Biology subjects

Huertas, J.

Publications and source records attributed to Huertas, J..

13 recordsLinked to original sources

bioRxiv2026

A Spectrum of Free Energy Landscape Topologies Encodes Chromatin Polymorphism and Phase Separation

Chromatin exhibits structural polymorphism, yet how such substantial structural heterogeneity is encoded in its underlying free-energy landscape remains unknown. Here we map the folding landscapes of tetra-nucleosome arrays using adaptive Markov state models constructed from near-atomistic coarse-grained simulations. We find that chromatin is not governed by a single folding funnel analogous to those of many globular proteins. Instead, it spans a continuum of free-energy landscape topologies, ranging from funnelled to rugged to flat. Remarkably, phase separation reshapes chromatins free-energy landscape by stabilising conformations that maximise intermolecular connectivity, demonstrating that the landscape is determined not only by intrinsic molecular parameters but also by the environment and intermolecular interactions. Linker DNA geometry selects among landscape regimes through geometric frustration of zig-zag nucleosome stacking. At short and intermediate linker lengths, canonical 10N bp linkers produce funnelled landscapes dominated by compact zig-zag structures, whereas 10N +5 bp linkers generate rugged landscapes with multiple competing minima. Long linkers relieve frustration and flatten the landscape into a broad entropic basin. This control operates with single-base-pair precision, such that changing linker length by one base pair is sufficient to reorganise both the thermodynamics and kinetics of chromatin folding. DNA sequence provides a second level of control by modulating linker deformability, but only when zig-zag geometric frustration is present. These results establish free-energy landscapes as a framework for understanding the physical origin of chromatin polymorphism and its coupling to phase separation.

biophysics↗

bioRxiv2026

Single-molecule nucleosome spacing coordinates chromatin fiber interactions

Nucleosome spacing influences higher-order chromatin fiber organization in vitro but how this relates to cellular chromosome structure remains contentious. To address this, we developed Ligation Analysis of Single-molecule Sequence Interactions (LASSI), a single-molecule epigenomic method that combines proximity ligation with near-nucleotide resolution, long-read adenine methyltransferase footprinting. LASSI measures nucleosome spacing patterns on interacting segments of chromatin genome-wide in cells, quantifying coupled chromosome organization in 1D and 3D. Applying LASSI to mouse embryonic stem cells (mESCs), we discover a genome-wide structural pattern we term fiber homotypy, where chromatin fibers with similar nucleosome spacing patterns interact more frequently in 3D. This pattern persists over long intrachromosomal distances in cis and interchromosomally in trans. Fiber homotypy negatively scales with genomic distance, though differently than contact probability, implying distinct mechanisms. It is further promoted by topologically associated domains (TADs), A/B compartments, and shared histone modification domains, suggesting instructive roles for each of these processes. Coarse-grain molecular dynamics simulations of chromatin fibers informed by LASSI reveal that the intrinsically heterogeneous spacing of nucleosomes along chromatin fibers in cells is a key regulator of homotypy. This variability tunes the free energy landscape of chromatin fiber interactions, promoting the compartmentalization of similar 1D chromatin structures via specific types of fiber-fiber interaction. Further linking compartmentalization and fiber homotypy, we demonstrate that loop extrusion antagonizes this phenomenon. Depletion of the cohesin subunit RAD21 in mESCs increases fiber homotypy genome-wide, while depletion of the cohesin unloader WAPL decreases fiber homotypy, consistent with effects seen on A/B compartmentalization. Our results demonstrate that fiber-fiber interactions driven by shared nucleosome spacing patterns instruct higher-order chromosome organization. Moreover, we show clear structural interdependence across cellular chromatin length-scales, likely tuned by processes ranging from nucleosome positioning to loop extrusion.

genomics↗

bioRxiv2026

A Goldilocks zone of DNA flexibility defines stable yet plastic nucleosomes, tuned by histone chemistry

Nucleosomes regulate DNA accessibility through partial DNA unwrapping driven by thermal fluctuations or forces exerted by molecular motors. Despite substantial chemical and DNA sequence diversity in vivo, much of our mechanistic understanding of nucleosome unwrapping is derived from canonical constructs. Here, we use a near-atomistic coarse-grained model to quantify the energetics and mechanisms of force-induced nucleosome unwrapping across over 40 nucleosomes spanning genomic and synthetic DNA sequences, histone variants, and post-translational modifications. We find that the force barriers to nucleosome unwrapping are shaped by histone composition and DNA flexibility, and arise from the formation of topologically protected, partially unwrapped intermediates. Strong nucleosome-positioning and genomic sequences consistently fall within a Goldilocks zone of intermediate DNA flexibility, which enables the formation of thermodynamically stable yet mechanically plastic nucleosomes that undergo controlled deformation under applied force. Limited mechanical variation within this zone results in modest DNA sequence-dependent effects on nucleosome deformability. In contrast, histone variants and post-translational modifications modulate nucleosome stability and plasticity in a strongly non-additive manner, with predominant contributions from H3 and H2A core arginines and H3 and H2B tail lysines. Together, our results provide a physical framework linking DNA sequence, histone composition, and nucleosome geometry to nucleosome stability, plasticity and DNA accessibility.

biophysics↗

bioRxiv2025

Near-atomistic simulations reveal the molecular principles that control chromatin structure and phase separation

Understanding how chromatins physicochemical properties shape its emergent organisation is central to deciphering genome function. To address this, we present OpenCGChromatin, a high-performance coarse-grained model that achieves near-atomistic simulations of chromatin systems an order of magnitude larger than previously possible, spanning biomolecular condensates and fibers tens of kilobases in length. OpenCGChromatin simulations independently predict, from physicochemical principles, the linker-DNA-dependent chromatin structures observed by cryo-ET and the relative thermodynamic stability of condensates inferred from biochemical assays. Crucially, OpenCGChromatin resolves histone-tail dynamics and interaction networks that remain inaccessible experimentally, explaining how linker-DNA length controls histone tail accessibility and the resulting multiscale structure of chromatin condensates. Extending simulations to 108-nucleosome fibers shows that acetylation disrupts chromatin compaction in a pattern-specific manner by weakening key tail-mediated interactions, with H4K16 and H3K9 emerging as the most energetically disruptive modifications. These results position OpenCGChromatin as a powerful framework for linking molecular detail to emergent chromatin organization.

biophysics↗

bioRxiv2025

An electrostatic repulsion model of centromere organisation

During cell division, chromosomes reorganise into compact bodies in which centromeres localise precisely at the chromatin surface1-4 to enable kinetochore-microtubule interactions essential for genome segregation5-8. The physical principles guiding this centromere positioning remain unknown. Here, we reveal that human core centromeres are directed to the chromatin surface by repulsion of centromere-associated proteins - independent of condensin-mediated loop extrusion and microtubule engagement. Using cellular perturbations, biochemical reconstitution, and multiscale molecular dynamics simulations, we show that chromatin surface localisation emerges from repulsion between condensed chromatin and both the kinetochore and the highly negatively charged centromere protein, CENP-B. Together, these elements form a centromeric region composed of two domains with opposing affinities, one favouring integration within the mitotic chromosome and the other favouring exposure to the surrounding cytoplasm, thereby driving surface positioning. Tethering synthetic negatively charged proteins to chromatin was sufficient to recapitulate this surface localisation in cells and in vitro, indicating that electrostatic repulsion is a key determinant of surface localisation. These findings demonstrate that centromere layering is not hardwired by chromatin folding patterns but instead emerges from phase separation in chromatin. Our work uncovers electrostatic polarity as a general and programmable mechanism to spatially organise chromatin.

cell biology↗

bioRxiv2025

Linker histone H1 functions as a liquid-like glue to organize chromatin in living human cells

Linker histone H1, the most abundant chromatin protein, condenses chromatin, modulates DNA transactions such as transcription and DNA replication/repair, and participates in differentiation, development and tumorigenesis. While recent studies indicate that nucleosomes are clustered as condensed chromatin domains in higher eukaryotic cells, how histone H1 mechanically condenses chromatin remains unclear. Here, using a combination of direct visualization of single-H1 molecules in living human cells and multiscale molecular dynamics simulations, we demonstrate that the majority of H1 behaves like a liquid inside chromatin domains, rather than binding stably to nucleosomes as suggested by the traditional model. H1 functions as a liquid-like "glue", mediating dynamic multivalent electrostatic interactions between nucleosomes within chromatin domains. Consistently, rapid-depletion of H1.2 leads to decondensed chromatin domains both in cells and in silico. Our findings suggest that the H1 "glue" condenses chromatin domains while keeping them fluid and accessible, thereby supporting essential DNA transactions.

cell biology↗

bioRxiv2025

Cold-induced nucleosome dynamics linked to silencing of Arabidopsis FLC

Temperature influences nucleosome dynamics, and thus chromatin, to regulate gene expression. Such mechanisms underlie the epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC) by prolonged cold. Here, we show a temperature-dependent transition in local chromatin structure at the H3K27me3 nucleation region, from a modality active for transcription to a state that can be Polycomb silenced. In vivo chromatin measurements and coarse-grained simulations at near-atomistic resolution show the active transcription state is characterized by a highly dynamic nucleosome arrangement that exposes the FLC TSS. Cold exposure then changes the chromatin structure by reducing nucleosome dynamics and re-positioning the +1 nucleosome, leading to transcriptional repression. This local chromatin transition partially depends on VRN1, a non-sequence-specific DNA-binding protein. Loss of VRN1 results in hyperaccumulation of H2A.Z, more dynamic nucleosomes and an inability to accumulate H2Aub and H3K27me3. Our work highlights how local nucleosome dynamics link to chromatin structure transitions to integrate temperature inputs into epigenetic switching mechanisms in plants.

molecular biology↗

bioRxiv2025

Multi-scale structure of chromatin condensates rationalizes phase separation and material properties

Biomolecular condensates, compartments that concentrate molecules without surrounding membranes, are integral to numerous cellular processes. The structure and interaction networks of molecules within condensates remain poorly understood. Using cryo-electron tomography and molecular dynamics simulations we elucidated the structure of phase separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules both within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which in turn governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose non-random organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules ultimately determines physical properties of chromatin condensates, with implications for cellular chromatin organization.

biophysics↗

bioRxiv2024

Nucleosome Spacing Can Fine-Tune Higher Order Chromatin Assembly

Cellular chromatin displays heterogeneous structure and dynamics, properties that control diverse nuclear processes. Models invoke phase separation of conformational ensembles of chromatin fibers as a mechanism regulating chromatin organization in vivo. Here we combine biochemistry and molecular dynamics simulations to examine, at single base-pair resolution, how nucleosome spacing controls chromatin phase separation. We show that as DNA linkers extend from 25 bp to 30 bp, as examplars of 10N+5 and 10N (integer N) bp lengths, chromatin condensates become less thermodynamically stable and nucleosome mobility increases. Simulations reveal that this is due to trade-offs between inter- and intramolecular nucleosome stacking, favored by rigid 10N+5 and 10N bp linkers, respectively. A remodeler can induce or inhibit phase separation by moving nucleosomes, changing the balance between intra- and intermolecular stacking. The intrinsic phase separation capacity of chromatin enables fine tuning of compaction and dynamics, likely contributing to heterogeneous chromatin organization in vivo.

biophysics↗

bioRxiv2024

Tandem-repeat proteins introduce tuneable properties to engineered biomolecular condensates

The cells ability to rapidly partition biomolecules into biomolecular condensates is linked to a diverse range of cellular functions. Understanding how the structural attributes of biomolecular condensates are linked with their biological roles can be facilitated by the development synthetic condensate systems that can be manipulated in a controllable and predictable way. Here, we design and characterise a tuneable synthetic biomolecular condensate platform fusing modular consensus-designed tetratricopeptide repeat (CTPR) proteins to intrinsically-disordered domains. Trends between the CTPR structural attributes and condensate propensity were recapitulated across different experimental conditions and by in silico modelling, demonstrating that the CTPR domain can systematically affect the condensates in a predictable manner. Moreover, we show that incorporating short binding motifs into the CTPR domain results in specific target-protein recruitment into the condensates. Our model system can be rationally designed in a versatile manner to both tune condensate propensity and endow the condensates with new functions.

biophysics↗

bioRxiv2021

OCT4 interprets and enhances nucleosome flexibility

Pioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4s impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 propagates and stabilizes open nucleosome conformations by specific sequence recognition and nonspecific DNA exploration. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.

biophysics↗

bioRxiv2020

Histone tails cooperate to control the breathing of genomic nucleosomes

Genomic DNA is packaged in chromatin, a dynamic fiber variable in size and compaction. In chromatin, repeating nucleosome units wrap 146 DNA basepairs around histone proteins. Genetic and epigenetic regulation of genes relies on structural transitions in chromatin which are driven by intra- and internucleosome dynamics and modulated by chemical modifications of the unstructured terminal tails of histones. Here we demonstrate how the interplay between histone H3 and H2A tails control ample nucleosome breathing motions. We monitored large openings of two genomic nucleosomes, and only moderate breathing of an engineered nucleosome in atomistic molecular simulations amounting to 18s. Transitions between open and closed nucleosome conformations were driven by the displacement and changes in compaction of the two histone tails. These motions involved changes in the DNA interaction profiles of clusters of epigenetic regulatory aminoacids in the tails. Histone tail modulated nucleosome breathing is a key mechanism of chromatin dynamics.

biophysics↗

bioRxiv2019

Nucleosomal DNA dynamics mediate Oct4 pioneer factor binding

Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence specific manner. Here we reveal the structural dynamics of nucleosomes that mediate Oct4 binding. Nucleosome mobility and the amplitude of nucleosome motions such as breathing and twisting correlate with the number of Oct4 binding sites available. Moreover, the regions around the binding sites display higher local mobility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations display stable protein-DNA interactions and are compatible with the DNA curvature and DNA-histone interactions.

biophysics↗