Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.

Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

Genome folding through loop extrusion by SMC complexes.

Genomic DNA is folded into loops and topologically associating domains (TADs), which serve important structural and regulatory roles. It has been proposed that these genomic structures are formed by a loop extrusion process, which is mediated by structural maintenance of chromosomes (SMC) protein complexes. Recent single-molecule studies have shown that the SMC complexes condensin and cohesin are indeed able to extrude DNA into loops. In this Review, we discuss how the loop extrusion hypothesis can explain key features of genome architecture; cellular functions of loop extrusion, such as separation of replicated DNA molecules, facilitation of enhancer-promoter interactions and immunoglobulin gene recombination; and what is known about the mechanism of loop extrusion and its regulation, for example, by chromatin boundaries that depend on the DNA binding protein CTCF. We also discuss how the loop extrusion hypothesis has led to a paradigm shift in our understanding of both genome architecture and the functions of SMC complexes.

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms.

Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesin's cohesion and loop extrusion activities can be partially separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wild-type cohesin. This suggests that cohesion establishment and cohesin's interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.

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

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)2,3 that have important roles in gene regulation and recombination during development and disease1,4-7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear8. Here, to address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF is dependent on DNA tension. Moreover, CTCF regulates cohesin's loop-extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

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

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

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

Cohesin and CTCF do not assemble TADs in Xenopus sperm and male pronuclei.

Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.

Conformation of sister chromatids in the replicated human genome.

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3-7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

Wapl repression by Pax5 promotes V gene recombination by Igh loop extrusion.

Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments1 and topologically associated domains (TADs)2,3 consisting of chromatin loops4. TADs are formed by chromatin-loop extrusion5-7, which depends on the loop-extrusion function of the ring-shaped cohesin complex8-12. Conversely, the cohesin-release factor Wapl13,14 restricts loop extension10,15. The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination16, which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax517,18. However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.

Transcription shapes 3D chromatin organization by interacting with loop extrusion.

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.

Sororin mediates sister chromatid cohesion by antagonizing Wapl.

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.

Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.

Cornelia de Lange syndrome mutations in NIPBL can impair cohesin-mediated DNA loop extrusion.

Cornelia de Lange syndrome (CdLS) is a developmental multisystem disorder frequently associated with mutations in NIPBL. CdLS is thought to arise from developmental gene regulation defects, but how NIPBL mutations cause these is unknown. Here we show that several NIPBL mutations impair the DNA loop extrusion activity of cohesin. Because this activity is required for the formation of chromatin loops and topologically associating domains, which have important roles in gene regulation, our results suggest that defects in cohesin-mediated loop extrusion contribute to the etiology of CdLS by altering interactions between developmental genes and their enhancers.

A General Platform for Visible Light Sulfonylation Reactions Enabled by Catalytic Triarylamine EDA Complexes.

Catalytic electron donor-acceptor (EDA) complexes have recently emerged as a powerful and sustainable alternative to iridium- and ruthenium-based photoredox synthetic methods. Yet, these complexes remain underexplored and reliant on the use of meticulously designed acceptors that require previous installation. Herein, we report a novel EDA complex employing tris(4-methoxyphenyl) amine as a catalytic donor for the sulfonylation of alkenes using inexpensive and readily available sulfonyl chlorides. Applying this operationally simple, visible-light-mediated general platform, we report both the redox-neutral and net-reductive functionalization of more than 60 substrates, encompassing vinylic or allylic sulfonylation, hydrosulfonylation, and sulfamoylation of activated and unactivated alkenes and alkynes.

Distribution and Mobility of Amines Confined in Porous Silica Supports Assessed via Neutron Scattering, NMR, and MD Simulations: Impacts on CO2 Sorption Kinetics and Capacities.

ConspectusSolid-supported amines are a promising class of CO2 sorbents capable of selectively capturing CO2 from diverse sources. The chemical interactions between the amine groups and CO2 give rise to the formation of strong CO2 adducts, such as alkylammonium carbamates, carbamic acids, and bicarbonates, which enable CO2 capture even at low driving force, such as with ultradilute CO2 streams. Among various solid-supported amine sorbents, oligomeric amines infused into oxide solid supports (noncovalently supported) are widely studied due to their ease of synthesis and low cost. This method allows for the construction of amine-rich sorbents while minimizing problems, such as leaching or evaporation, that occur with supported molecular amines.Researchers have pursued improved sorbents by tuning the physical and chemical properties of solid supports and amine phases. In terms of CO2 uptake, the amine efficiency, or the moles of sorbed CO2 per mole of amine sites, and uptake rate (CO2 capture per unit time) are the most critical factors determining the effectiveness of the material. While structure-property relationships have been developed for different porous oxide supports, the interaction(s) of the amine phase with the solid support, the structure and distribution of the organic phase within the pores, and the mobility of the amine phase within the pores are not well understood. These factors are important, because the kinetics of CO2 sorption, particularly when using the prototypical amine oligomer branched poly(ethylenimine) (PEI), follow an unconventional trend, with rapid initial uptake followed by a very slow, asymptotic approach to equilibrium. This suggests that the uptake of CO2 within such solid-supported amines is mass transfer-limited. Therefore, improving sorption performance can be facilitated by better understanding the amine structure and distribution within the pores.In this context, model solid-supported amine sorbents were constructed from a highly ordered, mesoporous silica SBA-15 support, and an array of techniques was used to probe the soft matter domains within these hybrid materials. The choice of SBA-15 as the model support was based on its ordered arrangement of mesopores with tunable physical and chemical properties, including pore size, particle lengths, and surface chemistries. Branched PEI─the most common amine phase used in solid CO2 sorbents─and its linear, low molecular weight analogue, tetraethylenepentamine (TEPA), were deployed as the amine phases. Neutron scattering (NS), including small angle neutron scattering (SANS) and quasielastic neutron scattering (QENS), alongside solid-state NMR (ssNMR) and molecular dynamics (MD) simulations, was used to elucidate the structure and mobility of the amine phases within the pores of the support. Together, these tools, which have previously not been applied to such materials, provided new information regarding how the amine phases filled the support pores as the loading increased and the mobility of those amine phases. Varying pore surface-amine interactions led to unique trends for amine distributions and mobility; for instance, hydrophilic walls (i.e., attractive to amines) resulted in hampered motions with more intimate coordination to the walls, while amines around hydrophobic walls or walls with grafted chains that interrupt amine-wall coordination showed recovered mobility, with amines being more liberated from the walls. By correlating the structural and dynamic properties with CO2 sorption properties, novel relationships were identified, shedding light on the performance of the amine sorbents, and providing valuable guidance for the design of more effective supported amine sorbents.

Pronounced Self-Induced Diastereomeric Anisochronism in Anisidine Amino Acid Diamides.

The emergent properties resulting from selective supramolecular interactions are of significant importance for materials and chemical systems. For the directed use of such properties, a fundamental understanding of the interaction mechanism and the resulting mode of function is necessary for a tailored design. The self-induced diastereomeric anisochronism effect (SIDA), which occurs in the intermolecular interaction of chiral molecules, generates unique properties such as chiral self-recognition and nonlinear effects. Here we show that anisidine amino acid diamides lead to extraordinary signal splitting in NMR spectra through supramolecular interaction and homochiral self-recognition. By systematic experiments we have investigated the underlying SIDA effect, explored its limits and finally successfully utilized it in the determination of enantiomeric ratios by NMR spectroscopy of chiral 'SIDA-inactive' compounds such as thalidomide.