Biology subjects

Mirny, L. A.

Publications and source records attributed to Mirny, L. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Live-cell imaging of enhancer-promoter dynamics reveals transient contact-driven gene activation

Enhancers are key regulators of mammalian gene expression, yet how they interact with promoters in space (contact vs. action-at-a-distance) and in time (transient vs. stable) remains poorly understood. Recent studies suggest that enhancers can activate promoters across distances exceeding 200 nanometers, challenging classical contact models, but limited spatiotemporal resolution has obscured the mechanistic details of enhancer-promoter (E-P) interactions and their link to transcription. Here, we engineered a synthetic biology platform optimized for the simultaneous visualization of E-P 3D distance and nascent transcription using super-resolution live-cell imaging. By applying five complementary approaches integrating imaging, 3D genomics, and gene expression data across cell lines, we estimate that transcriptional activation is mediated by [~]25-42 nanometer contacts on the seconds timescale. Our results support a transient contact mechanism for E-P-mediated gene activation.

biophysics↗

bioRxiv2024

Dynamics of microcompartment formation at the mitosis-to-G1 transition

As cells exit mitosis and enter G1, mitotic chromosomes decompact and transcription is reestablished. Previously, Hi-C studies showed that essentially all interphase 3D genome features including A/B-compartments, TADs, and CTCF loops, are lost during mitosis. However, Hi-C remains insensitive to features such as microcompartments, nested focal interactions between cis-regulatory elements (CREs). We therefore applied Region Capture Micro-C to cells from mitosis to G1. Unexpectedly, we observe microcompartments in prometaphase, which further strengthen in ana/telophase before gradually weakening in G1. Loss of loop extrusion through condensin depletion differentially impacts microcompartments and large A/B-compartments, suggesting that they are partially distinct. Using polymer modeling, we show that microcompartment formation is favored by chromatin compaction and disfavored by loop extrusion activity, explaining why ana/telophase likely provides a particularly favorable environment. Our results suggest that CREs exhibit intrinsic homotypic affinity leading to microcompartment formation, which may explain transient transcriptional spiking observed upon mitotic exit.

biophysics↗

bioRxiv2024

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at [~]1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

molecular biology↗

bioRxiv2023

Unique territorial and sub-chromosomal organization revealed in the holocentric moth Bombyx mori

Hallmarks of multicellular eukaryotic genome organization are chromosome territories, compartments, and loop-extrusion-mediated structures, including TADs. However, these are mainly observed in model organisms, and most eukaryotes remain unexplored. Using Hi-C in the silkworm Bombyx mori we discover a novel chromatin folding structure, compartment S, which is "secluded" from the rest of the chromosome. This compartment exhibits loop extrusion features and a unique genetic and epigenetic landscape, and it localizes towards the periphery of chromosome territories. While euchromatin and heterochromatin display preferential compartmental contacts, S domains are remarkably devoid of contacts with other regions, including with other S domains. Polymer simulations show that this contact pattern can only be explained by high loop-extrusion activity within compartment S, combined with low extrusion elsewhere through the genome. This unique, targeted extrusion represents a novel phenomenon and underscores how evolutionarily conserved mechanisms--compartmentalization and loop extrusion--can be repurposed to create new 3D genome architectures.

genomics↗

bioRxiv2023

Extrusion fountains are hallmarks of chromosome organization emerging upon zygotic genome activation

The initiation of gene expression during development, known as zygotic genome activation (ZGA), is accompanied by massive changes in chromosome organization. However, the earliest events of chromosome folding and their functional roles remain unclear. Using Hi-C on zebrafish embryos, we discovered that chromosome folding begins early in development with the formation of "fountains", a novel element of chromosome organization. Emerging preferentially at enhancers, fountains exhibit an initial accumulation of cohesin, which later redistributes to CTCF sites at TAD borders. Knockouts of pioneer transcription factors driving ZGA enhancers result in the specific loss of fountains, establishing a causal link between enhancer activation and fountain formation. Polymer simulations demonstrate that fountains may arise as sites of facilitated cohesin loading, requiring two-sided but desynchronized loop extrusion, potentially caused by cohesin collisions with obstacles or internal switching. Moreover, we detected similar fountain patterns at enhancers in mouse cells. Fountains disappear upon acute cohesin depletion, as well as during mitosis, and reappear with cohesin loading in early G1. Altogether, fountains represent the first known enhancer-specific elements of chromosome organization and constitute starting points for chromosome folding during development, likely through facilitated cohesin loading.

molecular biology↗

bioRxiv2023

Tunable DNMT1 degradation reveals cooperation of DNMT1 and DNMT3B in regulating DNA methylation dynamics and genome organization

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present untransformed and cancer cell models that allow inducible and reversible global modulation of DNAme through DNMT1 depletion. By dynamically assessing the effects of induced passive demethylation through cell divisions at both the whole genome and locus-specific level, we reveal a cooperative activity between DNMT1 and DNMT3B to maintain and control DNAme. Moreover, we show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin abundance, compartmentalization, and peripheral localization. DNA methylation loss coincided with a gradual reduction of cell fitness due to G1 arrest, but with minor level of mitotic failure. Altogether, this powerful system allows DNMT and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNA methylation dysfunction and human disease.

molecular biology↗

bioRxiv2022

Chromosome decompaction and cohesin direct Topoisomerase II activity to establish and maintain an unentangled interphase genome

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations and multi-contact 3C, and propose that, in contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a Topoisomerase II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias Topoisomerase II activity towards decatenation. At the second stage, the loops are released, and formation of new entanglements is prevented by lower Topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed, in experiment and models, a normal interphase state cannot be acquired.

genomics↗

bioRxiv2022

Design principles of 3D epigenetic memory systems

The epigenetic state of a cell is associated with patterns of chemical modifications of histones ("marks") across the genome, with different marks typical of active (euchromatic) and inactive (heterochromatic) genomic regions. These mark patterns can be stable over many cell generations--a form of epigenetic memory--despite their constant erosion due to replication and other processes. Enzymes that place histone marks are often stimulated by the same marks, as if "spreading" marks between neighboring histones. But this positive feedback may not be sufficient for stable memory, raising the question of what is. In this work, we show how 3D genome organization--in particular, the compartmental segregation of euchromatin and heterochromatin-- could serve to stabilize an epigenetic memory, as long as (1) there is a large density difference between the compartments, (2) the modifying enzymes can spread marks in 3D, and (3) the enzymes are limited in abundance relative to their histone substrates. We introduce a biophysical model stylizing chromatin and its dynamics through the cell cycle, in which enzymes spread self-attracting marks on a polymer. We find that marks localize sharply and stably to the denser compartment, but over several cell generations, the model generically exhibits uncontrolled spread or global loss of marks. Strikingly, imposing limitation of the modifying enzymes--a plausible but oft-neglected element--totally changes this picture, yielding an epigenetic memory system, stable for hundreds of cell generations. Our model predicts a rich phenomenology to compare to experiments, and reveals basic design principles of putative epigenetic memory systems relying on compartmentalized 3D genome structure for their function.

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

Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we present a novel approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observe viscoelastic displacements over microns within minutes in response to near-picoNewton forces, which are well captured by a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing the minor role of crosslinks and topological effects and challenging the view that interphase chromatin is a gel-like material. Our new technology opens avenues for future research, from chromosome mechanics to genome functions.

biophysics↗

bioRxiv2020

DNA-loop extruding SMC complexes can traverse one another in vivo

The spatial organization of chromosomes by structural maintenance of chromosomes (SMC) complexes is vital to organisms from bacteria to humans 1,2. SMC complexes were recently found to be motors that extrude DNA loops 3-11. It remains unclear, however, what happens when multiple SMC complexes encounter one another in vivo on the same DNA, how encounters are resolved, or how interactions help organize an active genome 12. Here, we set up a "crash-course track" system to study what happens when SMC complexes encounter one another. Using the parS/ParB system, which loads SMC complexes in a targeted manner13-17, we engineered the Bacillus subtilis chromosome to have multiple SMC loading sites. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of never-before-seen chromosome folding patterns. Polymer simulations indicate these patterns require SMC complexes to traverse past each other in vivo, contrary to the common assumption that SMC complexes mutually block each others extrusion activity 18. Our quantitative model of bypassing predicted that increasing the numbers of SMCs on the chromosome could overwhelm the bypassing mechanism, create SMC traffic jams, and lead to major chromosome reorganization. We validated these predictions experimentally. We posit that SMC complexes traversing one another is part of a larger phenomenon of bypassing large steric barriers which enables these loop extruders to spatially organize a functional and busy genome.

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

The interplay between asymmetric and symmetric DNA loop extrusion

Compaction of chromosomes is essential for reliable transmission of genetic information. Experiments suggest that this [~] 1000-fold compaction is driven by condensin complexes that extrude chromatin loops, i.e., progressively collect chromatin fiber from one or both sides of the complex to form a growing loop. Theory indicates that symmetric two-sided loop extrusion can achieve such compaction, but recent single-molecule studies observed diverse dynamics of condensins that perform one-sided, symmetric two-sided, and asymmetric two-sided extrusion. We use simulations and theory to determine how these molecular properties lead to chromosome compaction. High compaction can be achieved if even a small fraction of condensins have two essential properties: a long residence time and the ability to perform two-sided (not necessarily symmetric) extrusion. In mixtures of condensins I and II, coupling of two-sided extrusion and stable chromatin binding by condensin II promotes compaction. These results provide missing connections between single-molecule observations and chromosome-scale organization.

biophysics↗

bioRxiv2020

Limited evidence of tumour mutational burden as a biomarker of response to immunotherapy

Cancer immunotherapy by checkpoint blockade (ICB) is effective for various cancer types, yet its clinical use is encumbered by a high variability of patient response. Several studies have reported that the number of non-synonymous mutations (Tumor Mutational Burden, TMB), can predict patient response to ICB. This belief has become widespread and led to the FDA approval of immunotherapy patient prioritization based on TMB levels. The notion that TMB is predictive of response to immunotherapy is rooted in the neoantigen theory. It stipulates that cancer-specific mutations can form neoantigens recognized by the immune system; the more mutations a tumor has, the more likely the immune response is triggered. Here we revisit the data underlying the reported association of TMB with response, and the neoantigen theory. First we assembled the largest pan-cancer dataset of immunotherapy patients with sequencing and clinical data. Surprisingly, we find little evidence that TMB is predictive of response to ICB. We demonstrate that associations similar to the ones reported previously can be observed in shuffled data, suggesting that previous studies suffered from a lack of correction for multiple hypotheses testing and confounding disease subtypes. Second, we develop a model that expands the neoantigen theory and can be consistent with both immunogenicity of neoantigens and the lack of association between TMB and response. Our analysis shows that the use of TMB in clinical practice is not supported by available data and can deprive patients of treatment to which they are likely to respond.

genomics↗

bioRxiv2019

Molecular basis of CTCF binding polarity in genome folding

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin proteins (Merkenschlager & Nora, 2016; Fudenberg, Abdennur, Imakaev, Goloborodko, & Mirny, 2017). While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize DNA loops (de Wit et al., 2015; Guo et al., 2015; Rao et al., 2014; Vietri Rudan et al., 2015), the molecular basis of this polarity remains mysterious. Here we report that CTCF positions cohesin but does not control its overall binding or dynamics on chromatin by single molecule live imaging. Using an inducible complementation system, we found that CTCF mutants lacking the N-terminus cannot insulate TADs properly, despite normal binding. Cohesin remained at CTCF sites in this mutant, albeit with reduced enrichment. Given that the orientation of the CTCF motif presents the CTCF N-terminus towards cohesin as it translocates from the interior of TADs, these observations provide a molecular explanation for how the polarity of CTCF binding sites determines the genomic distribution of chromatin loops.

molecular biology↗

bioRxiv2019

Chromosome organization by one-sided and two-sided loop extrusion

SMC complexes, such as condensin or cohesin, organize chromatin throughout the cell cycle by a process known as loop extrusion. SMC complexes reel in DNA, extruding and progressively growing DNA loops. Modeling assuming two-sided loop extrusion reproduces key features of chromatin organization across different organisms. In vitro single-molecule experiments confirmed that yeast condensins extrude loops, however, they remain anchored to their loading sites and extrude loops in a "one-sided" manner. We therefore simulate one-sided loop extrusion to investigate whether "one-sided" complexes can compact mitotic chromosomes, organize interphase domains, and juxtapose bacterial chromosomal arms, as can be done by "two-sided" loop extruders. While one-sided loop extrusion cannot reproduce these phenomena, variants can recapitulate in vivo observations. We predict that SMC complexes in vivo constitute effectively two-sided motors or exhibit biased loading and propose relevant experiments. Our work suggests that loop extrusion is a viable general mechanism of chromatin organization. Impact statementWe reconcile seemingly contradictory findings of single-molecule and in vivo experiments on a major mechanism of chromosome organization by computationally investigating mechanisms of loop extrusion that are consistent with both.

biophysics↗

bioRxiv2019

RNA polymerases as moving barriers to condensin loop extrusion

To separate replicated sister chromatids during mitosis, eukaryotes and prokaryotes have structural maintenance of chromosome (SMC) condensin complexes that were recently shown to organize chromosomes by a process known as DNA loop extrusion. In rapidly dividing bacterial cells, the process of separating sister chromatids occurs concomitantly with ongoing transcription. How transcription interferes with the condensin loop extrusion process is largely unexplored, but recent experiments show that sites of high transcription may directionally affect condensin loop extrusion. We quantitatively investigate different mechanisms of interaction between condensin and elongating RNA polymerases (RNAP) and find that RNAPs are likely steric barriers that can push and interact with condensins. Supported by new Hi-C and ChIP-seq data for cells after transcription inhibition and RNAP degradation, we argue that translocating condensins must bypass transcribing RNAPs within ~2 seconds of an encounter at rRNA genes and within ~10 seconds at protein coding genes. Thus, while individual RNAPs have little effect on the progress of loop extrusion, long, highly transcribed operons can significantly impede the extrusion process. Our data and quantitative models further suggest that bacterial condensin loop extrusion occurs by two independent, uncoupled motor activities; the motors translocate on DNA in opposing directions and function together to enlarge chromosomal loops, each independently bypassing steric barriers in their path. Our study provides a quantitative link between transcription and 3D genome organization and proposes a mechanism of interactions between SMC complexes and elongating transcription machinery relevant from bacteria to higher eukaryotes.

biophysics↗

bioRxiv2018

Limits of chromosome compaction by loop-extruding motors

During mitosis, human chromosomes are linearly compacted ~ 1000-fold by loop-extruding motors. Recent experiments have shown that condensins extrude DNA loops, but in a \"one-sided\" manner. We explore whether one-sided extrusion can compact chromosomes by developing a mean-field model for polymer compaction by motors that actively extrude loops and turnover. The model establishes an upper bound of only ~ 10-fold for compaction by one-sided extrusion. Thus, it cannot be the sole mechanism of chromosome compaction. However, other, effectively two-sided mechanisms can achieve sufficient compaction.

biophysics↗