A replication fork determinant for the establishment of sister chromatid cohesion.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Cohesion establishment requires acetylation of conserved cohesin lysine residues by Eco1 acetyltransferase. Here, we explore how cohesin acetylation is linked to DNA replication. Biochemical reconstitution of replication-coupled cohesin acetylation reveals that transient DNA structures, which form during DNA replication, control the acetylation reaction. As polymerases complete lagging strand replication, strand displacement synthesis produces DNA flaps that are trimmed to result in nicked double-stranded DNA. Both flaps and nicks stimulate cohesin acetylation, while subsequent nick ligation to complete Okazaki fragment maturation terminates the acetylation reaction. A flapped or nicked DNA substrate constitutes a transient molecular clue that directs cohesin acetylation to a window behind the replication fork, next to where cohesin likely entraps both sister chromatids. Our results provide an explanation for how DNA replication is linked to sister chromatid cohesion establishment.

De novo deletions and duplications at recombination hotspots in mouse germlines.

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

Establishment of DNA-DNA Interactions by the Cohesin Ring.

The ring-shaped structural maintenance of chromosome (SMC) complexes are multi-subunit ATPases that topologically encircle DNA. SMC rings make vital contributions to numerous chromosomal functions, including mitotic chromosome condensation, sister chromatid cohesion, DNA repair, and transcriptional regulation. They are thought to do so by establishing interactions between more than one DNA. Here, we demonstrate DNA-DNA tethering by the purified fission yeast cohesin complex. DNA-bound cohesin efficiently and topologically captures a second DNA, but only if that is single-stranded DNA (ssDNA). Like initial double-stranded DNA (dsDNA) embrace, second ssDNA capture is ATP-dependent, and it strictly requires the cohesin loader complex. Second-ssDNA capture is relatively labile but is converted into stable dsDNA-dsDNA cohesion through DNA synthesis. Our study illustrates second-DNA capture by an SMC complex and provides a molecular model for the establishment of sister chromatid cohesion.

The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms.

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.

Sister chromatid cohesion halts DNA loop expansion.

Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.

A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.

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.

Chromosome organization by fine-tuning an ATPase.

Cohesin is an ATPase that drives chromosome organization through the generation of intramolecular loops and sister chromatid cohesion. Cohesin's ATPase is stimulated by Scc2 binding but attenuated by acetylation of its Smc3 subunit. In this issue of Genes & Development, Boardman and colleagues (pp. 277-290) take a genetic approach to generate a mechanistic model for the opposing regulation of cohesin's ATPase by Scc2 and Smc3 acetylation. Their findings provide in vivo insight into how this important genome organizer functions in vivo.

A model for Scc2p stimulation of cohesin's ATPase and its inhibition by acetylation of Smc3p.

The evolutionarily conserved cohesin complex mediates sister chromatid cohesion and facilitates mitotic chromosome condensation, DNA repair, and transcription regulation. These biological functions require cohesin's two ATPases, formed by the Smc1p and Smc3p subunits. Cohesin's ATPase activity is stimulated by the Scc2p auxiliary factor. This stimulation is inhibited by Eco1p acetylation of Smc3p at an interface with Scc2p. It was unclear how cohesin's ATPase activity is stimulated by Scc2p or how acetylation inhibits Scc2p, given that the acetylation site is distal to cohesin's ATPase active sites. Here, we identify mutations in budding yeast that suppressed the in vivo defects caused by Smc3p acetyl-mimic and acetyl-defective mutations. We provide compelling evidence that Scc2p activation of cohesin ATPase depends on an interface between Scc2p and a region of Smc1p proximal to cohesin's Smc3p ATPase active site. Furthermore, substitutions at this interface increase or decrease ATPase activity to overcome ATPase modulation by acetyl-mimic and acetyl-null mutations. Using these observations and an existing cryo-EM structure, we propose a model for regulating cohesin ATPase activity. We suggest that Scc2p binding to Smc1p causes the adjacent Smc1p residues and ATP to shift, stimulating Smc3p's ATPase. This stimulatory shift is inhibited through acetylation of the distal Scc2p-Smc3p interface.

Asymmetric Histone Inheritance: Establishment, Recognition, and Execution.

The discovery of biased histone inheritance in asymmetrically dividing Drosophila melanogaster male germline stem cells demonstrates one means to produce two distinct daughter cells with identical genetic material. This inspired further studies in different systems, which revealed that this phenomenon may be a widespread mechanism to introduce cellular diversity. While the extent of asymmetric histone inheritance could vary among systems, this phenomenon is proposed to occur in three steps: first, establishment of histone asymmetry between sister chromatids during DNA replication; second, recognition of sister chromatids carrying asymmetric histone information during mitosis; and third, execution of this asymmetry in the resulting daughter cells. By compiling the current knowledge from diverse eukaryotic systems, this review comprehensively details and compares known chromatin factors, mitotic machinery components, and cell cycle regulators that may contribute to each of these three steps. Also discussed are potential mechanisms that introduce and regulate variable histone inheritance modes and how these different modes may contribute to cell fate decisions in multicellular organisms.

Coordination of cohesin and DNA replication observed with purified proteins.

Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1-3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.

A walk through the SMC cycle: From catching DNAs to shaping the genome.

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.

Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release.

The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11 The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.

A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres.

The 16-subunit Constitutive Centromere-associated Network (CCAN)-based inner kinetochore is well-known for connecting centromeric chromatin to the spindle-binding outer kinetochore. Here, we report a non-canonical role for the inner kinetochore in directly regulating sister-chromatid cohesion at centromeres. We provide biochemical, X-ray crystal structure, and intracellular ectopic localization evidence that the inner kinetochore directly binds cohesin, a ring-shaped multi-subunit complex that holds sister chromatids together from S-phase until anaphase onset. This interaction is mediated by binding of the 5-subunit CENP-OPQUR sub-complex of CCAN to the Scc1-SA2 sub-complex of cohesin. Mutation in the CENP-U subunit of the CENP-OPQUR complex that abolishes its binding to the composite interface between Scc1 and SA2 weakens centromeric cohesion, leading to premature separation of sister chromatids during delayed metaphase. We further show that CENP-U competes with the cohesin release factor Wapl for binding the interface of Scc1-SA2, and that the cohesion-protecting role for CENP-U can be bypassed by depleting Wapl. Taken together, this study reveals an inner kinetochore-bound pool of cohesin, which strengthens centromeric sister-chromatid cohesion to resist metaphase spindle pulling forces.

An extrinsic motor directs chromatin loop formation by cohesin.

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

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.

High-Resolution Whole-Genome Analysis of Sister-Chromatid Contacts.

Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners.

Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.

The 3D Topography of Mitotic Chromosomes.

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.