RESUMO
The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.
Assuntos
Cromossomos de Mamíferos/química , Animais , Fator de Ligação a CCCTC , Ciclo Celular , Cromatina/metabolismo , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Ácidos Indolacéticos/farmacologia , Camundongos , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Assuntos
Cromossomos , DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo II/genética , Cromossomos/genética , Mitose/genética , Interfase/genética , PolímerosRESUMO
Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.
Assuntos
Cromatina/metabolismo , Lâmina Nuclear/metabolismo , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Cromatina/química , Cromossomos/química , Cromossomos/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Hibridização in Situ Fluorescente , InterfaseRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , DNA , Proteínas de Manutenção de Minicromossomo , Animais , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Fase G1 , Células HCT116 , Humanos , Camundongos , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Conformação de Ácido Nucleico , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of â¼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.
Assuntos
Cromossomos Humanos/ultraestrutura , Animais , Fator de Ligação a CCCTC/metabolismo , Células Cultivadas , Cromatina/química , Cromossomos de Mamíferos/ultraestrutura , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Humanos , Masculino , Mamíferos/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Razão Sinal-RuídoRESUMO
Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.
Assuntos
Epigênese Genética , Células Germinativas , Animais , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Epigenômica , Feminino , Células Germinativas/metabolismo , Masculino , Mamíferos/genética , Camundongos , EspermatogôniasRESUMO
The cell fate decision between interferon-producing plasmacytoid DC (pDC) and antigen-presenting classical DC (cDC) is controlled by the E protein transcription factor TCF4 (E2-2). We report that TCF4 comprises two transcriptional isoforms, both of which are required for optimal pDC development in vitro. The long Tcf4 isoform is expressed specifically in pDCs, and its deletion in mice impaired pDCs development and led to the expansion of non-canonical CD8+ cDCs. The expression of Tcf4 commenced in progenitors and was further upregulated in pDCs, correlating with stage-specific activity of multiple enhancer elements. A conserved enhancer downstream of Tcf4 was required for its upregulation during pDC differentiation, revealing a positive feedback loop. The expression of Tcf4 and the resulting pDC differentiation were selectively sensitive to the inhibition of enhancer-binding BET protein activity. Thus, lineage-specifying function of E proteins is facilitated by lineage-specific isoform expression and by BET-dependent feedback regulation through distal regulatory elements.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem da Célula , Imunoprecipitação da Cromatina , Células Dendríticas/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Fator de Transcrição 4 , TranscriptomaRESUMO
Compared to noncoding RNAs (ncRNAs), such as rRNAs and ribozymes, for which high-resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNA particles (mRNPs), but the manner of this mRNA compaction is unknown. Here, we developed and implemented RIPPLiT (RNA immunoprecipitation and proximity ligation in tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably associated with defined proteins, in this case, exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton-sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNPs, wherein strong locus-specific structures predominate, mRNPs behave more like flexible polymers. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that nascent and pre-translational mammalian mRNAs are compacted by their associated proteins into linear rod-like structures.
Assuntos
Precursores de RNA/ultraestrutura , Ribonucleoproteínas/genética , Ribonucleoproteínas/ultraestrutura , Núcleo Celular , Éxons , Células HEK293 , Humanos , Imunoprecipitação/métodos , Processamento de Proteína Pós-Traducional , Precursores de RNA/genética , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/ultraestrutura , RNA não Traduzido , Spliceossomos , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Animais , Camundongos , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Mamíferos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Mamíferos/genéticaRESUMO
Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.
Assuntos
Linhagem da Célula/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Diferenciação Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Interferons/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Células Mieloides/fisiologiaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery1,2. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina2,4, preferential attraction of similar chromatin to each other1,4-12, higher levels of chromatin mobility in active chromatin13-15 and transcription-related clustering of euchromatin16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals19,20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases.
Assuntos
Compartimento Celular , Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Animais , Compartimento Celular/genética , Núcleo Celular/genética , Eucromatina/genética , Eucromatina/metabolismo , Heterocromatina/genética , Camundongos , Modelos Biológicos , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismo , Fatores de TempoRESUMO
Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.
Assuntos
Cromatina/química , Cromossomos Humanos/química , Reagentes de Ligações Cruzadas/química , Técnicas Genéticas , Linhagem Celular , Cromatina/metabolismo , Bases de Dados Factuais , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , HumanosRESUMO
Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin 'ground state' could potentially provide insights into reprogramming cells to a state of totipotency.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Posicionamento Cromossômico , Oócitos/citologia , Análise de Célula Única/métodos , Zigoto/citologia , Animais , Núcleo Celular/genética , Transdiferenciação Celular , Reprogramação Celular , Cromatina/química , Cromatina/genética , Feminino , Haploidia , Interfase , Herança Materna/genética , Camundongos , Conformação de Ácido Nucleico , Oócitos/metabolismo , Herança Paterna/genética , Processos Estocásticos , Células-Tronco Totipotentes/citologia , Células-Tronco Totipotentes/metabolismo , Zigoto/metabolismoRESUMO
This corrects the article DOI: 10.1038/nature23884.
RESUMO
The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
Assuntos
Núcleo Celular/genética , Núcleo Celular/fisiologia , Genoma , Modelos Moleculares , Imagem Molecular/métodos , Análise Espaço-Temporal , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , Genômica/métodos , Genômica/organização & administração , Objetivos , Humanos , Disseminação de Informação , Camundongos , Modelos Biológicos , Reprodutibilidade dos Testes , Análise de Célula ÚnicaRESUMO
Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Epigênese Genética , Animais , Linhagem Celular , Posicionamento Cromossômico , Drosophila melanogaster/citologia , Repressão Epigenética , Fractais , Genoma/genética , Proteínas do Grupo Polycomb/metabolismo , Transcrição GênicaRESUMO
To separate replicated sister chromatids during mitosis, eukaryotes and prokaryotes have structural maintenance of chromosome (SMC) condensin complexes that were recently shown to organize chromosomes by a process known as DNA loop extrusion. In rapidly dividing bacterial cells, the process of separating sister chromatids occurs concomitantly with ongoing transcription. How transcription interferes with the condensin loop-extrusion process is largely unexplored, but recent experiments have shown that sites of high transcription may directionally affect condensin loop extrusion. We quantitatively investigate different mechanisms of interaction between condensin and elongating RNA polymerases (RNAPs) and find that RNAPs are likely steric barriers that can push and interact with condensins. Supported by chromosome conformation capture and chromatin immunoprecipitation for cells after transcription inhibition and RNAP degradation, we argue that translocating condensins must bypass transcribing RNAPs within â¼1 to 2 s of an encounter at rRNA genes and within â¼10 s at protein-coding genes. Thus, while individual RNAPs have little effect on the progress of loop extrusion, long, highly transcribed operons can significantly impede the extrusion process. Our data and quantitative models further suggest that bacterial condensin loop extrusion occurs by 2 independent, uncoupled motor activities; the motors translocate on DNA in opposing directions and function together to enlarge chromosomal loops, each independently bypassing steric barriers in their path. Our study provides a quantitative link between transcription and 3D genome organization and proposes a mechanism of interactions between SMC complexes and elongating transcription machinery relevant from bacteria to higher eukaryotes.