ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL.
Absolute quantification of cohesin, CTCF and their regulators in human cells.
Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.
View details for DOI 10.7554/eLife.52091
View details for PubMedID 32065581
Experimental and computational framework for a dynamic protein atlas of human cell division
2018; 561 (7723): 411-+
The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase there are ~250,000 nuclear cohesin complexes, of which ~160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.
View details for DOI 10.7554/eLife.46269
View details for PubMedID 31204999
Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation.
The Plant cell
Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, the timing of interactions and changes in cellular structure are all crucial to ensure the correct assembly, function and regulation of protein complexes1-4. Imaging of live cells can reveal protein distributions and dynamics but experimental and theoretical challenges have prevented the collection of quantitative data, which are necessary for the formulation of a model of mitosis that comprehensively integrates information and enables the analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries. Here we generate a canonical model of the morphological changes during the mitotic progression of human cells on the basis of four-dimensional image data. We use this model to integrate dynamic three-dimensional concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here to generate a dynamic protein atlas of human cell division is generic; it can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types, and can be conceptually transferred to other cellular functions.
View details for PubMedID 30202089
The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion.
The EMBO journal
Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer and reduced fertility. So far, nineteen genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homologue of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG 4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE 81 (AtMUS81), known for its role in the formation of non-interfering COs. AtFANCD2 promotes non-interfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.
View details for DOI 10.1105/tpc.17.00745
View details for PubMedID 29352063
Rapid movement and transcriptional re-localization of human cohesin on DNA
2016; 35 (24): 2671-2685
Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.
View details for PubMedID 29930102
MIS12/MIND Control at the Kinetochore.
2016; 167 (4): 889-891
The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.
View details for DOI 10.15252/embj.201695402
View details for Web of Science ID 000390948700008
View details for PubMedID 27799150
View details for PubMedCentralID PMC5167347
Sororin actively maintains sister chromatid cohesion
2016; 35 (6): 635-653
Kinetochores are complex multiprotein machines that link chromosomes to dynamic microtubules for chromosome segregation. Two studies in Cell reveal the structure of the human MIS12 and budding yeast MIND kinetochore complexes and the regulatory mechanisms that enable them to link chromosomes to microtubules during mitosis.
View details for DOI 10.1016/j.cell.2016.10.036
View details for PubMedID 27814517
Characterization of a DNA exit gate in the human cohesin ring.
2014; 346 (6212): 968-972
Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome-spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin-induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin-chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.
View details for Web of Science ID 000372254700007
View details for PubMedID 26903600
SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs
2014; 33 (22): 2643-2658
Chromosome segregation depends on sister chromatid cohesion mediated by cohesin. The cohesin subunits Smc1, Smc3, and Scc1 form tripartite rings that are thought to open at distinct sites to allow entry and exit of DNA. However, direct evidence for the existence of open forms of cohesin is lacking. We found that cohesin's proposed DNA exit gate is formed by interactions between Scc1 and the coiled-coil region of Smc3. Mutation of this interface abolished cohesin's ability to stably associate with chromatin and to mediate cohesion. Electron microscopy revealed that weakening of the Smc3-Scc1 interface resulted in opening of cohesin rings, as did proteolytic cleavage of Scc1. These open forms may resemble intermediate states of cohesin normally generated by the release factor Wapl and the protease separase, respectively.
View details for DOI 10.1126/science.1256904
View details for PubMedID 25414306
Cohesin's ATPase Activity Couples Cohesin Loading onto DNA with Smc3 Acetylation
2014; 24 (19): 2228-2237
Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing "cohesion fatigue". Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.
View details for Web of Science ID 000345299300007
View details for PubMedID 25257309
APC15 mediates CDC20 autoubiquitylation by APC/C-MCC and disassembly of the mitotic checkpoint complex
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2012; 19 (11): 1116-?
Cohesin mediates sister chromatid cohesion by topologically entrapping sister DNA molecules inside its ring structure. Cohesin is loaded onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on the adenosine triphosphatase (ATPase) activity of cohesin's Smc1 and Smc3 subunits. Subsequent cohesion establishment during DNA replication depends on Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which "locks" cohesin on DNA by inactivating the cohesin release factor Wapl.Human cohesin ATPase mutants associate transiently with DNA in a manner that depends on the loading complex but cannot be stabilized on chromatin by depletion of Wapl. These mutants cannot be acetylated, fail to interact with sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the ATPase mutants is not a consequence of their transient association with DNA but is directly caused by their inability to hydrolyze ATP because acetylation of wild-type cohesin also depends on ATP hydrolysis.Our data indicate that cohesion establishment involves the following steps. First, cohesin transiently associates with DNA in a manner that depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to entrapment of DNA and converts Smc3 into a state that can be acetylated. Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl, and stabilization of cohesin on DNA. Our finding that cohesin's ATPase activity is required for both cohesin loading and Smc3 acetylation raises the possibility that cohesion establishment is directly coupled to the reaction in which cohesin entraps DNA.
View details for DOI 10.1016/j.cub.2014.08.011
View details for Web of Science ID 000342747600017
View details for PubMedID 25220052
View details for PubMedCentralID PMC4188815
Sororin Mediates Sister Chromatid Cohesion by Antagonizing Wapl
2010; 143 (5): 737-749
The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/C(CDC20)) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/C(MCC))-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/C(CDC20) and APC/C(CDH1). Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/C(CDC20) and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.
View details for DOI 10.1038/nsmb.2412
View details for Web of Science ID 000310788800010
View details for PubMedID 23007861
View details for PubMedCentralID PMC3498062
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.
View details for DOI 10.1016/j.cell.2010.10.031
View details for Web of Science ID 000284583500016
View details for PubMedID 21111234