RESUMO
SMC (structural maintenance of chromosomes) protein complexes are an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. Here, we describe the roles of SMCs in chromosome biology and particularly review in vitro single-molecule studies that have recently advanced our understanding of SMC proteins. We describe the mechanistic biophysical aspects of loop extrusion that govern genome organization and its consequences.
Assuntos
Proteínas Cromossômicas não Histona , Complexos Multiproteicos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
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.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , DNA , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Técnicas In Vitro , CoesinasRESUMO
Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.
Assuntos
DNA Bacteriano , DNA Super-Helicoidal , Transcrição Gênica , DNA/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNARESUMO
In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.
Assuntos
Cromossomos Bacterianos , Bactérias/genética , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismoRESUMO
Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes, and whether this response relies on coordinated chromatin movement is still unclear. Here, we introduce an approach called Dense Flow reConstruction and Correlation (DFCC), to quantify correlation of chromatin motion with sub-pixel sensitivity at the level of the whole nucleus. DFCC reconstructs dense global flow fields of fluorescent images acquired in real-time. We applied our approach to analyze stochastic movements of DNA and histones, based on direction and magnitude at different time lags in human cells. We observe long-range correlations extending over several µm between coherently moving regions over the entire nucleus. Spatial correlation of global chromatin dynamics was reduced by inhibiting elongation by RNA polymerase II, and abolished in quiescent cells. Furthermore, quantification of spatial smoothness over time intervals up to 30 s points to clear-cut boundaries between distinct regions, while smooth transitions in small (<1 µm) neighborhoods dominate for short time intervals. Rough transitions between regions of coherent motion indicate directed squeezing or stretching of chromatin boundaries, suggestive of changes in local concentrations of actors regulating gene expression. The DFCC approach hence allows characterizing stochastically forming domains of nuclear activity.
Assuntos
Cromatina , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Transcrição Gênica , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/genética , DNA/análise , Histonas/análise , Humanos , Movimento (Física) , RNA Polimerase II/metabolismo , Processos EstocásticosRESUMO
Membrane abscission, the final cut of the last connection between emerging daughter cells, is an indispensable event in the last stage of cell division and in other cellular processes such as endocytosis, virus release or bacterial sporulation. However, its mechanism remains poorly understood, impeding its application as a cell-division machinery for synthetic cells. Here we use fluorescence microscopy and fluorescence recovery after photobleaching measurements to study the in vitro reconstitution of the bacterial protein dynamin A inside liposomes. Upon external reshaping of the liposomes into dumbbells, dynamin A self-assembles at the membrane neck, resulting in membrane hemi-scission and even full scission. Dynamin A proteins constitute a simple one-component division machinery capable of splitting dumbbell-shaped liposomes, marking an important step towards building a synthetic cell.
Assuntos
Células Artificiais , Lipossomos , Dinaminas/metabolismo , Endocitose , Divisão Celular , Bactérias/metabolismoRESUMO
DNA loop extrusion by structural-maintenance-of-chromosome (SMC) complexes has emerged as a primary organizing principle for chromosomes. The mechanism by which SMC motor proteins extrude DNA loops is still unresolved and much debated. The ring-like structure of SMC complexes prompted multiple models where the extruded DNA is topologically or pseudotopologically entrapped within the ring during loop extrusion. However, recent experiments showed the passage of roadblocks much bigger than the SMC ring size, suggesting a nontopological mechanism. Recently, attempts were made to reconcile the observed passage of large roadblocks with a pseudotopological mechanism. Here we examine the predictions of these pseudotopological models and find that they are not consistent with new experimental data on SMC roadblock encounters. Particularly, these models predict the formation of two loops and that roadblocks will reside near the stem of the loop upon encounter-both in contrast to experimental observations. Overall, the experimental data reinforce the notion of a nontopological mechanism for extrusion of DNA.
Assuntos
Cromossomos , DNA , Cromossomos/metabolismo , DNA/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Gene transcription by RNA polymerase II (RNAPol II) is a tightly regulated process in the genomic, temporal, and spatial context. Recently, we have shown that chromatin exhibits spatially coherently moving regions over the entire nucleus, which is enhanced by transcription. Yet, it remains unclear how the mobility of RNA Pol II molecules is affected by transcription regulation and whether this response depends on the coordinated chromatin movement. We applied our Dense Flow reConstruction and Correlation method to analyze nucleus-wide coherent movements of RNA Pol II in living human cancer cells. We observe a spatially coherent movement of RNA Pol II molecules over ≈1 µm, which depends on transcriptional activity. Inducing transcription in quiescent cells decreased the coherent motion of RNA Pol II. We then quantify the spatial correlation length of RNA Pol II in the context of DNA motion. RNA Pol II and chromatin spatially coherent motions respond oppositely to transcriptional activities. Our study holds the potential of studying the chromatin environment in different nuclear processes.
Assuntos
Cromatina , RNA Polimerase II , Núcleo Celular/metabolismo , DNA , Humanos , RNA Polimerase II/genética , Transcrição GênicaRESUMO
Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.
Assuntos
Nanopartículas , Nucleossomos , Proteínas de Ciclo Celular , Cromatina , Saccharomyces cerevisiaeRESUMO
The ParABS system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS, as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Ligação ProteicaRESUMO
The eukaryotic genome is hierarchically structured yet highly dynamic. Regulating transcription in this environment demands a high level of coordination to permit many proteins to interact with chromatin fiber at appropriate sites in a timely manner. We describe how recent advances in quantitative imaging techniques overcome caveats of sequencing-based methods (Hi-C and related) by enabling direct visualization of transcription factors and chromatin at high resolution, from single genes to the whole nucleus. We discuss the contribution of fluorescence imaging to deciphering the principles underlying this coordination within the crowded nuclear space in living cells and discuss challenges ahead.
Assuntos
Genoma , Transcrição Gênica , Núcleo Celular/genética , Cromatina/metabolismo , Cromossomos , Células Eucarióticas , Imagem Óptica , Fatores de Transcrição/metabolismoRESUMO
Chromatin 'blobs' were recently identified by live super-resolution imaging of labeled nucleosomes as pervasive but fleeting structural entities. However, the mechanisms leading to the formation of these blobs and their functional implications are unknown. We explore here whether causal relationships exist between parameters that characterize the chromatin blob dynamics and structure, by adapting a framework for spatio-temporal Granger-causality inference. Our analysis reveals that chromatin dynamics is a key determinant for both blob area and local density. Such causality, however, could be demonstrated only in 10-20% of the nucleus, suggesting that chromatin dynamics and structure at the nanometer scale are dominated by stochasticity. We show that the theory of active semiflexible polymers can be invoked to provide potential mechanisms leading to the organization of chromatin into blobs. Our results represent a first step toward elucidating the mechanisms that govern the dynamic and stochastic organization of chromatin in the cell nucleus.
Assuntos
Cromatina/metabolismo , Genoma , Nanoestruturas/química , Cromatina/química , Montagem e Desmontagem da Cromatina , Aprendizado Profundo , Humanos , Modelos Moleculares , Processos Estocásticos , Células Tumorais CultivadasRESUMO
Bulk chromatin motion has not been analyzed at high resolution. We present Hi-D, a method to quantitatively map dynamics of chromatin and abundant nuclear proteins for every pixel simultaneously over the entire nucleus from fluorescence image series. Hi-D combines reconstruction of chromatin motion and classification of local diffusion processes by Bayesian inference. We show that DNA dynamics in the nuclear interior are spatially partitioned into 0.3-3-µm domains in a mosaic-like pattern, uncoupled from chromatin compaction. This pattern was remodeled in response to transcriptional activity. Hi-D can be applied to any dense and bulk structures opening new perspectives towards understanding motion of nuclear molecules.