Loop Extrusion Research Articles

Cohesin distribution alone predicts chromatin organization in yeast via conserved-current loop extrusion.

Inhomogeneous patterns of chromatin-chromatin contacts within 10-100-kb-sized regions of the genome are a generic feature of chromatin spatial organization. These features, termed topologically associating domains (TADs), have led to the loop extrusion factor (LEF) model. Currently, our ability to model TADs relies on the observation that in vertebrates TAD boundaries are correlated with DNA sequences that bind CTCF, which therefore is inferred to block loop extrusion. However, although TADs feature prominently in their Hi-C maps, non-vertebrate eukaryotes either do not express CTCF or show few TAD boundaries that correlate with CTCF sites. In all of these organisms, the counterparts of CTCF remain unknown, frustrating comparisons between Hi-C data and simulations. To extend the LEF model across the tree of life, here, we propose the conserved-current loop extrusion (CCLE) model that interprets loop-extruding cohesin as a nearly conserved probability current. From cohesin ChIP-seq data alone, we derive a position-dependent loop extrusion rate, allowing for a modified paradigm for loop extrusion, that goes beyond solely localized barriers to also include loop extrusion rates that vary continuously. We show that CCLE accurately predicts the TAD-scale Hi-C maps of interphase Schizosaccharomyces pombe, as well as those of meiotic and mitotic Saccharomyces cerevisiae, demonstrating its utility in organisms lacking CTCF. The success of CCLE in yeasts suggests that loop extrusion by cohesin is indeed the primary mechanism underlying TADs in these systems. CCLE allows us to obtain loop extrusion parameters such as the LEF density and processivity, which compare well to independent estimates.

The cohesin complex: structure and principles of interaction with dna

Accurate duplication and separation of long linear genomic DNA molecules is associated with a number of purely mechanical problems. SMC complexes are key components of the cellular machinery that ensures decatenation of sister chromosomes and compaction of genomic DNA during division. Cohesin, one of the essential eukaryotic SMC complexes, has a typical ring structure with intersubunit pore through which DNA molecules can be threaded. The capacity of cohesin for such topological entrapment of DNA is crucial for the phenomenon of post-replicative association of sister chromatids better known as cohesion. Recently, it became apparent that cohesin and other SMC complexes are in fact motor proteins with a very peculiar movement pattern leading to the formation of DNA loops. This specific process was called loop extrusion. Extrusion underlies multiple cohesin’s functions beyond cohesion, but the molecular mechanism of the process remains a mystery. In this review, we have summarized data on the molecular architecture of cohesin, the influence of ATP hydrolysis cycle on this architecture, and the known modes of cohesin–DNA interactions. Many of the seemingly disparate facts presented here will probably be incorporated in a unified mechanistic model of loop extrusion in a not so far future.

Cohesin-dependent loop extrusion: molecular mechanics and role in cell physiology

The most prominent representatives of multisubunit SMC-complexes, cohesin and condensin, are best known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bacterial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on the process of SMC-dependent DNA looping, also known as loop extrusion. This review briefly summarizes the current understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its involvement in sister chromatid cohesion.

The chromosome folding problem and how cells solve it.

Every cell must solve the problem of how to fold its genome. We describe how the folded state of chromosomes is the result of the combined activity of multiple conserved mechanisms. Homotypic affinity-driven interactions lead to spatial partitioning of active and inactive loci. Molecular motors fold chromosomes through loop extrusion. Topological features such as supercoiling and entanglements contribute to chromosome folding and its dynamics, and tethering loci to sub-nuclear structures adds additional constraints. Dramatically diverse chromosome conformations observed throughout the cell cycle and across the tree of life can be explained through differential regulation and implementation of these basic mechanisms. We propose that the first functions of chromosome folding are to mediate genome replication, compaction, and segregation and that mechanisms of folding have subsequently been co-opted for other roles, including long-range gene regulation, in different conditions, cell types, and species.

Endogenous neuronal DNA double-strand breaks are not sufficient to drive brain aging and neurodegeneration.

Loss of genomic information due to the accumulation of somatic DNA damage has been implicated in aging and neurodegeneration 1-3 . Somatic mutations in human neurons increase with age 4 , but it is unclear whether this is a cause or a consequence of brain aging. Here, we clarify the role of endogenous, neuronal DNA double-strand breaks (DSBs) in brain aging and neurodegeneration by generating mice with post-developmental inactivation of the classical non-homologous end-joining (C-NHEJ) core factor Xrcc4 in forebrain neurons. Xrcc4 is critical for the ligation step of C-NHEJ and has no known function outside of DSB repair 5,6 . We find that, unlike their wild-type counterparts, C-NHEJ-deficient neurons accumulate high levels of DSB foci with age, indicating that neurons undergo frequent DSBs that are typically efficiently repaired by C-NHEJ across their lifespan. Genome-wide mapping reveals that endogenous neuronal DSBs preferentially occur in promoter regions and other genic features. Analysis of 3-D genome organization shows intra-chromosomal clustering and loop extrusion of neuronal DSB regions. Strikingly, however, DSB accumulation caused by loss of C-NHEJ induces only minor epigenetic alterations and does not significantly affect gene expression, 3-D genome organization, or mutational outcomes at neuronal DSBs. Despite extensive aging-associated accumulation of neuronal DSBs, mice with neuronal Xrcc4 inactivation do not show neurodegeneration, neuroinflammation, reduced lifespan, or impaired memory and learning behavior. We conclude that the formation of spontaneous neuronal DSBs caused by normal cellular processes is insufficient to cause brain aging and neurodegeneration, even in the absence of C-NHEJ, the principal neuronal DSB repair pathway.

DNA Replication and Polymer Chain Duplication Reshape the Genome in Space and Time

In eukaryotes, DNA replication constitutes a complex process whereby multiple origins are stochastically fired, and from which the replication machinery proceeds along chromosomes to achieve the faithful synthesis of two identical copies of the genome during the S phase of the cell cycle. Experimental evidence shows a functional correlation between the dynamics of replication and the spatial organization of the genome inside cell nuclei, suggesting that the process of replicating DNA may impact chromosome folding. However, the theoretical and mechanistic bases of such a hypothesis remain elusive. To address that question, we propose a quantitative, minimal framework that integrates the dynamics of replication along a polymer chain by accounting explicitly for the progression of the replication machinery and the resulting formation of sister chromatids. By systematically characterizing the 3D structural consequences of replication, and of possible interactions between active replication machineries, we show that the formation of transient loops may potentially impact chromosome organization across multiple temporal and spatial scales, from the level of individual origins to that of the global polymer chain. Comparison with available microscopy and chromosome conformation capture data in yeast suggests that a replication-dependent loop extrusion process may be acting , and may shape chromosomes as loose polymer bottle brushes during the S phase. Lastly, we explore the postreplication relative organization of sister chromatids and demonstrate the emergence of catenations and intertwined structures, which are regulated by the density of fired origins. Published by the American Physical Society 2024

Core enhancers of the 3'RR optimize IgH nuclear position and loop conformation for successful oriented class switch recombination.

In B lymphocytes, class switch recombination (CSR) is an essential process that adapts immunoglobulin (Ig) subtypes to antigenresponse. Taking place within the Ig heavy chain (IgH) locus, CSR needs controlled transcription of targeted regions governed by the IgH 3'regulatory region (3'RR). This super-enhancer is composed of four core enhancers surrounded by inverted repeated sequences, forming a quasi-palindrome. In addition to transcription, nuclear organization appears to be an important level in CSR regulation. While it is now established that chromatin loop extrusion takes place within IgH locus to facilitate CSR by bringing the donor and acceptor switch regions closer together, the underlying mechanism that triggers CSR loop formation remains partially understood. Here, by combining DNA 3Dfluorescence in situhybridizationwith various high-throughput approaches, we deciphered critical functions for the 3'RR core enhancer element in nuclear addressing, accessibility and chromatin looping of the IgH locus. We conclude thatthe 3'RR core enhancers are necessary and sufficient to pre-organize the position and conformation of IgH loci in resting B-cell nuclei to enable the deletional recombination events required for productive successful CSR in activated B-cell nuclei.

Dynamic barriers modulate cohesin positioning and genome folding at fixed occupancy.

In mammalian interphase cells, genomes are folded by cohesin loop extrusion limited by directional CTCF barriers. This interplay leads to the enrichment of cohesin at barriers, isolation between neighboring topologically associating domains, and elevated contact frequency between convergent CTCF barriers across the genome. However, recent in vivo measurements present a puzzle: reported residence times for CTCF on chromatin are in the range of a few minutes, while lifetimes for cohesin are much longer. Can the observed features of genome folding result from the action of relatively transient barriers? To address this question, we developed a dynamic barrier model, where CTCF sites switch between bound and unbound states with rates that can be directly compared with biophysical measurements. Using this model, we investigated how barrier dynamics would impact observables for a range of experimental genomic and imaging datasets, including ChIP-seq, Hi-C, and microscopy. We found the interplay of CTCF and cohesin binding timescales influence the strength of each of these features, leaving a signature of barrier dynamics even in the population-averaged snapshots offered by genomic datasets. First, in addition to barrier occupancy, barrier bound times are crucial for instructing features of genome folding. Second, the ratio of boundary to extruder lifetime greatly alters simulated ChIP-seq and simulated Hi-C. Third, large-scale changes in chromosome morphology observed experimentally after increasing extruder lifetime require dynamic barriers. By integrating multiple sources of experimental data, our biophysical model argues that CTCF barrier bound times effectively approach those of cohesin extruder lifetimes. Together, we demonstrate how models that are informed by biophysically measured protein dynamics broaden our understanding of genome folding.

Effects of the auxin-dependent degradation of the cohesin and condensin complexes on the repair of distant DNA double-strand breaks in mouse embryonic stem cells.

The SMC protein family, including cohesin and condensin I/II, plays a pivotal role in maintaining the topological structure of chromosomes and influences many cellular processes, notably the repair of double-stranded DNA breaks (DSBs). The cohesin complex impacts DSB repair by spreading γH2AX signal and containing DNA ends in close proximity by loop extrusion. Cohesin supports DNA stability by sister chromatid cohesion during the S/G2 phase, which limits DNA end mobility. Cohesin knockdown was recently shown to stimulate frequencies of genomic deletions produced by distant paired DSBs, but does not affect DNA repair of a single or close DSBs. We examined how auxin-inducible protein degradation of Rad21 (cohesin) or Smc2 (condensins I+II) changes the frequencies of rearrangements between paired distant DSBs in mouse embryonic stem cells (mESCs). We used Cas9 RNP nucleofection to generate deletions and inversions with high efficiency without additional selection. We determined optimal Neon settings and deletion appearance timings. Two strategies for auxin addition were tested (4 independent experiments in total). We examined deletion/inversion frequencies for two regions spanning 3.5 and 3.9 kbp in size. Contrary to expectations, in our setting, Rad21 depletion did not increase deletion/inversion frequencies, not even for the region with an active Ctcf boundary. We actually observed a 12 % decrease in deletions (but not inversions). At the same time, double condensin depletion (Smc2 degron line) demonstrated high biological variability between experiments, complicating the analysis, and requires additional examination in the future. TIDE analysis revealed that editing frequency was consistent (30-50 %) for most experiments with a minor decrease after auxin addition. In the end, we discuss the Neon/ddPCR method for deletion generation and detection in mESCs.

Permeable TAD boundaries and their impact on genome-associated functions.

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin-mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter-TAD loop formation. Based on the reanalysis of Nano-C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read-through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter-TAD loops is restricted though. We speculate that the DNA-encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer-promoter loop formation and other genomic processes.

Cohesin organizes 3D DNA contacts surrounding active enhancers in C. elegans.

In mammals, cohesin and CTCF organize the 3D genome into topologically associated domains (TADs) to regulate communication between cis-regulatory elements. Many organisms, including S. cerevisiae, C. elegans, and A. thaliana contain cohesin but lack CTCF. Here, we used C. elegans to investigate the function of cohesin in 3D genome organization in the absence of CTCF. Using Hi-C data, we observe cohesin-dependent features called "fountains", which are also reported in zebrafish and mice. These are population average reflections of DNA loops originating from distinct genomic regions and are ~20-40 kb in C. elegans. Hi-C analysis upon cohesin and WAPL depletion support the idea that cohesin is preferentially loaded at NIPBL occupied sites and loop extrudes in an effectively two-sided manner. ChIP-seq analyses show that cohesin translocation along the fountain trajectory depends on a fully intact complex and is extended upon WAPL-1 depletion. Hi-C contact patterns at individual fountains suggest that cohesin processivity is unequal on each side, possibly due to collision with cohesin loaded from surrounding sites. The putative cohesin loading sites are closest to active enhancers and fountain strength is associated with transcription. Compared to mammals, average processivity of C. elegans cohesin is ~10-fold shorter and NIPBL binding does not depend on cohesin. We propose that preferential loading and loop extrusion by cohesin is an evolutionarily conserved mechanism that regulates the 3D interactions of enhancers in animal genomes.

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.

Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent.

Structural maintenance of chromosome (SMC) complexes organize and regulate genomes via DNA loop extrusion. During this process, the complexes increase the loop size by reeling in DNA from one or both sides of the loop. The factors governing this symmetry remain unclear. Here, we combine single-molecule analysis and molecular dynamic simulations to investigate the symmetry of loop extrusion of various SMC complexes. We find that whereas monomeric condensin and cohesin are one-sided extruders, the symmetry of dimeric SMCs, such as Smc5/6 and Wadjet, is DNA tension dependent. At low DNA tension (< 0.1pN), Smc5/6 and Wadjet extrude DNA from both sides of the loop. At higher tension, however, they transition to a behavior akin to one-sided extruders, yet still capable of extruding from one or the other side thereby switching the direction of extrusion. Our simulations further reveal that thermal fluctuations significantly influence loop extrusion symmetry, causing variations in DNA reeling rates between the two motors in the dimeric complexes and their direction switching at stalling tensions. Our findings challenge the previous view of loop extrusion symmetry as a fixed characteristic, revealing its dynamic nature and regulation by both intrinsic protein properties and extrinsic factors.

Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development.

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

YY1-controlled regulatory connectivity and transcription are influenced by the cell cycle.

Few transcription factors have been examined for their direct roles in physically connecting enhancers and promoters. Here acute degradation of Yin Yang 1 (YY1) in erythroid cells revealed its requirement for the maintenance of numerous enhancer-promoter loops, but not compartments or domains. Despite its reported ability to interact with cohesin, the formation of YY1-dependent enhancer-promoter loops does not involve stalling of cohesin-mediated loop extrusion. Integrating mitosis-to-G1-phase dynamics, we observed partial retention of YY1 on mitotic chromatin, predominantly at gene promoters, followed by rapid rebinding during mitotic exit, coinciding with enhancer-promoter loop establishment. YY1 degradation during the mitosis-to-G1-phase interval revealed a set of enhancer-promoter loops that require YY1 for establishment during G1-phase entry but not for maintenance in interphase, suggesting that cell cycle stage influences YY1's architectural function. Thus, as revealed here for YY1, chromatin architectural functions of transcription factors can vary in their interplay with CTCF and cohesin as well as by cell cycle stage.

The physical chemistry of interphase loop extrusion.

Loop extrusion constitutes a universal mechanism of genome organization, whereby structural maintenance of chromosomes (SMC) protein complexes load onto the chromatin fiber and generate DNA loops of increasingly-larger sizes until their eventual release. In mammalian interphase cells, loop extrusion is mediated by the cohesin complex, which is dynamically regulated by the interchange of multiple accessory proteins. Although these regulators bind the core cohesin complex only transiently, their disruption can dramatically alter cohesin dynamics, gene expression, chromosome morphology and contact patterns. Still, a theory of how cohesin regulators and their molecular interplay with the core complex modulate genome folding remains at large. Here we derive a model of cohesin loop extrusion from first principles, based on in vivo measurements of the abundance and dynamics of cohesin regulators. We systematically evaluate potential chemical reaction networks that describe the association of cohesin with its regulators and with the chromatin fiber. Remarkably, experimental observations are consistent with only a single biochemical reaction cycle, which results in a unique minimal model that may be fully parameterized by quantitative protein measurements. We demonstrate how distinct roles for cohesin regulators emerge simply from the structure of the reaction network, and how their dynamic exchange can regulate loop extrusion kinetics over time-scales that far exceed their own chromatin residence times. By embedding our cohesin biochemical reaction network within biophysical chromatin simulations, we evidence how variations in regulatory protein abundance can alter chromatin architecture across multiple length- and time-scales. Predictions from our model are corroborated by biophysical and biochemical assays, optical microscopy observations, and Hi-C conformation capture techniques. More broadly, our theoretical and numerical framework bridges the gap between in vitro observations of extrusion motor dynamics at the molecular scale and their structural consequences at the genome-wide level.

LDB1 establishes multi-enhancer networks to regulate gene expression.

How specific enhancer-promoter pairing is established is still mostly unclear. Besides the CTCF/cohesin machinery, only a few nuclear factors have been studied for a direct role in physically connecting regulatory elements. Here, we show via acute degradation experiments that LDB1 directly and broadly promotes enhancer-promoter loops. Most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin, or YY1 as determined via the use of multiple degron systems. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1 occupied sites but may aid the formation of a subset of LDB1 anchored loops. Leveraging the dynamic reorganization of nuclear architecture during the transition from mitosis to G1-phase, we establish a relationship between LDB1-dependent interactions in the context of TAD organization and gene activation. Lastly, Tri-C and Region Capture Micro-C reveal that LDB1 organizes multi-enhancer networks to activate transcription. This establishes LDB1 as a direct driver of regulatory network inter-connectivity.

Effect of active loop extrusion on the two-contact correlations in the interphase chromosome.

The population-averaged contact maps generated by the chromosome conformation capture technique provide important information about the average frequency of contact between pairs of chromatin loci as a function of the genetic distance between them. However, these datasets do not tell us anything about the joint statistics of simultaneous contacts between genomic loci in individual cells. This kind of statistical information can be extracted using the single-cell Hi-C method, which is capable of detecting a large fraction of simultaneous contacts within a single cell, as well as through modern methods of fluorescent labeling and super-resolution imaging. Motivated by the prospect of the imminent availability of relevant experimental data, in this work, we theoretically model the joint statistics of pairs of contacts located along a line perpendicular to the main diagonal of the single-cell contact map. The analysis is performed within the framework of an ideal polymer model with quenched disorder of random loops, which, as previous studies have shown, allows us to take into account the influence of the loop extrusion process on the conformational properties of interphase chromatin.

An extrinsic motor directs chromatin loop formation by cohesin.

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation.

Fluidification of entangled polymers by loop extrusion

Loop extrusion is one of the main processes shaping chromosome organization across the cell cycle, yet its role in regulating deoxyribonucleic acid (DNA) entanglement and nucleoplasm viscoelasticity remains overlooked. We simulate entangled solutions of linear polymers under the action of generic loop extruding factors (LEFs) with a model that fully accounts for topological constraints and LEF-DNA uncrossability. We discover that extrusion drives the formation of bottlebrushlike structures which significantly lower the entanglement and effective viscosity of the system through an active fluidification mechanism. Interestingly, this fluidification displays an optimum at one LEF every 300–3000 base pairs. In marked contrast with entangled linear chains, the viscosity of extruded chains scales linearly with polymer length, yielding up to 1000-fold fluidification in our system. Our results illuminate how intrachain loop extrusion contributes to actively modulate genome entanglement and viscoelasticity . Published by the American Physical Society 2024