could also lead to new approaches for intervention by targeting these epigenetic modifications.

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD’s individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25–40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

Significance As nucleosomes prevent binding of transcription factors (TFs) to DNA, their position needs to be actively modulated. Nucleosomes can also reposition spontaneously, but this process and its effect on TF binding have not been extensively studied. Here, we developed a method based on single-molecule optical tweezers to simultaneously measure nucleosome diffusion and TF binding to the same DNA molecule. We show that nucleosomes undergo confined diffusion on the DNA, and that the confinement is relieved upon incorporation of the histone variant H2A.Z leading to an increase in TF binding, which then further biases nucleosome diffusion. Our results shed light on a previously uncharacterized mechanism of transcriptional regulation.

Characterizing the interactions between colloidal particles is important, both from a fundamental perspective as well as due to its technological importance. However, current methods to measure the interaction forces between two colloids have significant limitations. Here we describe a method that exploits the fluctuation spectra of two optically trapped microspheres in order to extract, and decouple, the conservative forces acting between them and their hydrodynamic coupling. We demonstrate the proposed method with two silica microspheres, and find good agreement between our results and previous predictions for the hydrodynamic and electrostatic interactions between the spheres.

RecBCD is highly processive and very fast DNA helicase-nuclease, responsible for the initiation of double-stranded break repair in E. coli. By employing a combination of equilibrium and time-resolved unwinding and binding experiments, both ensemble and single-molecule, we previously demonstrated the existence of auxiliary binding sites in RecBCD, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. We showed that RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites located in RecB and RecD subunits. Equilibrium dialysis indicated the number of nucleotide-binding sites to be at least four, and their likely location in the RecC subunit. In this current work, we provide further support for the functional role of the auxiliary sites, by abolishing the nucleotide-binding sites in RecC. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking and mass spectrometry, as well as molecular docking studies. Based on these predictions, several mutations in RecC were carefully designed, producing unique RecBCmutD. We have successfully cloned and purified RecBCmutD and shown that RecBCmutD has DNA-activated ATPase activity. Furthermore, an equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same binding isotherm as the WT RecBCD. Based on these observations, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.

Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.