RESUMO
X chromosome inactivation (XCI) generates clonal heterogeneity within XX individuals. Combined with sequence variation between human X chromosomes, XCI gives rise to intra-individual clonal diversity, whereby two sets of clones express mutually exclusive sequence variants present on one or the other X chromosome. Here we ask whether such clones merely co-exist or potentially interact with each other to modulate the contribution of X-linked diversity to organismal development. Focusing on X-linked coding variation in the human STAG2 gene, we show that Stag2variant clones contribute to most tissues at the expected frequencies but fail to form lymphocytes in Stag2WT Stag2variant mouse models. Unexpectedly, the absence of Stag2variant clones from the lymphoid compartment is due not solely to cell-intrinsic defects but requires continuous competition by Stag2WT clones. These findings show that interactions between epigenetically diverse clones can operate in an XX individual to shape the contribution of X-linked genetic diversity in a cell-type-specific manner.
Assuntos
Cromossomos Humanos X , Genes Ligados ao Cromossomo X , Variação Genética , Inativação do Cromossomo X , Humanos , Animais , Inativação do Cromossomo X/genética , Camundongos , Cromossomos Humanos X/genética , Feminino , Proteínas de Ciclo Celular/genética , Antígenos Nucleares/genética , Linfócitos/metabolismo , Cromossomo X/genética , CoesinasRESUMO
Cohesin holds together the sister chromatids from DNA replication onwards. How cohesion is established has long remained a black box. Through recent studies, a model is emerging in which a replisome-cohesin encounter results in the establishment of cohesive linkages at sites of replication termination.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Replicação do DNA , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromátides/metabolismoRESUMO
In this issue of Molecular Cell, two papers provide insight into atypical structural maintenance of chromosomes protein complexes (SMCs). Jeppsson et al.1 link Smc5/6 to supercoiled DNA, and Roisné-Hamelin et al.2 show how Wadjet SMC bends and cleaves invading DNAs.
Assuntos
Proteínas de Ciclo Celular , Cromossomos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromossomos/metabolismo , DNA , Reparo do DNA , Proteínas de Ligação a DNA/genéticaRESUMO
A potential mechanism of DNA loop extrusion by molecular motors is discussed.
RESUMO
Many cellular processes require large-scale rearrangements of chromatin structure. Structural maintenance of chromosomes (SMC) protein complexes are molecular machines that can provide structure to chromatin. These complexes can connect DNA elements in cis, walk along DNA, build and processively enlarge DNA loops and connect DNA molecules in trans to hold together the sister chromatids. These DNA-shaping abilities place SMC complexes at the heart of many DNA-based processes, including chromosome segregation in mitosis, transcription control and DNA replication, repair and recombination. In this Review, we discuss the latest insights into how SMC complexes such as cohesin, condensin and the SMC5-SMC6 complex shape DNA to direct these fundamental chromosomal processes. We also consider how SMC complexes, by building chromatin loops, can counteract the natural tendency of alike chromatin regions to cluster. SMC complexes thus control nuclear organization by participating in a molecular tug of war that determines the architecture of our genome.
Assuntos
Cromatina , Cromossomos , Cromossomos/genética , Cromossomos/metabolismo , Cromatina/genética , DNA/genética , Replicação do DNA/genética , Mitose , Proteínas de Ciclo Celular/químicaRESUMO
In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1-cohesin binding is maintained until the formation of microtubule-kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.
Assuntos
Proteínas de Ciclo Celular , Centrômero , Humanos , Células HeLa , Centrômero/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinetocoros , Mitose , Segregação de Cromossomos , Cromátides/metabolismoRESUMO
Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin's SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the three-dimensional genome in human cells. ESCO1 restricts the length of chromatin loops, and of architectural stripes emanating from CTCF sites. HDAC8 conversely promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation controls the interaction of cohesin with PDS5A to restrict chromatin loop length. Our data support a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Acetilação , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Histona Desacetilases/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , CoesinasRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Mitose/genéticaRESUMO
The genome consists of regions of transcriptionally active euchromatin and more silent heterochromatin. We reveal that the formation of heterochromatin domains requires cohesin turnover on DNA. Stabilization of cohesin on DNA through depletion of its release factor WAPL leads to a near-complete loss of heterochromatin domains. We observe the opposite phenotype in cells deficient for subunits of the Mediator-CDK module, with an almost binary partition of the genome into dense H3K9me3 domains, and regions devoid of H3K9me3 spanning the rest of the genome. We suggest that the Mediator-CDK module might contribute to gene expression by limiting the formation of dense heterochromatin domains. WAPL deficiency prevents the formation of heterochromatin domains, and allows for gene expression even in the absence of the Mediator-CDK subunit MED12. We propose that cohesin and Mediator affect heterochromatin in different ways to enable the correct distribution of epigenetic marks, and thus to ensure proper gene expression.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Complexo Mediador/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Epigênese Genética , Técnicas de Inativação de Genes , Humanos , Complexo Mediador/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , RNA-Seq , CoesinasRESUMO
Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise chromosome conformation capture (3C) data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that ΔSA1 cells gain intra-TAD interactions and increase compartmentalisation. ΔSA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesinSA1 and cohesinSA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.
RESUMO
Cohesin and CTCF are key to the 3D folding of interphase chromosomes. Cohesin forms chromatin loops via loop extrusion, a process that involves the formation and enlargement of DNA loops. The architectural protein CTCF controls this process by acting as an anchor for chromatin looping. How CTCF controls cohesin has long been a mystery. Recent work shows that CTCF dictates chromatin looping via a direct interaction of its N-terminus with cohesin. CTCF's ability to regulate chromatin looping turns out to also be partially dependent on several RNA-binding domains. In this review, we discuss recent insights and consider how cohesin and CTCF together may orchestrate the folding of the genome into chromosomal loops.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/metabolismo , CoesinasRESUMO
Cohesin catalyses the folding of the genome into loops that are anchored by CTCF1. The molecular mechanism of how cohesin and CTCF structure the 3D genome has remained unclear. Here we show that a segment within the CTCF N terminus interacts with the SA2-SCC1 subunits of human cohesin. We report a crystal structure of SA2-SCC1 in complex with CTCF at a resolution of 2.7 Å, which reveals the molecular basis of the interaction. We demonstrate that this interaction is specifically required for CTCF-anchored loops and contributes to the positioning of cohesin at CTCF binding sites. A similar motif is present in a number of established and newly identified cohesin ligands, including the cohesin release factor WAPL2,3. Our data suggest that CTCF enables the formation of chromatin loops by protecting cohesin against loop release. These results provide fundamental insights into the molecular mechanism that enables the dynamic regulation of chromatin folding by cohesin and CTCF.
Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Humanos , Ligantes , Modelos Moleculares , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , CoesinasRESUMO
Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Trifosfato de Adenosina/química , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Cromossomos/fisiologia , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Complexos Multiproteicos/fisiologia , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo , CoesinasRESUMO
From the dynamic interphase genome to compacted mitotic chromosomes, DNA is organized by the conserved SMC complexes cohesin and condensin. The picture is emerging that these complexes structure the genome through a shared basic principle that involves the formation and processive enlargement of chromatin loops. This appears to be an asymmetric process, in which the complex anchors at the base of a loop and then enlarges the loop in a one-sided manner. We discuss the latest insights into how ATPase-driven conformational changes within these complexes may enlarge loops, and consider how asymmetric DNA reeling can bring together genomic elements in a symmetric manner.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Animais , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Humanos , Mamíferos , Complexos Multiproteicos/genéticaRESUMO
Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the ß-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect 'cohesin traffic jams'. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.
Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos/genética , Alelos , Animais , Fator de Ligação a CCCTC/genética , Camundongos , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Globinas beta/genéticaRESUMO
What drives the formation of chromatin loops has been a long-standing question in chromosome biology. Recent work provides major insight into the basic principles behind loop formation. Structural maintenance of chromosomes (SMC) complexes, that are conserved from bacteria to humans, are key to this process. The SMC family includes condensin and cohesin, which structure chromosomes to enable mitosis and long-range gene regulation. We discuss novel insights into the mechanism of loop formation and the implications for how these complexes ultimately shape chromosomes. A picture is emerging in which these complexes form small loops that they then processively enlarge. It appears that SMC complexes act by family-wide basic principles, with complex-specific levels of control.
Assuntos
Proteínas de Transporte/genética , Cromatina/genética , Cromossomos/genética , Mitose/genética , Proteínas Nucleares/genética , Adenosina Trifosfatases/genética , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Humanos , Complexos Multiproteicos/genética , CoesinasRESUMO
To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos/metabolismo , Replicação do DNA , Proteínas de Transporte/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA , Recuperação de Fluorescência Após Fotodegradação , Humanos , Interfase , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S , CoesinasRESUMO
The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Acetiltransferases/metabolismo , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Elongases de Ácidos Graxos , Edição de Genes , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Repressoras/metabolismo , CoesinasRESUMO
Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin's Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin's association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase-independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation.