A theory of the dynamics of DNA loop initiation in condensin/cohesin complexes

The structural maintenance of chromosome complexes exhibit the remarkable ability to actively extrude DNA, which has led to the appealing and popular "loop extrusion" model to explain one of the most important processes in biology: the compaction of chromatin during the cell cycle. A potential mechanism for the action of extrusion is the classic Brownian ratchet, which requires short DNA loops to overcome an initial enthalpic barrier to bending, before favoured entropic growth of longer loops. We present a simple model of the constrained dynamics of DNA loop formation based on a frictional worm like chain, where for circular loops of order, or smaller than the persistence length, internal friction to bending dominates solvent dynamics. Using Rayleigh's dissipation function, we show how bending friction can be translated to simple one dimensional diffusion of the angle of the loop resulting in a Smoluchowski equation with a coordinate dependent diffusion constant. This interplay between Brownian motion, bending dissipation and geometry of loops leads to a qualitatively new phenomenon, where the friction vanishes for bends with an angle of exactly 180°, due to a decoupling between changes in loop curvature and angle. Using this theory and given current parameter uncertainties, we tentatively predict mean first passage times of between 1 and 10 seconds, which is of order the cycle time of ATP, suggesting spontaneous looping could be sufficient to achieve efficient initiation of looping.

Deficiency of Polη in Saccharomyces cerevisiae reveals the impact of transcription on damage-induced cohesion

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.

An Inter-strand Loop Extrusion Model

Loop extrusion convincingly describes how certain Structural Maintenance of Chromosome (SMC) proteins mediate the formation of large DNA loops. Yet, most of the existing computational models cannot reconcile the recent observations that, while per-forming cis-extrusion, condensins can traverse each other and bypass large roadblocks in vitro. In this work, we propose an inter-strand model for loop extrusion which not only reproduces the experimental features of loop extrusion by one SMC complex, but also predicts the formation of so-called “Z-loops” via the interaction of two or more SMCs extruding along the same DNA substrate. By performing Molecular Dynamics simulations of this model we discover that the experimentally observed asymmetry in the different types of Z-loops is a natural consequence of the DNA tethering in vitro. Intriguingly, our model predicts this bias to disappear in absence of tethering and a third type of Z-loop, which has not yet been identified in experiments, to appear. We conclude discussing the implications of inter-strand loop extrusion on entangled DNA.

Downregulation of SMC4 Inhibits the Progression of Cardia Adenocarcinoma via PI3K/ AKT Signaling Pathway Regulation

Background: Cardia adenocarcinoma (CA) is a subtype of gastric cancer with a high rate of local and distal recurrence and few targeted therapies. Structural maintenance of chromosome protein 4 (SMC4) is involved in the occurrence and progression of numerous malignancies, but its role and mechanism in CA are unknown.Methods: Through the Western Blot and qRT-PCR, the level of SMC4 expression was determined in CA. SMC4 knockout cells were then generated by stable transduction of BGC-823 and SGC-7901 cells. Cell proliferation was evaluated by MTT and clone formation test, Scratch and transwell tests were used to investigate cell migration as well as invasion, while through the flow cytometry, we examined the cell apoptosis and progression of the cell cycle. The regulatory effects of the epithelial-mesenchymal transition (EMT) and the PI3K/AKT pathway were investigated using Western Blot.Results: This study showed overexpression of SMC4 in various CA cells. SMC4 knockout significantly caused the inhibition of proliferation, migration, and invasion of BGC-823 and SGC-7901, and stimulate the process of apoptosis and cell cycle arrest in the G0/G1 phase. In addition, down-regulation of SMC4 resulted in decreased expression of Bcl-2, Cyclin D1, CDK4, CDK6, N-cadherin and Vimentin, with an increased level of proteins i.e Bax, caspase3, Cleaved-caspase3, P21, and E-cadherin. SMC4 knockout also reduced the phosphorylated protein levels of AKT, PI3K, and mTOR, while keeping their total protein levels constant.Conclusion: Downregulation of SMC4 can inhibit the biological progression of CA, suggesting that SMC4 could be a potential therapeutic target for the disease.

Bridging-induced phase separation induced by cohesin SMC protein complexes

Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo

ObjectiveTherapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo.DesignHBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events.ResultsBoth siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6.ConclusionThese results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Hit the brakes – a new perspective on the loop extrusion mechanism of cohesin and other SMC complexes

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.

Condensin complexes: understanding loop extrusion one conformational change at a time

Condensin and cohesin, both members of the structural maintenance of chromosome (SMC) family, contribute to the regulation and structure of chromatin. Recent work has shown both condensin and cohesin extrude DNA loops and most likely work via a conserved mechanism. This review focuses on condensin complexes, highlighting recent in vitro work characterising DNA loop formation and protein structure. We discuss similarities between condensin and cohesin complexes to derive a possible mechanistic model, as well as discuss differences that exist between the different condensin isoforms found in higher eukaryotes.

DNA break formation induces Scc2/cohesin-dependent recruitment of condensin to meiotic chromosomes

Meiotic chromosome pairing, recombination, and fertility depends on the conserved loop-axis architecture of meiotic chromosomes. This architecture is modulated by condensin, a structural maintenance of chromosome (SMC) complex that catalyzes chromatin loop formation. Here, we investigated how condensin is recruited to meiotic chromosomes in Saccharomyces cerevisiae. We show that double-strand-break (DSB) formation, the initiating event of meiotic recombination, causes condensin redistribution from the nucleolus to DSB hotspots, pericentromeric regions, and axis attachment sites. Hotspot association of condensin correlates weakly with break probability but does not depend on local DSB formation, whereas association with axis sites and pericentromeric regions depends on the Scc2-associated pool of cohesin, another SMC complex. Intriguingly, Scc2 distribution also changes in response to DSB formation. As condensin and Scc2-cohesin both catalyze chromatin loop extrusion, their redistribution upon DSB formation implies a profound change in chromatin loop dynamics that may help promote proper chromosome pairing and DNA repair.