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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]

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]

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 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]

Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor β (TGF-β) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-β signaling in promoting RUNX3 expression for efficient IgA CSR. [Display omitted] • USP7 promotes IgA and inhibits IgG switching independent of its function in DDR • USP7 stabilizes RUNX3 through deubiquitination to promote Sα GLT and IgA CSR • Sα GLT deficiency can upregulate upstream Sγ GLT and CSR • TGF-β promotes Sα GLT via RUNX3 and reverses elevated Sγ GLT and CSR caused by USP7 deficiency Zhao et al. show that USP7 and TGF-β maintain high levels of Sα germline transcription and IgA switching by promoting RUNX3 expression at the posttranslational and transcriptional levels, respectively, thereby inhibiting upstream Sγ germline transcription and CSR. [ABSTRACT FROM AUTHOR]

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin. [Display omitted] • Transcription by RNAPII plays no major role in the loading of fission yeast condensin • RNAPII stalls chromosomal condensin • High RNAPII occupancy is a barrier for the folding of chromatin by condensin • Transcription termination limits the hindrance of RNAPII on condensin Condensin shapes mitotic chromosomes by folding chromatin into loops, but the underlying mechanisms remain unclear. Lebreton et al. show that trains of RNAPII stall fission yeast condensin and that transcription termination underlies condensin-mediated long-range intrachromosomal contacts and accurate chromosome segregation, consistent with an assembly of mitotic chromosomes by DNA-loop extrusion. [ABSTRACT FROM AUTHOR]

Chromatin structure is critically involved in gene regulation and cell fate determination. How this structure is established and maintained in distinct, terminally differentiated cells remains elusive. Winick-Ng et al. address this puzzle by applying immunoGAM in different brain cell types and reveal cell type-specific chromatin topologies, long gene decompaction, and the involvement of transcription factors (TFs). [ABSTRACT FROM AUTHOR]

Chromosomes are organized as chromatin loops that promote segregation, enhancer-promoter interactions, and other genomic functions. Loops were hypothesized to form by 'loop extrusion,' by which structural maintenance of chromosomes (SMC) complexes, such as condensin and cohesin, bind to chromatin, reel it in, and extrude it as a loop. However, such exotic motor activity had never been observed. Following an explosion of indirect evidence, recent single-molecule experiments directly imaged DNA loop extrusion by condensin and cohesin in vitro. These experiments observe rapid (kb/s) extrusion that requires ATP hydrolysis and stalls under pN forces. Surprisingly, condensin extrudes loops asymmetrically, challenging previous models. Extrusion by cohesin is symmetric but requires the protein Nipbl. We discuss how SMC complexes may perform their functions on chromatin in vivo. [ABSTRACT FROM AUTHOR]

The loop extrusion hypothesis postulated that extrusion of DNA loops through cohesin rings organizes genomes. Recent findings suggest that cohesin itself is a molecular motor that extrudes DNA. This has important implications not only for the organization of interphase chromatin but also for other processes where cohesin plays vital roles. [ABSTRACT FROM AUTHOR]

What drives the formation of chromatin loops has been a long-standing question in chromosome biology. Recent work provides major insight into the basic principles behind loop formation. Structural maintenance of chromosomes (SMC) complexes, that are conserved from bacteria to humans, are key to this process. The SMC family includes condensin and cohesin, which structure chromosomes to enable mitosis and long-range gene regulation. We discuss novel insights into the mechanism of loop formation and the implications for how these complexes ultimately shape chromosomes. A picture is emerging in which these complexes form small loops that they then processively enlarge. It appears that SMC complexes act by family-wide basic principles, with complex-specific levels of control. [ABSTRACT FROM AUTHOR]

Mammalian chromosomes are organized at different length scales within the cell nucleus. Topologically Associating Domains (TADs) are structural units of 3D genome organization with functions in gene regulation, DNA replication, recombination and repair. Whereas TADs were initially interpreted as insulated domains, recent studies are revealing that these domains should be interpreted as dynamic collections of actively extruding loops. This process of loop extrusion is subsequently blocked at dedicated TAD boundaries, thereby promoting intra-domain interactions over their surroundings. In this review, we discuss how mammalian TAD structure can emerge from this dynamic process and we discuss recent evidence that TAD boundaries can have regulatory functions. • TADs act as regulatory neighborhoods for genome-associated processes. • TADs are dynamic entities containing a relatively low-density of extruded loops. • TADs are actively involved in long intra-TAD enhancer-promoter loop formation. • TAD boundaries fine-tune loop extrusion via protein interactions and DNA sequence. [ABSTRACT FROM AUTHOR]

We create a computational framework that utilizes loop extrusion (LE) by multiple condensin I/II motors to predict changes in chromosome organization during mitosis. The theory accurately reproduces the experimental contact probability profiles for the mitotic chromosomes in HeLa and DT40 cells. The LE rate is smaller at the start of mitosis and increases as the cells approach metaphase. Condensin II-mediated mean loop size is about six times larger than loops because of condensin I. The loops, which overlap each other, are stapled to a central dynamically changing helical scaffold formed by the motors during the LE process. A polymer physics-based data-driven method that uses the Hi-C contact map as the only input shows that the helix is characterized as random helix perversions (RHPs) in which the handedness changes randomly along the scaffold. The theoretical predictions, which are testable using imaging experiments, do not contain any parameters. [Display omitted] • Active polymer model accounts for chromosome organization through mitosis • Asymmetric loop extrusion by condensin motors describes mitotic structures • Mitotic chromosome structures are helical with a dynamically changing helix axis • Helical structures have random changes in handedness along the helical scaffold Multiple condensin motors drive structural changes in chromosomes during mitosis. Dey et al. create a theoretical model to quantitatively predict these structures. The theory and a data-driven model quantitatively predict the experimental observations for two cell lines. The handedness of the helical mitotic structures changes randomly along the helical axis. [ABSTRACT FROM AUTHOR]

Class-switch recombination (CSR) involves replacement of the Cμ constant region with another downstream C H region. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated DNA breaks that are targeted to transcriptionally active switch (S) regions. S region promoters (Prs) direct synapsis by associating with the Eμ and 3′Eα enhancers that jointly anchor a chromatin loop. We report that asymmetric loop extrusion allows 3′Eα to track along the locus and form Pr-Pr-E interactions that mediate CSR between downstream S regions, followed by switching to donor Sμ. This alternative pathway bypasses sequential switching and creates immunoglobulin (Ig)E+ B cells in the absence of IgG1 expression. Based on the analysis of diagnostic CSR products in B cell subsets, we identify a BCR-negative cell intermediate that is pivotal to efficient CSR. [Display omitted] • Loop extrusion drives germline transcript promoter-enhancer contacts during CSR • Promoter-promoter-3′ enhancer contacts mediate reverse sequential switching • Use of the three CSR pathways is mouse strain- and Ig isotype-specific • During CSR B cells become surface BCRneg then re-express IgM or a switched isotype Shen et al. report that 3′Eα tracks along the Igh locus via unidirectional loop extrusion to form germline transcript promoter (Pr)-Pr-E interactions that mediate an alternative CSR pathway. B cell intermediates of CSR are identified, which are AID-dependent, surface BCR-negative, and in the G 1 phase of the cell cycle. [ABSTRACT FROM AUTHOR]

How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells. • Interaction between polycomb target genes in ESCs occurs independently of cohesin • Loop extrusion by cohesin disrupts interactions between polycomb target genes • Cohesin removal enhances repression at polycomb target genes with increased interactions Using Hi-C, Capture-C, and DNA-FISH, Rhodes et al. discover that interactions between polycomb target genes occur independently of cohesin in embryonic stem cells. This relies on PRC1, and these interactions are disrupted by cohesin-mediated loop extrusion. Upon removal of cohesin, gene repression is enhanced at polycomb-occupied genes with increased interactions. [ABSTRACT FROM AUTHOR]