Biology subjects

Marenduzzo, D.

Publications and source records attributed to Marenduzzo, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Cross-chemistry single-nucleus RNA-seq identifies gene length and CpG-island promoters as determinants of transcriptional noise.

Cell-to-cell transcriptional heterogeneity, or noise, is an intrinsic property of the transcriptome with implications for development, disease progression, and aging. Bulk RNA-seq masks this variability by averaging gene expression across cells, whereas single-cell RNA sequencing (scRNA-seq) resolves it. Nevertheless, separating biological noise from technical variance remains challenging, particularly across platforms with different chemistries. We benchmarked two widely adopted technologies, Evercode WT (SPLiT-seq, Parse Biosciences) and Chromium (10x Genomics), on human lymphoblastoid nuclei. Evercode WT achieved targeted sequencing depth and nuclei number far more reliably, and its random-hexamer priming yielded more intronic reads and non-coding RNA genes; Chromium recovered more cells and detected polyadenylated transcripts and cell-line markers more sensitively. Despite these opposing biases, the platforms showed comparable gene detection and strongly correlated expression profiles. Using datasets from both platforms, we defined a noise metric detrended from mean expression and showed that per-gene estimates were reproducible across chemistries. Noise was lower in G2M than in G1 and was most strongly associated with gene length rather than exonic length. Expression of genes with CpG-island promoters was less variable than that of those without. This study establishes a platform-independent basis for quantifying transcriptional noise and a framework for selecting an appropriate scRNA-seq platform.

genomics↗

bioRxiv2026

CpG islands act as topological sinks for transcription-induced DNA supercoiling

Strong evolutionary selection has maintained CpG-dense islands (CGIs) at the promoters of constitutively expressed genes throughout the vertebrate genome, suggesting an important role in regulating DNA topology. Here, using Twist-seq, a psoralen-based approach for quantitative genome-wide profiling of DNA supercoiling, we reveal distinct topological states across human gene promoters. We show that CGI promoters accumulate elevated levels of negative supercoiling relative to non-CGI promoters and define localised topological domains at highly transcribed genes. Integrating genome-wide analyses with reaction-diffusion modelling and coarse-grained molecular dynamics simulations, we find that this behaviour is encoded by the intrinsic physical properties of CGI DNA. The GC-rich sequence context promotes nucleosome depletion and focuses torsional stress onto embedded AT-rich pockets, driving localised DNA melting and plectoneme-tip bubble formation within promoter-proximal nucleosome-free regions. This provides an energetically favourable pathway for redistributing transcription-induced torsional stress through transient strand separation and writhe, consistent with increased ssDNA formation at CGI promoters observed by ssDNA-seq. We propose that CGIs function as sequence-encoded topological sinks that buffer supercoiling while maintaining a promoter architecture permissive for transcription initiation, thereby preserving promoter integrity and genome stability.

molecular biology↗

bioRxiv2026

Irregular nucleosome positioning governs a crystalline to liquid-like phase transition and tunes chromatin accessibility

Chromatin must fold tightly enough to protect the genome while being sufficiently accessible for DNA dependent processes such as transcription. The physical rules that balance these competing roles remain unclear, as DNA sequence encodes both biochemical information such as transcription factor binding sites, and biophysical cues that shape chromatin structure. Here, using synthetic chromatin fibres assembled from physiologically relevant DNA sequences, we show that nucleosome positioning dictates the material state of chromatin. Heterochromatin-like sequences produce compact fibres stabilised by nucleosome stacking, whereas euchromatin-like sequences generate irregular nucleosome positioning that yields disrupted, heterogeneous, and mechanically deformable fibres. Quantitative polymer modelling reveals that these irregular arrays are highly dynamic, continually sampling a broad ensemble of conformations as nucleosome stacking breaks down. We identify two previously unrecognised thresholds encoded by nucleosome positioning: minimal positional irregularity (2-3 bp) triggers a transition from an ordered paracrystalline state to a liquid-like phase, whereas an order of magnitude greater irregularity (~18 bp) is required to generate accessibility and mechanical fragility permissive for transcription factor binding. Euchromatin-like arrays reside at this accessibility threshold. These findings indicate that nucleosome positioning tunes chromatin toward or away from critical structural states that couple genome protection, chromatin dynamics, and transcriptional potential, providing a physical mechanism that helps connect DNA sequence to gene expression.

molecular biology↗

bioRxiv2026

Towards a database capturing chromosome structure and function: symbols and syntax

Existing databases of interphase chromosome conformations typically store three-dimensional coordinates of genomic segments. However, since interphase chromatin is highly dynamic, such databases are dominated by transient configurations and unstructured regions, whose positions vary continuously between cells and over time, unlike folded proteins such as globin, which adopt similar structures in every cell. These drawbacks motivated the inception of a database based on strion (a portmanteau of a string capturing structure and function). A strion concisely describes the structure and activity of all transcription units in one cell, by retaining only functionally relevant positional information. Sets of strions describing structures in different cells sampled at different times are compiled into a super-strion. Then, 46 super-strions summarise the range of structure and activity of a human cell type, including information on all transcription units, how often each co-fires and co-clusters with others in transcription factories/hubs, enhancer interactomes and small-world expression networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/724942v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13a1263org.highwire.dtl.DTLVardef@18d2c78org.highwire.dtl.DTLVardef@162865corg.highwire.dtl.DTLVardef@1631d65_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

3D confinement reshapes RNA folding and enhances circularisation in the Zika virus

Many RNA molecules function within confined environments, but the effect of confinement on RNA folding remains poorly understood. Proximity ligation experiments reveal altered long-range contacts in confined versus unconfined states, yet they do not explain how spatial constraints give rise to these differences. Here, we develop a physical modeling approach that incorporates proximity ligation data into coarse-grained molecular dynamics simulations to reconstruct RNA 3D structures under confinement. We test our model on the[~] 11 kb genome of the Zika virus, comparing the folding in virions (confined) and in cells (unconfined). We observe that the probability of contact between two regions of the genome vs. linear distance follows different scaling laws in the confined and unconfined cases, in agreement with proximity ligation experiments. We find that genome circularization - an interaction that regulates replication in Zika - occurs more frequently under confinement in both experiments and simulations. Our model reveals that the formation of long double-stranded stems through stacking confers local nematic liquid crystalline order to the RNA, and predicts pseudoknot topologies consistent with those seen in crystallographic structures of shorter RNAs. These results provide insight into the mechanism through which confinement alters the ensemble of 3D structures accessible to a long RNA molecule. Significance statementSqueezing a long string into a tight ball changes how it behaves: distant segments are forced into close contact, making ends more likely to meet. Similar principles apply to biopolymers, such as RNA, when confined within virus particles or other enclosures smaller than the polymer contour length, but there is no framework to model the effects of confinement on RNA folding. Here we develop such a framework, combining molecular dynamics simulations with experimental measurements of contacts within unconfined and confined RNA molecules. We find that RNA is glassy and exhibits liquid crystalline order, and that genome circularization, a long-range interaction between the two ends of the viral RNA important for virus replication, is facilitated under confinement.

biophysics↗

bioRxiv2024

Nuclear RNA forms an interconnected network of transcription-dependent and tunable microgels

Living cells are organised through active, non-equilibrium processes, yet the physical principles governing their material states remain unclear. The cell nucleus has been described as either fluid-like or solid-like, but these behaviours have not been reconciled within a single framework. Here we show that nascent RNA and the multivalent RNA-binding protein SAF-A assemble into a nucleus-spanning microgel network maintained near the sol-gel transition. This active network exhibits scale- and time-dependent viscoelasticity and naturally reconciles solid-like and fluid-like responses. By integrating super-resolution imaging with mechanistic polymer modelling, we identify RNA production and degradation as control parameters that tune network connectivity. The model predicts enhanced susceptibility to modest perturbations near criticality, which we confirm experimentally by shifting the system between sol-like and gel-like regimes. Measurements of molecular transport, combined with simulations, reveal size-dependent diffusion and transient trapping within gel pores, establishing direct physical consequences of the near-critical state. Our results identify RNA metabolism as an energy-driven regulator of nuclear material properties and position the nucleus as a living example of an active microgel poised near criticality.

molecular biology↗

bioRxiv2024

Cyclical MinD membrane affinity differences are not necessary for MinD gradient formation in Bacillus subtilis

Proteins can diffuse micrometers in seconds, yet bacterial cells are able to maintain stable protein gradients. The best studied bacterial protein gradient is the Min system of Escherichia coli. In rod-shaped bacteria the MinCD proteins prevent formation of minicells by inhibiting FtsZ polymerization close to the cell poles. In E. coli these proteins oscillate between cell poles within a minute, resulting in an increased MinCD concentration at the poles. This oscillation is caused by the interaction between MinD and the protein MinE, which form an ATP-driven reaction-diffusion system, whereby the ATPase MinD cycles between a monomeric cytosolic and a dimeric membrane attached states. Bacillus subtilis also has MinCD, but lacks MinE. In this case MinCD form a static gradient that requires the transmembrane protein MinJ, located at cell poles and cell division sites. A recent reaction-diffusion model was successful in recreating the MinD gradient in B. subtilis, assuming that MinD cycles between cytosol and membrane, like in E. coli. Here we show that the monomeric and dimeric states of B. subtilis MinD have comparable membrane affinities, that MinD interacts with MinJ as a dimer, and that MinJ is not required for membrane localization of MinD. Based on these new findings we tested different models, using kinetic Monte Carlo simulations, and found that a difference in diffusion rate between the monomer and dimer, rather than a difference in membrane affinity, is important for B. subtilis MinCD gradient formation.

microbiology↗

bioRxiv2023

Tunable living bacterial networks: A multi-scaled description of their self-assembly and mechanical behaviour

Bacterial aggregate formation and surface accumulation are increasingly viewed as alternative pathways for biofilm colonization. However, little is known about the dynamics of bacterial aggregate cluster-cluster assembly and their subsequent microstructural and mechanical properties. To this end, we studied experimentally and computationally an aggregating bacterial system that forms a space-spanning interconnected network via cluster-cluster assembly. By controllably inducing bacterial filamentation, we aimed to understand how cell length distribution and cell surface hydrophobicity control the dynamics of aggregation and sedimentation, as well as the microstructure and mechanics of the settled bacterial networks. We found that filamentation lowers the percolation threshold, leading to gelation at a lower number density with distinct assembly dynamics and lower network connectivity. Furthermore, we analyzed the mechanical properties of the bacterial networks. Static stress tests reveal three yielding modes: discrete cluster-cluster disassembly, collective delamination, and sub-regional network fracture. The yielding modes are consistent with the gel-like viscoelastic properties of the cluster-cluster assembled networks observed during macroscale rheometry. In particular, we observe a scaling relationship between the storage modulus and the volume fraction, characteristic of an attractive rod gel. Our experimental observations are supported by Langevin dynamic simulations, providing mechanistic insights into the factors determining network self-assembly and connectivity. Our findings elucidate the gel-like structure-function dynamics in cluster-cluster aggregated bacterial systems and underscore the fundamental importance of filamentation in their properties and mechanical behavior.

biophysics↗

bioRxiv2023

A multicolour polymer model for the prediction of 3D structure and transcription in human chromatin

Transcription is a fundamental cellular process, and the first step of gene expression. In human cells, it depends on the binding to chromatin of various proteins, including RNA polymerases and numerous transcription factors (TFs). Observations indicate that these proteins tend to form macromolecular clusters, known as transcription factories, whose morphology and composition is still debated. While some microscopy experiments have revealed the presence of specialised factories, composed of similar TFs transcribing families of related genes, sequencing experiments suggest instead that mixed clusters may be prevalent, as a panoply of different TFs binds promiscuously the same chromatin region. The mechanisms underlying the formation of specialised or mixed factories remain elusive. With the aim of finding such mechanisms, here we develop a chromatin polymer model mimicking the chromatin binding-unbinding dynamics of different types of complexes of TFs. Surprisingly, both specialised (i.e., demixed) and mixed clusters spontaneously emerge, and which of the two types forms depends mainly on cluster size. The mechanism promoting mixing is the presence of non-specific interactions between chromatin and proteins, which become increasingly important as clusters become larger. This result, that we observe both in simple polymer models and more realistic ones for human chromosomes, reconciles the apparently contrasting experimental results obtained. Additionally, we show how the introduction of different types of TFs strongly affects the emergence of transcriptional networks, providing a pathway to investigate transcriptional changes following gene editing or naturally occurring mutations.

biophysics↗

bioRxiv2022

Bridging-mediated compaction of mitotic chromosomes

Eukaryotic chromosomes compact during mitosis and meiosis into elongated cylinders - and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamics simulations involving two types of such proteins. The first anchors topologically-stable and long-lived chromatin loops to create bottlebrush structures. The second forms multivalent bridges between distant parts of these loops without entrapping them. We show bridging factors lead to the formation of shorter and stiffer mitotic-like cylinders, without requiring any energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes in agreement with experiments, and provides a mechanistic model to understand mitotic chromatin structure at common fragile sites.

biophysics↗

bioRxiv2022

Topological Gelation of Reconnecting Polymers

DNA recombination is a ubiquitous process that ensures genetic diversity. Contrary to textbook pictures, DNA recombination, as well as generic DNA translocations, occur in a confined and highly entangled environment. Inspired by this observation, here we investigate a solution of semiflexible polymer rings undergoing generic cutting and reconnection operations under spherical confinement. Our setup may be realised using engineered DNA in presence of recombinase proteins or by considering micelle-like components able to form living (or reversibly breakable) polymer rings. We find that in such systems there is a topological gelation transition, which can be triggered by increasing either the stiffness or concentration of the rings. Flexible or dilute polymers break into an ensemble of short, unlinked and segregated rings, whereas sufficiently stiff or dense polymers self-assemble into a network of long, linked and mixed loops, many of which are knotted. We predict the two phases should behave qualitatively differently in elution experiments monitoring the escape dynamics from a permeabilised container. Besides shedding some light on the biophysics and topology of genomes undergoing DNA reconnection in vivo, our findings could be leveraged in vitro to design polymeric complex fluids, e.g., DNA-based complex fluids or living polymer networks, with desired topologies.

biophysics↗

bioRxiv2022

Condensin reorganizes centromeric chromatin during mitotic entry into a bipartite structure stabilized by cohesin

The Structural Maintenance of Chromosomes (SMC) complexes cohesin and condensin establish the 3D organization of mitotic chromosomes1-3. Cohesin is essential to maintain sister chromatid pairing until anaphase onset4, while condensin is important for mitotic centromere structure and elastic resistance to spindle forces5-8. Both complexes are also important to form productive kinetochore-spindle attachments6, 8, 9. How condensin and cohesin work together to shape the mitotic centromere to ensure faithful chromosome segregation remains unclear. Here we show by super-resolution imaging, Capture-C analysis and polymer modeling that vertebrate centromeres are partitioned into two distinct condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres and also in human neocentromeres devoid of satellite repeats, and is therefore a fundamental feature of vertebrate centromere identity. Super-resolution imaging reveals that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to mitotic spindle forces. In its absence, separated bipartite kinetochores frequently engage in merotelic spindle attachments. Consistently, uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. The two-domain structure of vertebrate regional centromeres described here incorporates architectural roles for both condensin and cohesin and may have implications for avoiding chromosomal instability in cancer cells.

cell biology↗

bioRxiv2022

Gene structure heterogeneity drives transcription noise within human chromosomes

Classical observations have long suggested there is a link between 3D gene structure and transcription1-4. However, due to the many factors regulating gene expression, and to the challenge of visualizing DNA and chromatin dynamics at the same time in living cells, this hypothesis has been difficult to quantitatively test experimentally. Here we take an orthogonal approach and use computer simulations, based on the known biophysical principles of genome organisation5-7, to simultaneously predict 3D structure and transcriptional output of human chromatin genome wide. We validate our model by quantitative comparison with Hi-C contact maps, FISH, GRO-seq and single-cell RNA-seq data, and provide the 3DGene resource to visualise the panoply of structures adopted by any active human gene in a population of cells. We find transcription strongly correlates with the formation of protein-mediated microphase separated clusters of promoters and enhancers, associated with clouds of chromatin loops, and show that gene noise is a consequence of structural heterogeneity. Our results also indicate that loop extrusion by cohesin does not affect average transcriptional patterns, but instead impacts transcriptional noise. These findings provide a functional role for intranuclear microphase separation, and an evolutionary mechanism for loop extrusion halted at CTCF sites, to modulate transcriptional noise.

molecular biology↗

bioRxiv2021

Transcription modulates chromatin dynamics and locus configuration sampling

In living cells the 3D structure of gene loci is dynamic, but this is not revealed by 3C and FISH experiments in fixed samples, leaving a significant gap in our understanding. To overcome these limitations we applied the "highly predictive heteromorphic polymer" (HiP-HoP) model, validated by experiments, to determine chromatin fibre mobility at the Pax6 locus in three mouse cell lines with different transcription states. While transcriptional activity minimally affects the movement of 40 kbp regions, we observed that the motion of smaller 1 kbp regions depends strongly on local disruption to chromatin fibre structure marked by H3K27 acetylation. This also significantly influenced locus configuration dynamics by modulating promoter-enhancer loops associated with protein bridging. Importantly these simulations indicate that chromatin dynamics are sufficiently fast to sample all possible conformations of loci within minutes, generating wide dynamic variability of gene loci structure within single cells. Experiments inhibiting transcription change chromatin fibre structure subtly, yet we predict they should substantially affect mobility. This combination of simulation and experimental validation provide a novel insight and mechanistic model to explain how transcriptional activity influences chromatin structure and gene dynamics.

biophysics↗

bioRxiv2020

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to reestablish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation, super-resolution imaging and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon reentry into G1. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

genomics↗

bioRxiv2020

Phase separation induced by cohesin SMC protein complexes

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

biophysics↗

bioRxiv2020

Complex small-world regulatory networks emerge from the 3D organisation of the human genome

The discovery that overexpressing one or a few critical transcription factors can switch cell state suggests that gene regulatory networks are relatively simple. In contrast, genome-wide association studies (GWAS) point to complex phenotypes being determined by hundreds of loci that rarely encode transcription factors and which individually have small effects. Here, we use computer simulations and a simple fitting-free polymer model of chromosomes to show that spatial correlations arising from 3D genome organisation naturally lead to stochastic and bursty transcription plus complex small-world regulatory networks (where the transcriptional activity of each genomic region subtly affects almost all others). These effects require factors to be present at sub-saturating levels; increasing levels dramatically simplifies networks as more transcription units are pressed into use. Consequently, results from GWAS can be reconciled with those involving overexpression. We apply this pan-genomic model to predict patterns of transcriptional activity in whole human chromosomes, and, as an example, the effects of the deletion causing the diGeorge syndrome.

biophysics↗

bioRxiv2020

A nonequilibrium strategy for fast target search on the genome

Vital biological processes such as genome repair require fast and efficient binding of selected proteins to specific target sites on DNA. Here we propose an active target search mechanism based on "chromophoresis", the dynamics of DNA-binding proteins up or down gradients in the density of epigenetic marks, or colours (biochemical tags on the genome). We focus on a set of proteins that deposit marks from which they are repelled - a case which is only encountered away from thermodynamic equilibrium. For suitable ranges of kinetic parameter values, chromophoretic proteins can perform undirectional motion and are optimally redistributed along the genome. Importantly, they can also locally unravel a region of the genome which is collapsed due to self-interactions and "dive" deep into its core, for a striking enhancement of the efficiency of target search on such an inaccessible substrate. We discuss the potential relevance of chromophoresis for DNA repair.

biophysics↗