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In mammalian polymorphonuclear cells, polymorphonuclear structures are assembled by loop extrusion. Mega-loop anchors and interchromosomal hubs are stabilized by nuclear condensates. Diminished loop extrusion might alter nuclear deformability. Loop extrusion programs may instruct cellular architecture. It might be feasible to engineer artificial nuclear shapes and altered nuclear deformability to manipulate immune effector cell migration. Since the cardinal discovery by Metchnikoff that neutrophils assemble into polymorphonuclear structures, insights into the mechanisms that underpin the formation of such structures have remained rudimentary. Recent studies have demonstrated that in early mammalian hematopoiesis, mononuclear versus polymorphonuclear cell fate decisions are orchestrated by intricate competitive physical forces that involve loop extrusion and phase separation programs. Changing loop extrusion programs might enable the engineering of new nuclear shapes and artificial cytoplasmic architectures in effector immune cells and beyond. Immune cell fate decisions are regulated, at least in part, by nuclear architecture. Here, we outline how nuclear architecture instructs mammalian polymorphonuclear cell differentiation. We discuss how in neutrophils loop extrusion mechanisms regulate the expression of genes involved in phagocytosis and shape nuclear morphology. We propose that diminished loop extrusion programs also orchestrate eosinophil and basophil differentiation. We portray a new model in which competitive physical forces, loop extrusion, and phase separation, instruct mononuclear versus polymorphonuclear cell fate decisions. We posit that loop extrusion programs instruct the spatial organization of cytoplasmic organelles, including neutrophil granules, mitochondria, and endoplasmic reticulum. Finally, we suggest that changing loop extrusion programs might allow the engineering of new nuclear shapes and artificial cytoplasmic architectures. [ABSTRACT FROM AUTHOR]

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. Synopsis: The loop extrusion hypothesis has shaped current thinking about chromatin organization by SMC complexes. This article experimentally tests the hypothesis, with a negative outcome, and puts forward transcription as an alternative, external motor force promoting chromatin loop formation. Yeast cohesin variants unable to perform in vitro DNA loop extrusion readily form chromatin loops in vivo. Transcription occupies key roles in chromatin loop formation and growth. In a unified model, cohesin sequentially captures two DNAs to establish sister chromatid cohesion at DNA replication forks and chromatin loops at places of transcription. Cohesin variants defective in DNA loop extrusion in vitro can still form chromatin loops in living yeast cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

During the asymmetric loop extrusion of DNA by a condensin complex, one domain of the complex stably anchors to the DNA molecule, and another domain reels in the DNA strand into a loop. The DNA strand in the loop is fully relaxed, or there is no tension in the loop. Just outside of the loop, there is a tension that resists the extrusion of DNA. To maintain the extrusion of the DNA loop, the condensin complex must have a domain capable of generating a force to overcome the tension outside of the loop. This study proposes that the groove-shaped HEAT repeat domain Ycg1 plays the role of a molecular motor. A DNA molecule may bind to the groove electrostatically, and the weak binding force facilitates the random thermal motion of DNA molecules. A mechanical model that random collisions between DNA and the nonparallel inner surfaces of the groove may generate a directional force which is required for the loop extrusion to sustain. The hinge domain binds to the DNA molecule and acts as an anchor during asymmetric DNA loop extrusion. When the effects of ATP hydrolysis and the viscous drag of the fluid environment are considered, the motor–anchor model for the condensin complex and the mechanical model might explain the asymmetric loop extrusion, the formation of steps, the step size distribution in the loop extrusion, the tension-dependent extrusion speed, the interaction between coexisting loops on the DNA strand, and untying the knots during extrusion. This model can also explain the observed formation of the Z-loop. [ABSTRACT FROM AUTHOR]

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension D = 4 is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times (Δ t) longer than tens of minutes to be proportional to Δ t 1/3. We validate our results with hybrid molecular dynamics--Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation. [ABSTRACT FROM AUTHOR]

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. 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 formation of DNA loops. This specific process has been called loop extrusion. Extrusion underlies multiple functions of cohesin beyond cohesion, but molecular mechanism of the process remains a mystery. In this review, we summarized the data on molecular architecture of cohesin, effect of ATP hydrolysis cycle on this architecture, and 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 the not-so-distant future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

SMC (Structural maintenance of chromosomes) complexes are key participants in the spatial organization of DNA in all living organisms: in bacteria, archaea and eukaryotes. In bacteria, there are several homologues of SMC complexes that perform seemingly unrelated functions, but function through very similar, highly conserved mechanisms. In recent years, it has been established that SMC complexes are capable of forming loops from DNA (through the so-called loop extrusion), which allows them to be considered as a separate class of DNA translocases. This paper discusses bacterial SMC complexes in comparison with their homologues such as MukBEF, MksBEF, RecN, and Wadjet, as well as with eukaryotic SMC complexes. Their properties, role and functions in the key processes of the bacterial cell are discussed. [ABSTRACT FROM AUTHOR]

The connection between the patterns observed in 3C-type experiments and the modeling of polymers remains unresolved. This paper presents a simulation pipeline that generates thermodynamic ensembles of 3D structures for topologically associated domain (TAD) regions by loop extrusion model (LEM). The simulations consist of two main components: a stochastic simulation phase, employing a Monte Carlo approach to simulate the binding positions of cohesins, and a dynamical simulation phase, utilizing these cohesins' positions to create 3D structures. In this approach, the system's total energy is the combined result of the Monte Carlo energy and the molecular simulation energy, which are iteratively updated. The structural maintenance of chromosomes (SMC) protein complexes are represented as loop extruders, while the CCCTC-binding factor (CTCF) locations on DNA sequence are modeled as energy minima on the Monte Carlo energy landscape. Finally, the spatial distances between DNA segments from ChIA-PET experiments are compared with the computer simulations, and we observe significant Pearson correlations between predictions and the real data. LoopSage model offers a fresh perspective on chromatin loop dynamics, allowing us to observe phase transition between sparse and condensed states in chromatin. • We improved the already-existing LEM (loop extrusion model) algorithm for the prediction of 3D structure of chromatin. • We present a simulation pipeline that generates thermodynamic ensembles of 3D structures for topologically associated domain (TAD) regions. • The CCCTC-binding factor (CTCF) locations on DNA sequence are modeled as energy minima on the Monte Carlo energy landscape. • The spatial distances between DNA segments from ChIA-PET experiments show high correlation with the averaged simulated distances. • LoopSage allows us to observe phase transition between sparse and condensed states in chromatin. [ABSTRACT FROM AUTHOR]

The establishment, maintenance and dynamic regulation of three-dimensional (3D) chromatin structures provide an important means for partitioning of genome into functionally distinctive domains, which helps to define specialized gene expression programs associated with developmental stages and cell types. Increasing evidence supports critical roles for intrinsically disordered regions (IDRs) harbored within transcription factors (TFs) and chromatin-modulatory proteins in inducing phase separation, a phenomenon of forming membrane-less condensates through partitioning of biomolecules. Such a process is also critically involved in the establishment of high-order chromatin structures and looping. IDR- and phase separation-driven 3D genome (re)organization often goes wrong in disease such as cancer. This review discusses about recent advances in understanding how phase separation of intrinsically disordered proteins (IDPs) modulates chromatin looping and gene expression. [ABSTRACT FROM AUTHOR]

The 2 m-long human DNA is tightly intertwined into the cell nucleus of the size of 10 μm. The DNA packing is explained by folding of chromatin fiber. This folding leads to the formation of such hierarchical structures as: chromosomal territories, compartments; densely-packed genomic regions known as Topologically Associating Domains (TADs), or Chromatin Contact Domains (CCDs), and loops. We propose models of dynamical human genome folding into hierarchical components in human lymphoblastoid, stem cell, and fibroblast cell lines. Our models are based on explosive percolation theory. The chromosomes are modeled as graphs where CTCF chromatin loops are represented as edges. The folding trajectory is simulated by gradually introducing loops to the graph following various edge addition strategies that are based on topological network properties, chromatin loop frequencies, compartmentalization, or epigenomic features. Finally, we propose the genome folding model - a biophysical pseudo-time process guided by a single scalar order parameter. The parameter is calculated by Linear Discriminant Analysis of chromatin features. We also include dynamics of loop formation by using Loop Extrusion Model (LEM) while adding them to the system. The chromatin phase separation, where fiber folds in 3D space into topological domains and compartments, is observed when the critical number of contacts is reached. We also observe that at least 80% of the loops are needed for chromatin fiber to condense in 3D space, and this is constant through various cell lines. Overall, our in-silico model integrates the high-throughput 3D genome interaction experimental data with the novel theoretical concept of phase separation, which allows us to model event-based time dynamics of chromatin loop formation and folding trajectories.

Nuclei are central hubs for information processing in eukaryotic cells. The need to fit large genomes into small nuclei imposes severe restrictions on genome organization and the mechanisms that drive genome-wide regulatory processes. How a disordered polymer such as chromatin, which has vast heterogeneity in its DNA and histone modification profiles, folds into discernibly consistent patterns is a fundamental question in biology. Outstanding questions include how genomes are spatially and temporally organized to regulate cellular processes with high precision and whether genome organization is causally linked to transcription regulation. The advent of next-generation sequencing, super-resolution imaging, multiplexed fluorescent in situ hybridization, and single-molecule imaging in individual living cells has caused a resurgence in efforts to understand the spatiotemporal organization of the genome. In this review, we discuss structural and mechanistic properties of genome organization at different length scales and examine changes in higher-order chromatin organization during important developmental transitions.

Cohesin organizes genome topology thanks to its ability to extrude DNA loops and to hold together the sister chromatids. Genome folding and cohesion establishment by cohesin depend on NIPBL, but its requirement for chromatin association of cohesin is unclear. The interactions of NIPBL with chromatin remodelers, replication proteins, and transcriptional regulators have important implications for cohesin distribution and function. Cohesin complexes carrying Stromal Antigen 1 or 2 (STAG1 or STAG2) have different chromatin association dynamics and respond differently to low NIPBL levels. Altered gene expression in patients with Cornelia de Lange Syndrome with NIPBL mutations is likely the consequence of reduced loop extrusion. Cohesin folds the genome in dynamic chromatin loops and holds the sister chromatids together. NIPBLScc2 is currently considered the cohesin loader, a role that may need reevaluation. NIPBL activates the cohesin ATPase, which is required for topological entrapment of sister DNAs and to fuel DNA loop extrusion, but is not required for chromatin association. Mechanistic dissection of these processes suggests that both NIPBL and the cohesin STAG subunit bind DNA. NIPBL also regulates conformational switches of the complex. Interactions of NIPBL with chromatin factors, including remodelers, replication proteins, and the transcriptional machinery, affect cohesin loading and distribution. Here, we discuss recent research addressing how NIPBL modulates cohesin activities and how its mutation causes a developmental disorder, Cornelia de Lange Syndrome (CdLS). [ABSTRACT FROM AUTHOR]

BackgroundUbiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription.ResultsWe demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation.ConclusionThis study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

The active loop extrusion hypothesis proposes that chromatin threads through the cohesin protein complex into progressively larger loops until reaching specific boundary elements. We build upon this hypothesis and develop an analytical theory for active loop extrusion which predicts that loop formation probability is a nonmonotonic function of loop length and describes chromatin contact probabilities. We validate our model with Monte Carlo and hybrid Molecular Dynamics-Monte Carlo simulations and demonstrate that our theory recapitulates experimental chromatin conformation capture data. Our results support active loop extrusion as a mechanism for chromatin organization and provide an analytical description of chromatin organization that may be used to specifically modify chromatin contact probabilities. [ABSTRACT FROM AUTHOR]

Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by an inducible gene. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers capture mobilized cohesin demonstrates that transcription-driven complexes translocate processively in vivo. Together, this study reveals unexplored limitations to cohesin movement on chromosomes. [ABSTRACT FROM AUTHOR]

Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.

Abstract DNA loop extrusion emerges as a key process establishing genome structure and function. We introduce MoDLE, a computational tool for fast, stochastic modeling of molecular contacts from DNA loop extrusion capable of simulating realistic contact patterns genome wide in a few minutes. MoDLE accurately simulates contact maps in concordance with existing molecular dynamics approaches and with Micro-C data and does so orders of magnitude faster than existing approaches. MoDLE runs efficiently on machines ranging from laptops to high performance computing clusters and opens up for exploratory and predictive modeling of 3D genome structure in a wide range of settings.

The cohesin complex has a range of crucial functions in the cell. Cohesin is essential for mediating chromatid cohesion during mitosis, for repair of double‐strand DNA breaks, and for control of gene transcription. This last function has been the subject of intense research ever since the discovery of cohesin's role in the long‐range regulation of the cut gene in Drosophila. Subsequent research showed that the expression of some genes is exquisitely sensitive to cohesin depletion, while others remain relatively unperturbed. Sensitivity to cohesin depletion is also remarkably cell type‐ and/or condition‐specific. The relatively recent discovery that cohesin is integral to forming chromatin loops via loop extrusion should explain much of cohesin's gene regulatory properties, but surprisingly, loop extrusion has failed to identify a 'one size fits all' mechanism for how cohesin controls gene expression. This review will illustrate how early examples of cohesin‐dependent gene expression integrate with later work on cohesin's role in genome organization to explain mechanisms by which cohesin regulates gene expression. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosomes (SMC) complexes are essential proteins found in genomes of all cellular organisms. Essential functions of these proteins, such as mitotic chromosome formation and sister chromatid cohesion, were discovered a long time ago. Recent advances in chromatin biology showed that SMC proteins are involved in many other genomic processes, acting as active motors extruding DNA, which leads to the formation of chromatin loops. Some loops formed by SMC proteins are highly cell type and developmental stage specific, such as SMC-mediated DNA loops required for VDJ recombination in B-cell progenitors, or dosage compensation in Caenorhabditis elegans and X-chromosome inactivation in mice. In this review, we focus on the extrusion-based mechanisms that are common for multiple cell types and species. We will first describe an anatomy of SMC complexes and their accessory proteins. Next, we provide biochemical details of the extrusion process. We follow this by the sections describing the role of SMC complexes in gene regulation, DNA repair, and chromatin topology. [ABSTRACT FROM AUTHOR]

Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

Mammalian genomes are folded into spatial domains, which regulate gene expression by modulating enhancer-promoter contacts. Here, we review recent studies on the structure and function of Topologically Associating Domains (TADs) and chromatin loops. We discuss how loop extrusion models can explain TAD formation and evidence that TADs are formed by the ring-shaped protein complex, cohesin, and that TAD boundaries are established by the DNA-binding protein, CTCF. We discuss our recent genomic, biochemical and single-molecule imaging studies on CTCF and cohesin, which suggest that TADs and chromatin loops are dynamic structures. We highlight complementary polymer simulation studies and Hi-C studies employing acute depletion of CTCF and cohesin, which also support such a dynamic model. We discuss the limitations of each approach and conclude that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle.

In the human cell nucleus, dynamically organized chromatin is the substrate for gene regulation, DNA replication, and repair. A central mechanism of DNA loop formation is an ATPase motor cohesin-mediated loop extrusion. The cohesin complexes load and unload onto the chromosome under the control of other regulators that physically interact and affect motor activity. Regulation of the dynamic loading cycle of cohesin influences not only the chromatin structure but also genome-associated human disorders and aging. This review focuses on the recently spotlighted genome organizing factors and the mechanism by which their dynamic interactions shape the genome architecture in interphase. [ABSTRACT FROM AUTHOR]

Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP‐dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP‐mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of −0.4. This ∆Lk increases to −0.8 during ATP binding and resets to −0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin‐DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a "pinch and merge" mechanism implies that two DNA‐binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop. Synopsis How the structural maintenance of chromosomes (SMC) complex condensin extrudes DNA loops via ATPase activity remains unclear. Results showing that yeast condensin activity produces cyclic alterations in the topology of its interacting DNA suggest how it generates DNA translocation steps to achieve loop extrusion. Condensin activity restrains negative DNA supercoils upon ATP usage.The Ycg1 subunit is not required to restrain negative DNA supercoils.The ATP‐bound condensin pinches a short left‐handed loop of DNA.The transiently constrained DNA loop is not the extruding loop.A "pinch and merge" mechanism of DNA loop extrusion is proposed. [ABSTRACT FROM AUTHOR]

Each time a cell divides, it needs to duplicate the genome and then separate the two copies. In eukaryotes, which usually have more than one linear chromosome, this entails tethering the two newly replicated DNA molecules, a phenomenon known as sister chromatid cohesion (SCC). Cohesion ensures proper chromosome segregation to separate poles during mitosis. SCC is achieved by the presence of the cohesin complex. Besides its canonical function, cohesin is essential for chromosome organization and DNA damage repair. Surprisingly, yeast cohesin is loaded in G1 before DNA replication starts but only acquires its binding activity during DNA replication. Work in microorganisms, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe has greatly contributed to the understanding of cohesin composition and functions. In the last few years, much progress has been made in elucidating the role of cohesin in chromosome organization and compaction. Here, we discuss the different functions of cohesin to ensure faithful chromosome segregation and genome stability during the mitotic cell division in yeast. We describe what is known about its composition and how DNA replication is coupled with SCC establishment. We also discuss current models for the role of cohesin in chromatin loop extrusion and delineate unanswered questions about the activity of this important, conserved complex. [ABSTRACT FROM AUTHOR]

The compaction of linear DNA into micrometer-sized nuclear boundaries involves the establishment of specific three-dimensional (3D) DNA structures complexed with histone proteins that form chromatin. The resulting structures modulate essential nuclear processes such as transcription, replication, and repair to facilitate or impede their multi-step progression and these contribute to dynamic modification of the 3D-genome organization. It is generally accepted that protein–protein and protein–DNA interactions form the basis of 3D-genome organization. However, the constant generation of mechanical forces, torques, and other stresses produced by various proteins translocating along DNA could be playing a larger role in genome organization than currently appreciated. Clearly, a thorough understanding of the mechanical determinants imposed by DNA transactions on the 3D organization of the genome is required. We provide here an overview of our current knowledge and highlight the importance of DNA and chromatin mechanics in gene expression. [ABSTRACT FROM AUTHOR]

DNA loop extrusion plays a key role in the regulation of gene expression and the structural arrangement of chromatin. Most existing mechanistic models of loop extrusion depend on some type of ratchet mechanism, which should permit the elongation of loops while preventing their collapse, by enabling DNA to move in only one direction. STAG2 is already known to exert a role as DNA anchor, but the available structural data suggest a possible role in unidirectional DNA motion. In this work, a computational simulation framework was constructed to evaluate whether STAG2 could enforce such unidirectional displacement of a DNA double helix. The results reveal that STAG2 V-shape allows DNA sliding in one direction, but blocks opposite DNA movement via a linear ratchet mechanism. Furthermore, these results suggest that RAD21 binding to STAG2 controls its flexibility by narrowing the opening of its V-shape, which otherwise remains widely open in absence of RAD21. Therefore, in the proposed model, in addition to its already described role as a DNA anchor, the STAG2-RAD21 complex would be part of a ratchet mechanism capable of exerting directional selectivity on DNA sliding during loop extrusion. The identification of the molecular basis of the ratchet mechanism of loop extrusion is a critical step in unraveling new insights into a broad spectrum of chromatin activities and their implications for the mechanisms of chromatin-related diseases. • STAG2 could exert a molecular ratchet role in DNA loop extrusion. • This ratchet mechanism would allow DNA loop extension by unidirectional DNA sliding. • RAD21 binding stabilizes STAG2 in a conformation compatible with the ratchet role. [ABSTRACT FROM AUTHOR]

Cohesin regulates sister chromatid cohesion but also contributes to chromosome folding by promoting the formation of chromatin loops, a process mediated by loop extrusion. Although PDS5 regulates cohesin dynamics on chromatin, the exact function of PDS5 in cohesin‐mediated chromatin looping remains unclear. Two paralogs of PDS5 exist in vertebrates, PDS5A and PDS5B. Here we show that PDS5A and PDS5B co‐localize with RAD21 and CTCF at loop anchors. Rapid PDS5A or PDS5B degradation in liver cancer cells using an inducible degron system reduces chromatin loops and increases loop size. RAD21 enrichment at loop anchors is decreased upon depletion of PDS5A or PDS5B. PDS5B loss also reduces CTCF signals at loop anchors and has a stronger effect on loop enlargement compared with PDS5A. Co‐depletion of PDS5A and PDS5B reduces RAD21 levels at loop anchors although the amount of cohesin on chromatin is increased. Our study provides insight into how PDS5 proteins regulate cohesin‐mediated chromatin looping. Synopsis: PDS5 proteins stabilize cohesin at loop anchors, facilitate chromatin loop formation and restrict loop expansion in mammals.PDS5 proteins stabilize cohesin‐mediated chromatin loops.PDS5 proteins limit loop size.PDS5 proteins and CTCF together define loop anchors. [ABSTRACT FROM AUTHOR]

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB−/-). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E-), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr−/+, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr−/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB−/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E-and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr−/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr−/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements.

Condensins are molecular motors that compact DNA via linear translocation. In Caenorhabditis elegans, the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of recruitment elements on the X-chromosome (rex) and is required for the formation of topologically associating domains (TADs). We take advantage of autosomes that are largely devoid of condensin DC and TADs to address how rex sites and condensin DC give rise to the formation of TADs. When an autosome and X-chromosome are physically fused, despite the spreading of condensin DC into the autosome, no TAD was created. Insertion of a strong rex on the X-chromosome results in the TAD boundary formation regardless of sequence orientation. When the same rex is inserted on an autosome, despite condensin DC recruitment, there was no spreading or features of a TAD. On the other hand, when a ‘super rex’ composed of six rex sites or three separate rex sites are inserted on an autosome, recruitment and spreading of condensin DC led to the formation of TADs. Therefore, recruitment to and spreading from rex sites are necessary and sufficient for recapitulating loop-anchored TADs observed on the X-chromosome. Together our data suggest a model in which rex sites are both loading sites and bidirectional barriers for condensin DC, a one-sided loop-extruder with movable inactive anchor.

Cohesin, a structural maintenance of chromosome (SMC) complex, organizes chromatin into three-dimensional structures by threading chromatin into loops and stabilizing long-range chromatin interactions. Four subunits in a 1:1:1:1 ratio compose the cohesin core, which is regulated by auxiliary factors that interact with or modify the core subunits. An ongoing debate about cohesin’s mechanism of action regards its stoichiometry. Namely, is cohesin activity mediated by a single complex or cooperation between several complexes that organize into dimers or oligomers? Several investigations that used various experimental approaches have tried to resolve this dispute. Some have convincingly demonstrated that the cohesin monomer is the active unit. However, others have revealed the formation of cohesin dimers and higher-order clusters on and off chromosomes. Elucidating the biological function of cohesin clusters and determining what regulates their formation are just two of the many new questions raised by these findings. We briefly review the history of the argument about cohesin stoichiometry and the central evidence for cohesin activity as a monomer vs. an oligomer. Finally, we discuss the possible biological significance of cohesin oligomerization and present open questions that remain to be answered.

Abstract Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings.

It is now established that Bcl11b specifies T cell fate. Here, we show that in developing T cells, the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (thymocyte differentiation factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single-loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.

Cohesin folds chromosomes via DNA loop extrusion. Cohesin-mediated chromosome loops regulate transcription by shaping long-range enhancer–promoter interactions, among other mechanisms. Mutations of cohesin subunits and regulators cause human developmental diseases termed cohesinopathy. Vertebrate cohesin consists of SMC1, SMC3, RAD21, and either STAG1 or STAG2. To probe the physiological functions of cohesin, we created conditional knockout (cKO) mice with Stag2 deleted in the nervous system. Stag2 cKO mice exhibit growth retardation, neurological defects, and premature death, in part due to insufficient myelination of nerve fibers. Stag2 cKO oligodendrocytes exhibit delayed maturation and downregulation of myelination-related genes. Stag2 loss reduces promoter-anchored loops at downregulated genes in oligodendrocytes. Thus, STAG2-cohesin generates promoter-anchored loops at myelination-promoting genes to facilitate their transcription. Our study implicates defective myelination as a contributing factor to cohesinopathy and establishes oligodendrocytes as a relevant cell type to explore the mechanisms by which cohesin regulates transcription.

Structural maintenance of chromosomes (SMC) complexes are essential proteins found in genomes of all cellular organisms. Essential functions of these proteins, such as mitotic chromosome formation and sister chromatid cohesion, were discovered a long time ago. Recent advances in chromatin biology showed that SMC proteins are involved in many other genomic processes, acting as active motors extruding DNA, which leads to the formation of chromatin loops. Some loops formed by SMC proteins are highly cell type and developmental stage specific, such as SMC-mediated DNA loops required for VDJ recombination in B-cell progenitors, or dosage compensation in Caenorhabditis elegans and X-chromosome inactivation in mice. In this review, we focus on the extrusion-based mechanisms that are common for multiple cell types and species. We will first describe an anatomy of SMC complexes and their accessory proteins. Next, we provide biochemical details of the extrusion process. We follow this by the sections describing the role of SMC complexes in gene regulation, DNA repair, and chromatin topology.

SMC protein complexes are molecular machines that provide structure to chromosomes. These complexes bridge DNA elements and by doing so build DNA loops in cis and hold together the sister chromatids in trans. We discuss how drastic conformational changes allow SMC complexes to build such intricate DNA structures. The tight regulation of these complexes controls fundamental chromosomal processes such as transcription, recombination, repair, and mitosis. SMC complexes such as cohesin and condensin shape the DNA of all life on earth. Oldenkamp et al. consider general principles across SMC protein complexes. A chain of conformational states enables this family of molecular machines to structure DNAs and control fundamental chromosomal processes. [ABSTRACT FROM AUTHOR]

Condensin is a structural maintenance of chromosomes (SMC) complex needed for the compaction of DNA into chromatids during mitosis. Lengthwise DNA compaction by condensin is facilitated by ATPase-driven loop extrusion, a process that is believed to be the fundamental activity of most, if not all, SMC complexes. In order to obtain molecular insights, we obtained electron cryomicroscopy structures of yeast condensin in the presence of a slowly hydrolyzable ATP analog and linear as well as circular DNAs. The DNAs were shown to be "clamped" between the engaged heterodimeric SMC ATPase heads and the Ycs4 subunit, in a manner similar to previously reported DNA-bound SMC complex structures. Ycg1, the other non-SMC subunit, was only flexibly bound to the complex, while also binding DNA tightly and often remaining at a distance from the head module. In the clamped state, the DNA is encircled by the kleisin Brn1 and the two engaged head domains of Smc2 and Smc4. The Brn1/Smc2/Smc4 tripartite ring is closed at all interfaces, including at the neck of Smc2. We show that the neck gate opens upon head engagement in the absence of DNA, but it remains shut when DNA is present. Our work demonstrates that condensin and other SMC complexes go through similar conformations of the head modules during their ATPase cycle. In contrast, the behavior of the Ycg1 subunit in the condensin complex might indicate differences in the implementation of the extrusion reactions, and our findings will constrain further mechanistic models of loop extrusion by SMC complexes. [ABSTRACT FROM AUTHOR]

The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The DNA‐passage activity of topoisomerase II accidentally produces DNA knots and interlinks within and between chromatin fibers. Fortunately, these unwanted DNA entanglements are actively removed by some mechanism. Here we present an outline on DNA knot formation and discuss recent studies that have investigated how intracellular DNA knots are removed. First, although topoisomerase II is able to minimize DNA entanglements in vitro to below equilibrium values, it is unclear whether such capacity performs equally in vivo in chromatinized DNA. Second, DNA supercoiling could bias topoisomerase II to untangle the DNA. However, experimental evidence indicates that transcriptional supercoiling of intracellular DNA boosts knot formation. Last, cohesin and condensin could tighten DNA entanglements via DNA loop extrusion (LE) and force their dissolution by topoisomerase II. Recent observations indicate that condensin activity promotes the removal of DNA knots during interphase and mitosis. This activity might facilitate the spatial organization and dynamics of chromatin. [ABSTRACT FROM AUTHOR]

For decades, biochemical methods for the analysis of genome structure and function provided cell-population-averaged data that allowed general principles and tendencies to be disclosed. Microscopy-based studies, which immanently involve single-cell analysis, did not provide sufficient spatial resolution to investigate the particularly small details of 3D genome folding. Nevertheless, these studies demonstrated that mutual positions of chromosome territories within cell nuclei and individual genomic loci within chromosomal territories can vary significantly in individual cells. The development of new technologies in biochemistry and the advent of super-resolution microscopy in the last decade have made possible the full-scale study of 3D genome organization in individual cells. Maps of the 3D genome build based on C-data and super-resolution microscopy are highly consistent and, therefore, biologically relevant. The internal structures of individual chromosomes, loci, and topologically associating domains (TADs) are resolved as well as cell-cycle dynamics. 3D modeling allows one to investigate the physical mechanisms underlying genome folding. Finally, joint profiling of genome topology and epigenetic features will allow 3D genomics to handle complex cell-to-cell heterogeneity. In this review, we summarize the present state of studies into 3D genome organization in individual cells, analyze the technical problems of single-cell studies, and outline perspectives of 3D genomics. • C-based technologies and super-resolution microscopy provide consistent and complementary data on the individual 3D genomes. • Sparse single-cell Hi-C maps require sophisticated technical controls prior to the analysis of biological features. • 3D genome displays cell-to-cell variability generated by stochastic fluctuations and dynamic specific interactions. • Joint profiling of epigenome and 3D genome in the same cell and live-cell imaging are perspective trends the in 3D genomics. [ABSTRACT FROM AUTHOR]

Abstract Background Topologically associating domains (TADs) are genomic regions of self-interaction. Additionally, it is known that TAD boundaries are enriched in CTCF binding sites. In turn, CTCF sites are known to be asymmetric, whereby the convergent configuration of a pair of CTCF sites leads to the formation of a chromatin loop in vivo. However, to date, it has been unclear how to reconcile TAD structure with CTCF-based chromatin loops. Results We approach this problem by analysing CTCF binding site strengths and classifying clusters of CTCF sites along the genome on the basis of their relative orientation. Analysis of CTCF site orientation classes as a function of their spatial distribution along the human genome reveals that convergent CTCF site clusters are depleted while divergent CTCF clusters are enriched in the 5- to 100-kb range. We then analyse the distribution of CTCF binding sites as a function of TAD boundary conservation across seven primary human blood cell types. This reveals divergent CTCF site enrichment at TAD boundaries. Furthermore, convergent arrays of CTCF sites separate the left and right sections of TADs that harbour internal CTCF sites, resulting in unequal TAD ‘halves’. Conclusions The orientation-based CTCF binding site cluster classification that we present reconciles TAD boundaries and CTCF site clusters in a mechanistically elegant fashion. This model suggests that the emergent structure of nuclear chromatin in the form of TADs relies on the obligate alternation of divergent and convergent CTCF site clusters that occur at different length scales along the genome. Graphical abstract

Abstract Background Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. Results We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation. Conclusion This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

Abstract Background CTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem. Results Using CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin (Pcdh) and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain (Igh) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin “loop extrusion” model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice. Conclusions These findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.

Mammalian genome structure is closely linked to function. At the scale of kilobases to megabases, CTCF and cohesin organize the genome into chromatin loops. Mechanistically, cohesin is proposed to extrude chromatin loops bidirectionally until it encounters occupied CTCF DNA-binding sites. Curiously, loops form predominantly between CTCF binding sites in a convergent orientation. How CTCF interacts with and blocks cohesin extrusion in an orientation-specific manner has remained a mechanistic mystery. Here, we review recent papers that have shed light on these processes and suggest a multi-step interaction between CTCF and cohesin. This interaction may first involve a pausing step, where CTCF halts cohesin extrusion, followed by a stabilization step of the CTCF-cohesin complex, resulting in a chromatin loop. Finally, we discuss our own recent studies on an internal RNA-Binding Region (RBRi) in CTCF to elucidate its role in regulating CTCF clustering, target search mechanisms and chromatin loop formation and future challenges.

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB−/-). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E-), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr−/+, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr−/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB−/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E-and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr−/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr−/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements. [ABSTRACT FROM AUTHOR]

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6. SYNOPSIS: The Smc5/6 complex prevents accumulation of toxic DNA junctions during DNA replication and repair, in order to enable faithful chromosome segregation in mitosis and meiosis. Biochemical reconstitution of the yeast holo‐complex reveals a central role of the Nse5/6 sub‐complex in DNA substrate selection. Nse5/6 promotes ATP‐dependent, salt‐stable DNA association of Smc5/6.Nse5/6 inhibits the Smc5/6 ATPase by preventing productive ATP binding in the absence of DNA.Nse5/6 contacts the Smc5/6 hexamer via multiple interfaces including one at the SMC joint and one at the heads.Lysine cross‐linking (XL‐MS) and cysteine cross‐linking uncover major conformational changes upon Nse5/6 association.A crystal structure of the Nse5/6 core shows a HEAT‐repeat‐like organization. [ABSTRACT FROM AUTHOR]

Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings. [ABSTRACT FROM AUTHOR]

Cohesin and CTCF are key to the 3D folding of interphase chromosomes. Cohesin forms chromatin loops via loop extrusion, a process that involves the formation and enlargement of DNA loops. The architectural protein CTCF controls this process by acting as an anchor for chromatin looping. How CTCF controls cohesin has long been a mystery. Recent work shows that CTCF dictates chromatin looping via a direct interaction of its N-terminus with cohesin. CTCF's ability to regulate chromatin looping turns out to also be partially dependent on several RNA-binding domains. In this review, we discuss recent insights and consider how cohesin and CTCF together may orchestrate the folding of the genome into chromosomal loops. • Cohesin builds DNA loops to shape the interphase genome. • CTCF determines the loci where cohesin forms loops. • CTCF's N-terminus binds cohesin to enable the formation of CTCF-anchored loops. • CTCF also regulates chromatin looping via its RNA-binding domains. • CTCF might in addition regulate a looping-independent function of cohesin. [ABSTRACT FROM AUTHOR]

'Structural maintenance of chromosomes' (SMC) complexes are required for the folding of genomic DNA into loops. Theoretical considerations and single-molecule experiments performed with the SMC complexes cohesin and condensin indicate that DNA folding occurs via loop extrusion. Recent work indicates that this process is essential for the assembly of antigen receptor genes by V(D)J recombination in developing B and T cells of the vertebrate immune system. Here, I review how recent studies of the mouse immunoglobulin heavy chain locus Igh have provided evidence for this hypothesis and how the formation of chromatin loops by cohesin and regulation of this process by CTCF and Wapl might ensure that all variable gene segments in this locus (V H segments) participate in recombination with a re-arranged DJ H segment, to ensure generation of a maximally diverse repertoire of B-cell receptors and antibodies. [ABSTRACT FROM AUTHOR]