RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , Conformación de Ácido Nucleico , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Hidrólisis , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformación Proteica , CohesinasRESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , ADN/química , Genoma , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Biológicos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , CohesinasRESUMEN
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.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Cromátides , Humanos , Cromátides/genética , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , CohesinasRESUMEN
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.
Asunto(s)
Factor de Unión a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , ADN , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , ADN/metabolismo , Técnicas In Vitro , CohesinasRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , ADN , Proteínas de Mantenimiento de Minicromosoma , Animales , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , ADN/metabolismo , Fase G1 , Células HCT116 , Humanos , Ratones , Componente 3 del Complejo de Mantenimiento de Minicromosoma/química , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Conformación de Ácido Nucleico , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
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.
Asunto(s)
Cromátides , Proteínas Cromosómicas no Histona , Humanos , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mitosis , ADN , Fase G2 , CohesinasRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo II/química , ADN/genética , Complejos Multiproteicos/química , Proteínas de Unión a Poli-ADP-Ribosa/química , Rotura Cromosómica , Cromosomas/genética , ADN/química , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Etopósido/química , Conversión Génica/genética , Células HCT116 , Humanos , Cinética , Complejos Multiproteicos/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Torsión Mecánica , Transcripción Genética , Translocación Genética/genéticaRESUMEN
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.
Asunto(s)
Factor de Unión a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Cohesinas , Espermatozoides , Xenopus laevis , Animales , Masculino , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Espermatozoides/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Ratones , Humanos , Cromatina/metabolismo , Cromatina/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Genoma , Núcleo Celular/metabolismoRESUMEN
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.
Asunto(s)
Cromátides/química , Emparejamiento Cromosómico , Replicación del ADN , Genoma Humano/genética , Conformación de Ácido Nucleico , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/análisis , ADN/biosíntesis , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , CohesinasRESUMEN
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.
Asunto(s)
Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Factor de Transcripción PAX5/metabolismo , Proteínas/genética , Proteínas Represoras/metabolismo , Recombinación V(D)J/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Ratones , Complejo Represivo Polycomb 2/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Regiones Promotoras Genéticas/genética , CohesinasRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Cromatina , Animales , Ratones , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas de los Mamíferos/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Mamíferos/genéticaRESUMEN
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.
Asunto(s)
Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/química , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Humanos , Fase S , Xenopus/metabolismo , CohesinasRESUMEN
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.
Asunto(s)
Síndrome de Cornelia de Lange , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , ADN/genética , Síndrome de Cornelia de Lange/genética , Humanos , Mutación , CohesinasRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/metabolismo , Mitosis , Edición Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Imagenología Tridimensional , Microscopía Fluorescente , Imagen Molecular , Factores de TiempoRESUMEN
The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Segregación Cromosómica , Microscopía por Crioelectrón , Puntos de Control de la Fase M del Ciclo Celular , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Ubiquitina/metabolismo , Sitios de Unión , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/ultraestructura , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , UbiquitinaciónRESUMEN
Mammalian genomes are spatially organized by CCCTC-binding factor (CTCF) and cohesin into chromatin loops and topologically associated domains, which have important roles in gene regulation and recombination. By binding to specific sequences, CTCF defines contact points for cohesin-mediated long-range chromosomal cis-interactions. Cohesin is also present at these sites, but has been proposed to be loaded onto DNA elsewhere and to extrude chromatin loops until it encounters CTCF bound to DNA. How cohesin is recruited to CTCF sites, according to this or other models, is unknown. Here we show that the distribution of cohesin in the mouse genome depends on transcription, CTCF and the cohesin release factor Wings apart-like (Wapl). In CTCF-depleted fibroblasts, cohesin cannot be properly recruited to CTCF sites but instead accumulates at transcription start sites of active genes, where the cohesin-loading complex is located. In the absence of both CTCF and Wapl, cohesin accumulates in up to 70 kilobase-long regions at 3'-ends of active genes, in particular if these converge on each other. Changing gene expression modulates the position of these 'cohesin islands'. These findings indicate that transcription can relocate mammalian cohesin over long distances on DNA, as previously reported for yeast cohesin, that this translocation contributes to positioning cohesin at CTCF sites, and that active genes can be freed from cohesin either by transcription-mediated translocation or by Wapl-mediated release.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/metabolismo , Genoma/genética , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/genética , Animales , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/deficiencia , Proteoglicanos Tipo Condroitín Sulfato/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Cromosomas de los Mamíferos/genética , ADN/genética , ADN/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Masculino , Ratones , Transporte de Proteínas , Proteínas/genética , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Sitio de Iniciación de la Transcripción , CohesinasRESUMEN
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.
Asunto(s)
Acetiltransferasas/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Acetilación , Proteínas de Ciclo Celular/metabolismo , Humanos , Mitosis/genética , CohesinasRESUMEN
Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.