Biology subjects

Eeftens, J. M.

Publications and source records attributed to Eeftens, J. M..

7 recordsLinked to original sources

bioRxiv2026

Multiscale Physical Effects of CpG Methylation on DNA Mechanics, Nucleosome Wrapping, and Chromatin Condensates

DNA methylation at CpG dinucleotides is a central epigenetic modification linked to transcriptional repression and heterochromatin formation, yet its physical impact on chromatin organization remains incompletely understood. Here, we integrate single-molecule force spectroscopy, FRAP, and nanorheology to quantify how CpG methylation alters chromatin mechanics across scales. Optical tweezer-based measurements show that CpG methylation modestly increases DNA contour and persistence lengths, indicating a slightly extended and stiffer DNA helix. When assembled into nucleosomes, CpG methylation weakens both outer-and inner turn wrapping, reducing unwrapping forces and per-nucleosome unwrapping energy. On a higher-order assembly scale, although CpG methylated and unmethylated chromatin fibers display similar thresholds for salt-induced phase separation, methylated condensates exhibit markedly reduced internal mobility: FRAP reveals slower chromatin exchange, nanorheology shows increased viscoelasticity and longer relaxation times. Finally, CpG methylation enhances HP1-chromatin interactions, lowering the HP1 concentration required for condensation, despite no change in HP1 binding to naked DNA. Together, these results show that CpG methylation remodels chromatin mechanics by tuning DNA rigidity, nucleosome wrapping energetics, and condensate material properties, providing a multiscale physical framework for how DNA methylation contributes to heterochromatin.

biochemistry↗

bioRxiv2025

Condensate-driven chromatin organization via elastocapillary interactions

Biomolecular condensates are ubiquitous structures found throughout eukaryotic cells, with nuclear condensates playing a key role in the mesoscale organization and functionality of the genome1,2. Protein- and RNA-rich liquid-like condensates form through phase separation on and around chromatin, driving diverse condensate morphologies with varying sphericity and intra-condensate chromatin density3,4. However, a unifying set of physical principles underlying these varied interactions and their implications for chromatin organization remains elusive. Here, we develop and experimentally validate a mesoscopic model that bridges the physics of phase separation and chromatin mechanics. Specifically, by integrating computational modeling with experiments using two canonical condensate proteins, the heterochromatin protein HP1, and the euchromatin protein BRD4, we demonstrate that wetting properties and chromatin stiffness shape condensate morphology, while condensates remodel chromatin mechanics and organization. This two-way interplay is governed by elastocapillarity--the deformation of chromatin by condensate interfacial tension -- and resolves discrepancies in nuclear condensate behavior, with emergent behaviors that deviate from the simplest liquid-liquid phase separation (LLPS) models5-8. Our findings underscore that nuclear condensates and chromatin cannot be studied in isolation, as they are fundamentally interdependent, impacted by biomolecularly-defined wetting properties, with implications for genome organization, transcriptional regulation, and epigenetic control in diverse phenotypes, including cancer2,9,10. Beyond the nucleus, the methodologies we present offer a generalizable platform for exploring multiphase, multicomponent soft matter systems across a broad range of biological and synthetic contexts11.

biophysics↗

bioRxiv2024

Robust quantification of cellular mechanics using optical tweezers

Mechanical properties of cells are closely related to function and play a crucial role in many cellular processes, including migration, differentiation, and cell fate determination. Numerous methods have been developed to assess cell mechanics under various conditions, but they often lack accuracy on biologically relevant piconewton-range forces, or have limited control over the applied force. Here, we use optically-trapped polystyrene beads to accurately apply piconewton-range forces to adherent and suspended cells. We precisely apply a constant force to cells by means of a force-feedback system, allowing for quantification of deformation, cell stiffness and creep response from a single measurement. Using drug-induced perturbations of the cytoskeleton, we show that this approach is sensitive to detecting changes in cellular mechanical properties. Collectively, we provide a framework for using optical tweezers to apply highly accurate forces to adherent and suspended cells, and describe straightforward metrics to quantify cellular mechanical properties.

cell biology↗

bioRxiv2024

Interplay of condensation and chromatin binding underlies BRD4 targeting

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is under-explored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcription factor BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

molecular biology↗

bioRxiv2022

Viscoelastic RNA entanglement and advective flow underlie nucleolar form and function

The nucleolus facilitates transcription, processing, and assembly of ribosomal RNA (rRNA), the most abundant RNA in cells. Nucleolar function is facilitated by its multiphase liquid properties, but nucleolar fluidity and its connection to ribosome biogenesis remain unclear. Here, we used quantitative imaging, mathematical modeling, and pulse-chase nucleotide labelling to map nucleolar rRNA dynamics. Inconsistent with a purely diffusive process, rRNA steadily expands away from the transcriptional sites, moving in a slow (~1[A]/s), radially-directed fashion. This motion reflects the viscoelastic properties of a highly concentrated gel of entangled rRNA, whose constant polymerization drives steady outward flow. We propose a new viscoelastic rRNA release model, where nucleolar rRNA cleavage and processing reduce entanglement, fluidizing the nucleolar periphery to facilitate release of mature pre-ribosomal particles.

biophysics↗

bioRxiv2020

Epigenetic memory as a time integral over prior history of Polycomb phase separation

Structural organization of the genome into transcriptionally active euchromatin and silenced heterochromatin is essential for eukaryotic cell function. Heterochromatin is a more compact form of chromatin, and is associated with characteristic post-translational histone modifications and chromatin binding proteins. Phase-separation has recently been suggested as a mechanism for heterochromatin formation, through condensation of heterochromatin associated proteins. However, it is unclear how phase-separated condensates can contribute to stable and robust repression, particularly for heritable epigenetic changes. The Polycomb complex PRC1 is known to be key for heterochromatin formation, but the multitude of Polycomb proteins has hindered our understanding of their collective contribution to chromatin repression. Here, we take a quantitative live cell imaging approach to show that PRC1 proteins form multicomponent condensates through hetero-oligomerization. They preferentially seed at H3K27me3 marks, and subsequently write H2AK119Ub marks. Using optogenetics to nucleate local Polycomb condensates, we show that Polycomb phase separation can induce chromatin compaction, but phase separation is dispensable for maintenance of the compacted state. Our data are consistent with a model in which the time integral of historical Polycomb phase separation is progressively recorded in repressive histone marks, which subsequently drive chromatin compaction. These findings link the equilibrium thermodynamics of phase separation with the fundamentally non-equilibrium concept of epigenetic memory.

biophysics↗

bioRxiv2017

Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

Condensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

biophysics↗