Biology subjects

Wereszczynski, J.

Publications and source records attributed to Wereszczynski, J..

15 recordsLinked to original sources

bioRxiv2026

Determinants of centromeric nucleosome conformation

Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A, which forms a specialized nucleosome required for kinetochore assembly. Compared to canonical H3 nucleosomes, CENP-A nucleosomes exhibit an open DNA conformation that leaves an additional 13 base pairs of DNA accessible at the entry and exit sites. While the CENP-A N-helix has previously been implicated in promoting this enhanced DNA breathing, the contributions of the intrinsically disordered N-terminal tail and adjacent latch regions of CENP-A to nucleosome conformation remain unknown. The intrinsically disordered N-terminal regions of histone H3 are known to facilitate interactions with DNA to stabilize overall nucleosome conformation. Here, we utilized a combination of MNase digestion assays and coarse-grained molecular dynamics simulations of nucleosomes containing targeted swaps of the H3 and CENP-A tail, latch, and N-helix regions to systematically test the contribution of each N-terminal histone region to nucleosomal DNA conformation. Substitution of individual H3 N-terminal regions with those of CENP-A opened the DNA at the entry/exit sites and increased DNA accessibility. While any single CENP-A N-terminal region was sufficient to open the canonical nucleosomal DNA conformation, replacement of any single CENP-A N-terminal region with its H3 counterpart was insufficient to restore the closed DNA conformation characteristic of canonical H3 nucleosomes. Instead, progressive incorporation of multiple H3-derived regions produced increasingly closed DNA conformations, demonstrating that the H3 tail, latch, and N-helices act cooperatively to stabilize wrapped nucleosomal DNA. Taken together, these findings demonstrate that the more restricted DNA breathing of canonical nucleosomes arises from coordinated contributions across multiple N-terminal regions and suggest that the multi-region redundancy in the conformational flexibility of the centromeric nucleosome could emphasize the importance of retaining flexibility in the centromeric nucleosome, even upon post-translational modification and binding to structural proteins.

biophysics↗

bioRxiv2025

Molecular Mechanisms of Gain-of-Function Mutations in {lambda} Cro Revealed by Molecular Dynamics Simulations

Transcription factors regulate gene expression by coordinating complex networks in organisms ranging from bacteriophages to humans. Bacteriophage {lambda} Cro is a 66-residue repressor that binds DNA as a dimer to block transcription. Because of its small size, simple structure, and well-characterized function, Cro has long served as a model system for understanding the structure/function relationship in transcription factors. Experiments have shown that a small set of mutations can convert it into a dual-function transcription factor capable of both repression and activation. One engineered variant retains activity when truncated to 63 amino acids but loses function at 59, highlighting how little sequence is required for complex regulatory behavior. To probe the molecular basis of this adaptability, we performed multi-microsecond all-atom molecular dynamics simulations of wild-type Cro and two engineered variants, Act3 and Act8. The simulations reveal that minimal sequence changes can reorganize interaction surfaces, shift DNA-binding modes, modulate binding affinities, and redistribute intramolecular communication pathways. These effects on DNA binding occur alongside changes that may broaden regulatory potential, offering insight into how compact transcription factors evolve new functions. Together, these observations provide a mechanistic framework for understanding how transcription factor sequence, structure, and dynamics reshape gene regulatory function.

biophysics↗

bioRxiv2025

Viral Nucleosome-Like Particles Exhibit Dynamic Flexibility and Reduced Thermodynamic Stability

DNA packaging imposes fundamental physical constraints on genomes across the tree of life. However, most of our mechanistic understanding of these processes comes from the eukaryotic nucleosome, where highly basic histones, along with their flexible tails, coordinate DNA compaction and gene accessibility. Large DNA viruses challenge this paradigm by assembling nucleosome-like particles with a divergent histone architecture. These viral assemblies lack canonical histone tails, contain covalently fused domains linked by structured connectors, and exhibit altered surface electrostatics, features that collectively impose unique biophysical properties on viral chromatin. Here, we use multi-microsecond all-atom molecular dynamics simulations to dissect how histone fusion, tail loss, and connector architecture reshape the structural and thermo-dynamic behavior of the Melbournevirus nucleosome, a model system for studying viral chromatin organization. We find that viral systems exhibit elevated DNA unwrapping, weaker and more transient histone-DNA contacts, and localized flexibility at connector regions. Conformational adaptation at histone junctions partially offsets these effects, with structural shifts tuned to local DNA geometry during wrapping transitions. By capturing how nucleosome dynamics shift across time and sequence, our study reveals how conserved biophysical principles of chromatin architecture are reconfigured across life to meet distinct evolutionary demands.

biophysics↗

bioRxiv2025

Deciphering the Molecular Mechanisms of BPTF Interactions with Nucleosomes via Molecular Simulations

Many transcription factors regulate DNA accessibility and gene expression by recognizing post-translational modifications on histone tails within nucleosomes. These interactions are often studied in vitro using short peptide mimics of histone tails, which may overlook conformational changes that occur in the full nucleosomal context. Here, we employ molecular dynamics simulations to investigate the binding dynamics of the PHD finger and bromodomain of BPTF, both in solution and bound to either a histone H3 peptide or a full nucleosome. Our results show that BPTF adopts distinct conformational states depending on its binding context, with nucleosome engagement inducing compaction of the multidomain structure. PHD finger binding displaces the H3 tail from DNA, increasing H3 tail flexibility while promoting compensatory binding of the H4 tail to nucleosomal DNA. This redistribution of histone-DNA contacts weakens overall hydrogen bonding with DNA, suggesting localized destabilization of the nucleosome core. Despite electrostatic repulsion limiting direct reader-DNA contacts, strong Van der Waals interactions with the H3 tail stabilize binding. Our results provide atomistic insight into how BPTF engagement modulates nucleosome structure and may facilitate chromatin remodeling.

biophysics↗

bioRxiv2025

The Opposing Roles of H2A and H2B Ubiquitination on Nucleosome Dynamics Revealed by Molecular Simulations

Nucleosome ubiquitination at lysine 119 of histone H2A (H2AK119ub) and lysine 120 of histone H2B (H2BK120ub) are prominent post-translational modifications with opposing roles in chromatin regulation. Although H2AK119ub is associated with transcriptional repression and H2BK120ub with activation, the molecular basis for these contrasting effects has remained unclear. Here, we use microsecond all-atom and millisecond coarse-grained molecular dynamics simulations to reveal how the position of ubiquitin reshapes nucleosome structure and assembly. H2AK119ub rigidifies the histone core by indirectly reinforcing the L1-L1 interface between H2A histones, strengthening both tetramer-dimer and dimer-dimer interactions, and slowing complete nucleosome assembly. In contrast, H2BK120ub disrupts these interfaces, weakens the histone core, and favors partially assembled hexasome and tetrasome states. Both modifications cause dramatic slowdowns in nucleosome folding, with H2BK120ub producing an order-of-magnitude greater effect. These simulations establish clear molecular mechanisms by which site-specific ubiquitination alters nucleosome stability and assembly kinetics. Our findings quantitatively explain how H2A and H2B ubiquitination exert opposing effects on chromatin regulation. This mechanism is directly relevant to the opposing roles of these marks in transcriptional activation and repression, and may represent one way that combinations of histone modifications modulate chromatin function in vivo.

biophysics↗

bioRxiv2024

The Influence of Ionic Environment on Nucleosome-Mica Interactions Revealed via Molecular Dynamics Simulations

Mica serves as a crucial substrate in Atomic Force Microscopy (AFM) studies for visualizing and characterizing nucleosomes. Nucleosomes interact with the negatively charged mica surface via adsorbed cations. However, the specific influences of monovalent and divalent cations on nucleosome adsorption to the mica surface remain unclear. In this study, we investigated the binding of nucleosomes to the mica surface in the presence of monovalent potassium ions and divalent magnesium ions using molecular dynamics simulations. We also explored the impact of pre-treated mica surfaces on nucleosome binding and structure. Our findings reveal that nucleosome-mica interactions vary depending on the cations present, resulting in distinct effects on nucleosome structure. Notably, nucleosomes bind effectively to a mica surface in the presence of potassium ions with minimal structural perturbations.

biophysics↗

bioRxiv2023

Modeling Allosteric Mechanisms of Eukaryotic Type II Topoisomerases

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N- and C-terminals of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point towards specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular-level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process. SIGNIFICANCETypeII Topoisomerases (TopoIIs) are essential and ubiquitous for maintaining DNA topology in the nucleus. The mechanisms by which information about the nucleotide-binding state is transmitted from the ATPase domains throughout the TopoII complex remain poorly understood. We used microsecond timescale molecular dynamics simulations to probe the allosteric mechanisms underlying TopoII function. Results indicate remarkable flexibility of the ATPase domains on this timescale which is modulated by nucleotide binding and local and distant amino acid substitutions. Furthermore, we mapped the allosteric networks linking the ATPase and DNA cleavage domains, and connected them to the ATPase domain dynamics. Our findings provide molecular-level insights into how nucleotide binding and hydrolysis regulate the TopoII catalytic cycle and how mutations can disrupt these processes.

biophysics↗

bioRxiv2022

Conformational and Thermodynamic Differences Underlying Wild-type and Mutant YEATS Domain Specificity for Epigenetic Marks

Histone post-translational modifications (PTMs) are interpreted by multiple reader domains and proteins to regulate gene expression. The ENL YEATS domain is a prototypical PTM reader that recognizes multiple lysine acetylation marks on the histone H3 tails as a way of recruiting chromatin remodellers. Two ENL YEATS mutations have been identified which have been linked with leukemia, Wilms tumor, and other forms of cancer and result in either an insertion or deletion of residues in the loop connecting beta sheets distant from the protein active site. In vitro experiments have shown that these mutations modulate the selectivity of YEATS domains for various lysine acetylation marks, although different experiments have provided contrasting views on the ability of the insertion and deletion mutants to discern specific PTMs. Here, we have performed multiple molecular dynamics simulations of wild-type, insertion, and deletion mutant YEATS domains free from and in complex with two PTM peptides: one that is acetylated at K9 of H3 and the other that is acetylated at residue K27 of H3. Results show that these two peptides have distinct flexibilities and binding energetics when bound to YEATS domains, and that these properties are affected by interactions with residues within and outside of the peptide consensus motif. Furthermore, these properties are modulated by the YEATS insertion and deletion mutants, which results in disparate binding effects in these systems. Together, these results suggest that only the partial exposure of histone tails is sufficient in the context of nucleosomes for YEATS-mediated recognition of acetylation marks on histone tails. They also caution against the over-interpretation of results obtained from experiments on reader domainhistone peptide binding in isolation and not in the full-length nucleosome context.

biophysics↗

bioRxiv2022

The Effects of RNA.DNA-DNA Triple Helices on Nucleosome Structures and Dynamics

Non-coding RNAs (ncRNAs) are an emerging epigenetic factor and have been recognized as playing a key role in many gene expression pathways. Structurally, binding of ncRNAs to isolated DNA is strongly dependent on sequence complementary, and results in the formation of an RNA.DNA-DNA (RDD) triple helix. However, in vivo DNA is not isolated, but is packed in chromatin fibers, the fundamental unit of which is the nucleosome. Biochemical experiments have shown that ncRNA binding to nucleosomal DNA is elevated at DNA entry and exit sites and is dependent on the presence of the H3 N-terminal tails. However, the structural and dynamical bases for these mechanisms remains unknown. Here, we have examined the mechanisms and effects of RDD formation in the context of the nucleosome using a series of all-atom molecular dynamics simulations. Results highlight the importance of DNA sequence on complex stability, elucidate the effects of the H3 tails on RDD structures, show how RDD formation impacts the structure and dynamics of the H3 tails, and show how RNA alters the local and global DNA double helical structure. Together, our results suggest ncRNAs can modify nucleosome, and potentially higher-order chromatin, structures and dynamics as a means of exerting epigenetic control. SIGNIFICANCENon-coding RNAs (ncRNAs) play an essential role in gene regulation by binding to DNA and forming RNA.DNA-DNA (RDD) triple helices. In the cell, this occurs in the context where DNA is not a free double helix but is instead condensed into chromatin fibers. At the fundamental level, this compaction involves wrapping approximately 147 DNA basepairs around eight histone proteins to form the nucleosome. Here, we have used molecular dynamics simulations to understand the interplay between the structure and dynamics of RDD triple helices with the nucleosome. Results highlight the importance of RNA sequence on RDD stability regardless of its environment and suggest potential mechanisms for cross-talk between epigenetic factors and the effects of ncRNA binding on local and higher-order chromatin structures.

biophysics↗

bioRxiv2021

Directed inter-domain motions enable the IsdH Staphylococcus aureus receptor to rapidly extract heme from human hemoglobin

Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tridomain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globins heme pocket. Using molecular simulations, Markov modeling, and quantitative measurements of heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hbs pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

biophysics↗

bioRxiv2021

CENP-N promotes the compaction of centromeric chromatin

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. The arrangement of nucleosomes at the centromere into higher order structure is unknown. Here we demonstrate that the CENP-A interacting protein CENP-N promotes the stacking of CENP-A containing mono-nucleosomes and nucleosomal arrays through a previously undefined interaction between the 6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryoEM structures and biophysical characterization of such CENP-N mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere. One-Sentence SummaryThe centromere-associated protein CENP-N promotes centromere-specific nucleosome stacking and higher order structures in vitro and in the cell.

molecular biology↗

bioRxiv2020

Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways

Eukaryotes and many archaea package their DNA with histones. While the four eukaryotic histones wrap [~]147 DNA base pairs into nucleosomes, archaeal histones form "nucleosome-like" complexes that continuously wind between 60 - 500 base pairs of DNA ("archaeasomes"), suggested by crystal contacts and analysis of cellular chromatin. Solution structures of large archaeasomes (>90 DNA base pairs) have never been directly observed. Here, we utilize molecular dynamics simulations, analytical ultracentrifugation, and cryoEM to structurally characterize the solution state of archaeasomes on longer DNA. Simulations reveal dynamics of increased accessibility without disruption of DNA-binding or tetramerization interfaces. Mg2+ concentration influences compaction, and cryoEM densities illustrate that DNA is wrapped in consecutive substates arranged 90{degrees} out-of-plane with one another. Without ATP-dependent remodelers, archaea may leverage these inherent dynamics to balance chromatin packing and accessibility.

biophysics↗

bioRxiv2020

The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction. SignificanceLinker histones dynamically bind to DNA in chromatin fibers and serve as epigentic regulators. However, the extent to which they influence the gamut of chromatin architecture is still not well understood. Using molecular dynamics simulations, we studied compact octa-nucleosome arrays with and without the H1 linker histone to better understand the mechanisms dictating the structure of the chromatin fiber. Inclusion of H1 results in stabilization of the compact chromatin structure, while its removal results in a major conformational change towards an untwisted ladder-like state. The increased rigidity and correlations within the H1-bound array suggests that H1-saturated chromatin fibers are better suited to transferring long-range epigentic information.

biophysics↗

bioRxiv2020

WASP: A software package for correctly characterizing the topological development of ribbon structures

We introduce the Writhe Application Software Package (WASP) which can be used to characterise the topology of ribbon structures, the underlying mathematical model of DNA, Biopolymers, superfluid vorticies, elastic ropes and magnetic flux ropes. This characterisation is achieved by the general twist-writhe decomposition of both open and closed ribbons, in particular through a quantity termed the polar writhe. We demonstrate how this decomposition is far more natural and straightforward than artificial closure methods commonly utilized in DNA modelling. In particular, we demonstrate how the decomposition of the polar writhe in local and non-local components distinctly characterizes local helical structure and knotting/linking of the ribbon. This decomposition provides additional information not given by alternative approaches. As an example application, the WASP routines are used to characterise the evolving topology (writhe) of DNA minicircle and open ended plectoneme formation magnetic/optical tweezer simulations. Finally it is demonstrated that a number of well known alternative writhe expressions are actually simplifications of the polar writhe measure.

biophysics↗

bioRxiv2019

The Molecular Basis of Specific DNA Binding by the BRG1 AT-hook and Bromodomain

The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to identify the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.

biochemistry↗