Auswahl der wissenschaftlichen Literatur zum Thema „Chromatin loop extrusion“

It has been a puzzle how decondensed interphase chromosomes remain essentially unknotted. The natural expectation is that in the presence of type II DNA topoisomerases that permit passages of double-stranded DNA regions through each other, all chromosomes should reach the state of topological equilibrium. The topological equilibrium in highly crowded interphase chromosomes forming chromosome territories would result in formation of highly knotted chromatin fibres. However, Chromosome Conformation Capture (3C) methods revealed that the decay of contact probabilities with the genomic distance in interphase chromosomes is practically the same as in the crumpled globule state that is formed when long polymers condense without formation of any knots. To remove knots from highly crowded chromatin, one would need an active process that should not only provide the energy to move the system from the state of topological equilibrium but also guide topoisomerase-mediated passages in such a way that knots would be efficiently unknotted instead of making the knots even more complex. We perform coarse-grained molecular dynamics simulations of the process of chromatin loop extrusion involving knotted and catenated chromatin fibres to check whether chromatin loop extrusion may be involved in active unknotting of chromatin fibres. Our simulations show that the process of chromatin loop extrusion is ideally suited to actively unknot, decatenate and demix chromatin fibres in interphase chromosomes.

ABSTRACTThe three-dimensional structure of chromatin is determined by the action of protein complexes of the structural maintenance of chromosome (SMC) family. Eukaryotic cells contain three SMC complexes, cohesin, condensin, and a complex of Smc5 and Smc6. Initially, cohesin was linked to sister chromatid cohesion, the process that ensures the fidelity of chromosome segregation in mitosis. In recent years, a second function in the organization of interphase chromatin into topologically associated domains has been determined, and loop extrusion has emerged as the leading mechanism of this process. Interestingly, fundamental mechanistic differences exist between mitotic tethering and loop extrusion. As distinct molecular switches that aim to suppress loop extrusion in different biological contexts have been identified, we hypothesize here that loop extrusion is the default biochemical activity of cohesin and that its suppression shifts cohesin into a tethering mode. With this model, we aim to provide an explanation for how loop extrusion and tethering can coexist in a single cohesin complex and also apply it to the other eukaryotic SMC complexes, describing both similarities and differences between them. Finally, we present model-derived molecular predictions that can be tested experimentally, thus offering a new perspective on the mechanisms by which SMC complexes shape the higher-order structure of chromatin.

Mammalian chromatin is spatially organized at many scales showing two prominent features in interphase: (i) alternating regions (1–10 Mb) of active and inactive chromatin that spatially segregate into different compartments, and (ii) domains (<1 Mb), that is, regions that preferentially interact internally [topologically associating domains (TADs)] and are central to gene regulation. There is growing evidence that TADs are formed by active extrusion of chromatin loops by cohesin, whereas compartmentalization is established according to local chromatin states. Here, we use polymer simulations to examine how loop extrusion and compartmental segregation work collectively and potentially interfere in shaping global chromosome organization. A model with differential attraction between euchromatin and heterochromatin leads to phase separation and reproduces compartmentalization as observed in Hi-C. Loop extrusion, essential for TAD formation, in turn, interferes with compartmentalization. Our integrated model faithfully reproduces Hi-C data from puzzling experimental observations where altering loop extrusion also led to changes in compartmentalization. Specifically, depletion of chromatin-associated cohesin reduced TADs and revealed finer compartments, while increased processivity of cohesin strengthened large TADs and reduced compartmentalization; and depletion of the TAD boundary protein CTCF weakened TADs while leaving compartments unaffected. We reveal that these experimental perturbations are special cases of a general polymer phenomenon of active mixing by loop extrusion. Our results suggest that chromatin organization on the megabase scale emerges from competition of nonequilibrium active loop extrusion and epigenetically defined compartment structure.

Structural maintenance of chromosomes (SMC) complexes are essential proteins found in genomes of all cellular organisms. Essential functions of these proteins, such as mitotic chromosome formation and sister chromatid cohesion, were discovered a long time ago. Recent advances in chromatin biology showed that SMC proteins are involved in many other genomic processes, acting as active motors extruding DNA, which leads to the formation of chromatin loops. Some loops formed by SMC proteins are highly cell type and developmental stage specific, such as SMC-mediated DNA loops required for VDJ recombination in B-cell progenitors, or dosage compensation in Caenorhabditis elegans and X-chromosome inactivation in mice. In this review, we focus on the extrusion-based mechanisms that are common for multiple cell types and species. We will first describe an anatomy of SMC complexes and their accessory proteins. Next, we provide biochemical details of the extrusion process. We follow this by the sections describing the role of SMC complexes in gene regulation, DNA repair, and chromatin topology.

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 ∼1 to 2 s of an encounter at rRNA genes and within ∼10 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.

Abstract Enhancers are DNA sequences at a long genomic distance from target genes. Recent experiments suggest that enhancers are anchored to the surfaces of condensates of transcription machinery and that the loop extrusion process enhances the transcription level of their target genes. Here, we theoretically study the polymer dynamics driven by the loop extrusion of the linker DNA between an enhancer and the promoter of its target gene to calculate the contact probability of the promoter to the transcription machinery in the condensate. Our theory predicts that when the loop extrusion process is active, the contact probability increases with increasing linker DNA length. This finding reflects the fact that the relaxation time, with which the promoter stays in proximity to the surface of the transcriptional condensate, increases as the length of the linker DNA increases. This contrasts the equilibrium case for which the contact probability between the promoter and the transcription machineries is smaller for longer linker DNA lengths.

Transient DNA loops occur throughout the genome due to thermal fluctuations of DNA and the function of SMC complex proteins such as condensin and cohesin. Transient crosslinking within and between chromosomes and loop extrusion by SMCs have profound effects on high-order chromatin organization and exhibit specificity in cell type, cell cycle stage, and cellular environment. SMC complexes anchor one end to DNA with the other extending some distance and retracting to form a loop. How cells regulate loop sizes and how loops distribute along chromatin are emerging questions. To understand loop size regulation, we employed bead–spring polymer chain models of chromatin and the activity of an SMC complex on chromatin. Our study shows that (1) the stiffness of the chromatin polymer chain, (2) the tensile stiffness of chromatin crosslinking complexes such as condensin, and (3) the strength of the internal or external tethering of chromatin chains cooperatively dictate the loop size distribution and compaction volume of induced chromatin domains. When strong DNA tethers are invoked, loop size distributions are tuned by condensin stiffness. When DNA tethers are released, loop size distributions are tuned by chromatin stiffness. In this three-way interaction, the presence and strength of tethering unexpectedly dictates chromatin conformation within a topological domain.

We propose a model for cohesin-mediated loop extrusion, where the loop extrusion is driven entropically by the energy difference between supercoiled and torsionally relaxed chromatin fibers. Different levels of negative supercoiling are controlled by varying imposed friction between the cohesin ring and the chromatin fiber. The speed of generation of negative supercoiling by RNA polymerase associated with TOP1 is kept constant and corresponds to 10 rotations per second. The model was tested by coarse-grained molecular simulations for a wide range of frictions between 2 to 200 folds of that of generic fiber and the surrounding medium. The higher friction allowed for the accumulation of higher levels of supercoiling, while the resulting extrusion rate also increased. The obtained extrusion rates for the given range of investigated frictions were between 1 and 10 kbps, but also a saturation of the rate at high frictions was observed. The calculated contact maps indicate a qualitative improvement obtained at lower levels of supercoiling. The fits of mathematical equations qualitatively reproduce the loop sizes and levels of supercoiling obtained from simulations and support the proposed mechanism of entropically driven extrusion. The cohesin ring is bound on the fibers pseudo-topologically, and the model suggests that the topological binding is not necessary.

L’étude de l’organisation 3D du génome a révélé l’existence de boucles de chromatine et des Topologically Associating Domains (TADs) de l’ordre de plusieurs centaines de kilobases, créés par l’anneau de cohésine par le processus d’extrusion de boucle d’ADN. Cependant, ces structures ont été caractérisées presque exclusivement par des techniques de génomique et d’imagerie de cellules fixées, leur dynamique temporelle reste donc peu comprise. Par exemple, la durée des contacts créés par extrusion de boucles n’est pas définie et des paramètres majeurs de ce processus comme la durée de vie des contacts ancre-ancre et la vitesse d’extrusion in vivo sont toujours inconnus. Pour répondre à cette lacune, j’ai quantifié la dynamique de l’extrusion de boucle cohésine-dépendante en visualisant et suivant dans le temps plusieurs paires d’ancres de boucles dans des cellules humaines vivantes. Il est attendu que l’extrusion de boucle soit identifiée par une diminution progressive de la distance ancre-ancre. Cependant, cette signature est occultée par la dynamique stochastique de la chromatine, les ancres de boucles pouvant entrer en contact même sans extrusion. De plus, mesurer la distance ancre-ancre à partir d’images fluorescentes est rendu difficile par plusieurs sources d’erreurs comme les erreurs aléatoires liées à la localisation de points fluorescents. Pour estimer les conditions expérimentales qui permettent de détecter et quantifier l’extrusion de boucles malgré ces complications, j’ai utilisé des simulations de polymères et modélisé le processus d’extrusion de boucle in silico. De plus, j’ai testé et validé de nouvelles méthodes d’analyse pour quantifier les boucles de chromatine à partir d’images statiques (e. g. à partir d’images d’ancres de boucles acquises par DNA FISH), estimer la fraction, fréquence et durée de vie des contacts ancre-ancre, ainsi qu’estimer la vitesse d’extrusion effective in vivo à partir d’images dynamiques. En se basant sur les résultats des simulations de polymères, j’ai taggué par fluorescence de multiples ancres de boucles et TADs dans des cellules vivantes par le système CRISPR/Cas9. Nous avons conclu que les contacts entre les ancres étaient peu fréquents et de courte durée, par rapport à la durée du cycle cellulaire. Cependant, les boucles sont presque constamment soumises à l’extrusion par la cohésine. En comparant les résultats de modélisation et les expériences, nous avons pu estimer des paramètres biophysiques généraux de la dynamique d’extrusion de boucles. Ces résultats suggèrent que l’extrusion de boucles de chromatine cohésine-dépendante est un processus hautement dynamique, qui crée des interactions à longue portée transitoires plutôt que des contacts stables. Mes résultats aideront à comprendre quantitativement des processus biologiques fondamentaux qui utilisent les contacts transitoires mais à longue distance créés par l’extrusion de boucles, comme la réparation de l’ADN et la régulation de l’expression des gènes
Studies of spatial genome organization have revealed the existence of chromatin loops and Topologically Associating Domains (TADs) of several hundred kilobases in size, which are created by the cohesin ring complex through a process of DNA loop extrusion. However, these structures have been characterized almost exclusively by genomic techniques and fixed cell imaging, thus their temporal dynamics are still poorly understood. For example, it is not clear whether loop extrusion creates stable or transient contacts at loop anchors and key parameters of this process, including loop lifetime and extrusion speed, remain unknown. To address this gap, my thesis aims to quantify the dynamics of cohesin-dependent loop extrusion by visualizing and tracking in time pairs of anchors at several loops and TADs in living human cells. Extrusion is expected to manifest itself as a progressive decrease in anchor-anchor distances. However, this signature is obscured by stochastic motions of the chromatin, whereby anchors can occasionally come into contact even without extrusion. Furthermore, measuring the anchor-anchor distance from fluorescent images is complicated by several sources of uncertainties, such as unavoidable random errors in the computational localization of fluorescent spots. To evaluate the experimental conditions under which one can expect to detect and quantify loop extrusion despite such complications, I first performed an analysis in silico using polymer simulations that account for loop extrusion. Using these simulations, I also tested and validated novel analysis methods to quantify chromatin loop dynamics from static imaging (e. g. from DNA-FISH images of loop anchors), and to estimate the lifetime and frequency of anchor contacts, as well as the effective loop extrusion speed from dynamic imaging in vivo. Using the simulation results as guidelines, we fluorescently labelled multiple loop and TAD anchors in human cells using the CRISPR/Cas9 system and tracked the loop anchors by live-cell imaging. Based on our analysis of the imaging data, we found that contacts between the two loop anchors are infrequent and short-lived as compared to the cell cycle duration. However, loops were found to be almost constantly extruded by cohesin. By comparing simulations and experimental data, we could estimate key biophysical parameters of loop extrusion dynamics including loop lifetimes and extrusion speed. Our results suggest that cohesin-dependent loop extrusion is a highly dynamic process, which creates transient long-range interactions rather than stable contact s. Our findings will help to quantitatively understand biological processes that involve short-lived but long-range contacts created by loop extrusion, including mechanisms of DNA repair and gene regulation

La réparation des cassures double-brin de l'ADN (DSB) est essentielle pour préserver l'intégrité du génome. Suite à l'apparition de DSB dans le génome, la PI3K kinase ATM permet la phosphorylation du variant d'histone H2AX sur un large domaine chromatinien de l'ordre du mégabase, qui constituera ainsi un foyer de réparation. La façon dont ces foyers sont assemblés aussi rapidement pour établir un environnement nucléaire favorable à la réparation n'est pas encore connue. Les TAD (Topologically Associated Domains) correspondent à des régions chromatiniennes organisées en 3D dans le noyau et sont déjà connus comme étant impliqués dans des processus cellulaires tels que la transcription ou la réplication. Cependant leur rôle dans la réparation de l'ADN n'est pas encore connu à ce jour. Nous avons ainsi pu montrer que les TAD sont des unités fonctionnelles de la réponse cellulaire aux dommages à l'ADN puisqu'ils servent de matrice à la formation des foyers de réparation. En effet, nous avons montré que la phosphorylation de H2AX sur un TAD entier est permise grâce à un processus d'extrusion de boucle de chromatine dépendant des cohésines et ayant lieu de part et d'autre de chaque DSB. Ces travaux ont permis de montrer le rôle majeur de la conformation des chromosomes dans la maintenance de l'intégrité du génome tout en mettant en évidence pour la première fois un exemple de modification de la chromatine grâce au processus d'extrusion de boucle de chromatine. D'autre part, nous avons montré que les TAD du génome entier sont renforcés en réponse aux DSB et jouent un rôle majeur dans la répression transcriptionnelle qui a lieu en cis des DSB. Enfin, nous avons démontré que des TAD entiers contenant une DSB sont capables de se déplacer au sein du noyau pour se regrouper entre eux en G1. De façon importante, nous avons montré que ce regroupement des TAD endommagés peut conduire à la formation de translocations, évènement pouvant mener à l'apparition de cancers
DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity. Upon DSBs, the ATM PI3K kinase rapidly triggers the establishment of a megabase-sized, ƴH2AXdecorated chromatin domains which further act as seeds for the formation of DNA Damage Response (DDR) foci. How these foci are rapidly assembled in order to establish a "repairprone" environment within the nucleus is yet unclear. Topologically Associating Domains (TADs) are a key feature of 3D genome organization that regulate transcription and replication, but little is known about their contribution to DNA repair processes. We found that TADs are functional units of the DDR, instrumental for the correct establishment of ƴH2AX/53BP1 chromatin domains in a manner that involves cohesin-mediated loop extrusion on both sides of the DSB. Indeed, we showed that H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. This work highlights the critical impact of chromosome conformation in the maintenance of genome integrity and provides the first example of a chromatin modification established by loop extrusion. In another hand, we found that TADs of the wole genome are reinforced following DSB induction and that TADs play a major role in the down-regulation of the transcription which takes place in cis of DSBs. Finally, we found that damaged-TADs can move across the nucleus to cluster together in the G1 phase of the cell cycle. We also found that damaged-TADs clustering can lead to the formation of translocations, which are often at the origin of cancers

Following the discovery of the one-dimensional sequence of human DNA, much focus has been directed on microscopy and molecular techniques to learn about the spatial organization of chromatin in a 3D cell. The development of these powerful tools has enabled high-resolution, genome-wide analysis of chromosome structure under many different conditions. In this thesis, I focus on how the organization of interphase chromatin is established and maintained following mitosis. Mitotic chromosomes are folded into helical loop arrays creating short and condensed chromosomes, while interphase chromosomes are decondensed and folded into a number of structures at different length scales ranging from loops between CTCF sites, enhancers and promoters to topologically associating domains (TADs), and larger compartments. While the chromatin organization at these two very different states is well defined, the transition from a mitotic to interphase chromatin state is not well understood. The aim of this thesis is to determine how interphase chromatin is organized following mitotic chromosome decondensation and to interrogate factors potentially responsible for driving the transition. First, I determine the temporal order with which CTCF-loops, TADs, and compartments reform as cells exit mitosis, revealing a unique structure at the anaphase-telophase transition never observed before. Second, I test the role of transcription in reformation of 3D chromosome structure and show that active transcription is not required for the formation of most interphase chromatin features; instead, I propose that transcription relies on the proper formation of these structures. Finally, I show that RNA in the interphase nucleus can be degraded with only slight consequences on the overall chromatin organization, suggesting that once interphase chromatin structures are achieved, the structures are stable and RNA is only required to reduce the mixing of active and inactive compartments. Together, these studies further our understanding of how interphase structures form, how these structures relate to functional activities of the interphase cell, and the stability of chromatin structures over time.