Biology subjects

Peters, J.-M.

Publications and source records attributed to Peters, J.-M..

At least 19 recordsLinked to original sources

bioRxiv2026

A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organization

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor (IHF), condition-dependent changes in loop-like DNA structures in cohesin-CTCF-precocious dissociation of sisters 5A (PDS5A) reactions, and promoter-driven multimerization of GAGA factor (GAF) clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/708946v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1225883org.highwire.dtl.DTLVardef@1e1041dorg.highwire.dtl.DTLVardef@1d53d18org.highwire.dtl.DTLVardef@9e0a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Ultrafast CTCF dynamics control cohesin barrier function

Genomes are organized into chromatin loops through cohesin-mediated extrusion, with CTCF acting as a polar boundary element. As cohesin approaches CTCF at kilobase-per-second speeds, it must rapidly choose whether to stall or bypass. How CTCF encodes this probabilistic decision within a brief encounter window has remained unclear. Here we show that CTCF governs this probabilistic outcome by rapidly sampling a dynamic ensemble of conformations generated by spontaneous rearrangements of its DNA-binding zinc fingers. This ensemble is tuned by DNA sequence, CpG methylation, nearby nucleosomes, and the cohesin regulator PDS5A before cohesin engagement. Upon cohesin binding, PDS5A enhances loop-anchor mechanical stability, reinforcing orientation-dependent boundaries. These findings establish conformational ensemble tuning, rather than static occupancy, as a regulatory principle linking base pair-scale motions to megabase-scale genome organization. One sentence summaryChromatin boundary function is governed not by CTCF occupancy alone, but by a tunable ensemble of DNA-bound conformations that probabilistically gates cohesin capture.

biophysics↗

bioRxiv2025

Two CTCF motifs impede cohesin-mediated DNA loop extrusion

Cohesin extrudes DNA into loops and is positioned along the genome by stalling at CTCF upon encountering its N-terminal region (NTR). The mechanism underlying this stalling, however, is unresolved. Using single-molecule assays that monitor DNA loop extrusion (LE) in the presence of NTR fragments, we identify two amino acid motifs, YDF and KTYQR, that hinder LE. KTYQR is found to fully impede LE activity, while YDF hinders cohesin to complete LE step cycles and converts cohesin into a unidirectional extruder by strengthening the affinity of STAG1 to DNA. We thus identify two distinct NTR motifs that stall LE via different yet synergistic mechanisms, highlighting the multifaceted ways employed by CTCF to modulate LE to shape and regulate genomes. One-Sentence SummaryThe N-terminus of CTCF employs two independent motifs that synergistically stall cohesin-mediated DNA loop extrusion.

biophysics↗

bioRxiv2024

NIPBL and STAG1 enable loop extrusion by providing differential DNA-cohesin affinity

DNA loop extrusion by cohesin has emerged as a critical pathway for chromosome organization. In vitro single-molecule experiments indicate that loop extrusion requires the assembly of an heteropentameric complex consisting of the SMC1/SMC3 heterodimer, STAG1, NIPBL and the kleisin SCC1. The multimeric nature of this complex and regulatoin of its dynamic DNA interactions that are modulated by ATP binding-hydrolysis cycles make it challenging to reveal the molecular mechanism of loop extrusion. Here, we use mass photometry to quantify the key interactions responsible for cohesin assembly, DNA binding, and their modulation by ATP binding and hydrolysis. We find that STAG1 binds tightly to the trimeric complex formed by the SMC1/SMC3 heterodimer and SCC1, creating a DNA binding site whose strength is modulated by the presence of ATP. NIPBL binds strongly to both DNA and the STAG1-tetramer, enabling assembly of the holoenzyme in the absence of DNA or ATP. Taken together, these results suggest that NIPBL acts as a DNA anchor, while a dynamic STAG1-Trimer binding site drives DNA loop formation.

biophysics↗

bioRxiv2024

Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early Gl, Cohesin-STAGl and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in Gl, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. 0ur quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.

cell biology↗

bioRxiv2024

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step

Eukaryotes carry three types of Structural Maintenance of Chromosomes (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion. SMCs modulate DNA supercoiling, but it has remained incompletely understood how this is achieved. Here we present a single-molecule magnetic tweezers assay that directly measures how much twist is induced by an individual SMC in each loop-extrusion step. We demonstrate that all three SMC complexes induce the same large negative twist (i.e., a linking number change {Delta}Lk of -0.6 at each loop-extrusion step) into the extruded loop, independent of step size. Using ATP-hydrolysis mutants and non-hydrolysable ATP analogues, we find that ATP binding is the twist-inducing event during the ATPase cycle, which coincides with the force-generating loop-extrusion step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-loop-extrusion mechanism among these SMC complexes.

biophysics↗

bioRxiv2024

Cohesin supercoils DNA during loop extrusion

Cohesin extrudes genomic DNA into loops that promote chromatin assembly, gene regulation and recombination. Here we show that cohesin introduces negative supercoils into extruded DNA. Supercoiling requires engagement of cohesins ATPase heads, DNA clamping by these heads, and a DNA binding site on cohesins hinge, indicating that cohesin supercoils DNA when constraining it between the hinge and the clamp. Our results suggest that DNA extrusion stops once cohesin reaches its stall torque during supercoiling, and a cohesin mutant predicted to stall at lower torque forms shorter loops in cells. These results indicate that supercoiling is an integral part of the loop extrusion mechanism and that cohesin controls genome architecture not only by looping DNA but also by supercoiling it.

biochemistry↗

bioRxiv2023

SMC motor proteins extrude DNA asymmetrically and contain a direction switch

Structural Maintenance of Chromosomes (SMC) complexes organize the genome via DNA loop extrusion. While some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, while the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. STAG1 stabilizes NIPBL on cohesin, preventing NIPBL turnover and direction switches. The findings reveal that SMCs, surprisingly, contain a direction switch subunit. HighlightsO_LIAll eukaryotic SMC complexes extrude DNA asymmetrically. C_LIO_LIApparent symmetric loop extrusion is the result of frequent direction switches. C_LIO_LIn human cohesin, loop-extrusion direction changes require exchange of NIPBL. C_LIO_LISTAG1 stabilizes NIPBL on human cohesin. C_LI

molecular biology↗

bioRxiv2023

Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2 phase

Genetic information is stored in linear DNA molecules, which fold extensively inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in a highly intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule-based spindle can move them to opposite poles. While the spindle-mediated transport of sister chromatids has been studied in detail, the chromosome-intrinsic mechanics pre-segregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop-extruding activity of cohesin, but not that of condensins. Increasing cohesins looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis-specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.

cell biology↗

bioRxiv2022

SORORIN is an evolutionary conserved antagonist of WAPL

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORINs functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1{Delta} mutants display cohesion defects, which are partially alleviated by wpl1{Delta}. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.

cell biology↗

bioRxiv2022

CTCF is a DNA-tension-dependent barrier to cohesin-mediated DNA loop extrusion

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CTCF generates topologically associating domains (TADs)2-4 that play key roles in gene regulation and recombination during development and disease1,5-8. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unknown9. To address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, as well as to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted, but unexpectedly is dependent on DNA tension. Moreover, CTCF regulates cohesins loop extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, where the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

biophysics↗

bioRxiv2022

Can pseudotopological models for SMC-driven DNA loop extrusion explain the traversal of physical roadblocks bigger than the SMC ring size?

DNA loop extrusion by structural-maintenance-of-chromosome (SMC) complexes has emerged as a primary organizing principle for chromosomes. The mechanism by which SMC motor proteins extrude DNA loops is still unresolved and much debated. The ring-like structure of SMC complexes prompted multiple models where the extruded DNA is topologically or pseudotopologically entrapped within the ring during loop extrusion. However, recent experiments showed the passage of roadblocks much bigger than the SMC ring size, suggesting a nontopological mechanism. Recently, attempts were made to reconcile the observed passage of large roadblocks with a pseudotopological mechanism. Here we examine the predictions of these pseudotopological models, and find that they are not consistent with some experimental data on SMC roadblock encounters. Particularly, these models predict the formation of two loops and that roadblocks will reside near the stem of the loop upon encounter - both in contrast to experimental observations. Overall, the experimental data reinforce the notion of a nontopological mechanism for extrusion of DNA.

biophysics↗

bioRxiv2022

Transcription shapes 3D chromatin organization by interacting with loop-extruding cohesin complexes

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin, and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic knockouts of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were "moving barriers" to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is a new type of extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization. Significance StatementLoop extrusion by cohesin is critical to folding the mammalian genome into loops. Extrusion can be halted by CTCF proteins bound at specific genomic loci, which generates chromosomal domains and can regulate gene expression. However, the process of transcription itself can modulate cohesin, thus refolding chromosomes near active genes. Through experiments and simulations, we show that transcribing RNA polymerases (RNAPs) act as "moving barriers" to loop-extruding cohesins. Unlike stationary CTCF barriers, RNAPs actively relocalize cohesins, which generates characteristic patterns of spatial organization around active genes. Our model predicts that the barrier function of RNAP can explain why cohesin accumulates at active promoters and provides a mechanism for clustering active promoters. Through transcription-extrusion interactions, cells might dynamically regulate functional genomic contacts.

genomics↗

bioRxiv2021

SMC complexes can traverse physical roadblocks bigger than their ring size

The ring-shaped structural-maintenance-of-chromosomes (SMC) complexes condensin and cohesin extrude loops of DNA as a key motif in chromosome organization. It remains, how ever, unclear how these SMC motor proteins can extrude DNA loops in chromatin that is bound with proteins. Here, using in vitro single-molecule visualization, we show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to DNA loop extrusion by yeast condensin. Strikingly, we find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, can be translocated into DNA loops during condensin-driven extrusion. Similarly, human cohesin can pass 200 nm particles during loop extrusion, which even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open up to entrap DNA. These findings disqualify all common loop-extrusion models where DNA passes through the SMC rings (pseudo)topologically, and instead point to a nontopological mechanism for DNA loop extrusion. One-sentence summaryHuge DNA-bound roadblocks can be incorporated into SMC-extruded DNA loops, pointing to a nontopological mechanism for loop extrusion.

biophysics↗

bioRxiv2020

MCM complexes are barriers that restrict cohesin-mediated loop extrusion

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered. Little is known about whether loop extrusion is impeded by DNA-bound macromolecular machines. We demonstrate that the replicative helicase MCM is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C of one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Simulations are consistent with MCMs as abundant, random barriers. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes. One Sentence SummaryMCM complexes are obstacles that impede the formation of CTCF-anchored loops.

cell biology↗

bioRxiv2020

Sister-chromatid-sensitive Hi-C reveals the conformation of replicated human chromosomes

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1-3 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells4-11 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

cell biology↗

bioRxiv2020

Cohesin-dependent and independent mechanisms support chromosomal contacts between promoters and enhancers

It is currently assumed that 3D chromosomal organisation plays a central role in transcriptional control. However, recent evidence shows that steady-state transcription of only a minority of genes is affected by depletion of architectural proteins such as cohesin and CTCF. Here, we have used Capture Hi-C to interrogate the dynamics of chromosomal contacts of all human gene promoters upon rapid architectural protein degradation. We show that promoter contacts lost in these conditions tend to be long-range, with at least one interaction partner localising in the vicinity of topologically associated domain (TAD) boundaries. In contrast, many shorter-range chromosomal contacts, particularly those that connect active promoters with each other and with active enhancers remain unaffected by cohesin and CTCF depletion. We demonstrate that the effects of cohesin depletion on nascent transcription can be explained by changes in the connectivity of their enhancers. Jointly, these results provide a mechanistic explanation to the limited, but consistent effects of cohesin and CTCF on steady-state transcription and point towards the existence of alternative enhancer-promoter pairing mechanisms that are independent of these proteins.

genomics↗

bioRxiv2019

Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, as well as to the efficiency and success of structural studies requiring crystallization or computational alignment of individual molecules. Here, we show that mass photometry (MP) accurately reports on sample heterogeneity using minimal volumes with molecular resolution within minutes. We benchmark our approach by negative stain electron microscopy (nsEM), including workflows involving chemical crosslinking and multi-step purification of a multi-subunit ubiquitin ligase. When applied to proteasome stability, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP for rapid sample characterization, prioritization and optimization.

biophysics↗