The ParB clamp docks onto Smc for DNA loading via a joint-ParB interface

Chromosomes readily unlink from one another and segregate to daughter cells during cell division highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes mediate chromosome folding by DNA loop extrusion. In most bacteria, SMC complexes start loop extrusion at the ParB/parS partition complex formed near the replication origin. Whether they are recruited by recognizing a specific DNA structure in the partition complex or a protein component is unknown. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate chromosomes when transplanted into another organism. Using chimeric proteins and chemical cross-linking, we find that ParB binds to the Smc subunit directly. We map a binding interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex to initiate DNA loop extrusion.

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.

Proteins mediating different DNA topologies block RNAP elongation with different efficiency

Many DNA-binding proteins induce topological structures such as loops or wraps through binding to two or more sites along the DNA. Such topologies may regulate transcription initiation and may also be roadblocks for elongating RNA polymerase (RNAP). Remarkably, a lac repressor protein bound to a weak binding site (O2) does not obstruct RNAP in vitro but becomes an effective roadblock when securing a loop of 400 bp between two widely separated binding sites. To investigate whether topological structures mediated by proteins bound to closely spaced binding sites and interacting cooperatively also represent roadblocks, we compared the effect of the lambda CI and 186 CI repressors on RNAP elongation. Dimers of lambda CI can bind to two sets of adjacent sites separated by hundreds of bp and form a DNA loop via the interaction between their C-terminal domains. The 186 CI protein can form a wheel of seven dimers around which specific DNA binding sequences can wrap. Atomic force microscopy (AFM) was used to image transcription elongation complexes of DNA templates that contained binding sites for either the lambda or 186 CI repressor. While RNAP elongated past lambda CI on unlooped DNA, as well as past 186 CI-wrapped DNA, it did not pass the lambda CI-mediated loop. These results may indicate that protein-mediated loops with widely separated binding sites more effectively block transcription than a wrapped topology with multiple, closely spaced binding sites.

A hold-and-feed mechanism drives directional DNA loop extrusion by condensin

SMC protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop in two separate chambers. Single-molecule and cryo-electron microscopy provide evidence for a power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon ATP binding, while the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of 'motor' and 'anchor' chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the course of the SMC reaction cycle determines the directionality of DNA loop extrusion.

STAG2 promotes the myelination transcriptional program in oligodendrocytes

SUMMARYCohesin folds chromosomes via DNA loop extrusion. Cohesin-mediated chromosome loops regulate transcription by shaping long-range enhancer-promoter interactions, among other mechanisms. Mutations of cohesin subunits and regulators cause human developmental diseases termed cohesinopathy. Vertebrate cohesin consists of SMC1, SMC3, RAD21, and either STAG1 or STAG2. To probe the physiological functions of cohesin, we created conditional knockout (cKO) mice with Stag2 deleted in the nervous system. Stag2 cKO mice exhibit growth retardation, neurological defects, and premature death, in part due to insufficient myelination of nerve fibers. Stag2 cKO oligodendrocytes exhibit delayed maturation and downregulation of myelination-related genes. Stag2 loss reduces promoter-anchored loops at downregulated genes in oligodendrocytes. Thus, STAG2-cohesin generates promoter-anchored loops at myelination-promoting genes to facilitate their transcription. Our study implicates defective myelination as a contributing factor to cohesinopathy and establishes oligodendrocytes as a relevant cell type to explore the mechanisms by which cohesin regulates transcription.

A low Smc flux avoids collisions and facilitates chromosome organization in B. subtilis

SMC complexes are widely conserved ATP-powered DNA-loop-extrusion motors indispensable for organizing and faithfully segregating chromosomes. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength, and the distribution of Smc loading sites, the residency time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.

Chromatin insulation dynamics in glioblastoma: challenges and future perspectives of precision oncology

Glioblastoma (GBM) is the most aggressive primary brain tumor, having a poor prognosis and a median overall survival of less than two years. Over the last decade, numerous findings regarding the distinct molecular and genetic profiles of GBM have led to the emergence of several therapeutic approaches. Unfortunately, none of them has proven to be effective against GBM progression and recurrence. Epigenetic mechanisms underlying GBM tumor biology, including histone modifications, DNA methylation, and chromatin architecture, have become an attractive target for novel drug discovery strategies. Alterations on chromatin insulator elements (IEs) might lead to aberrant chromatin remodeling via DNA loop formation, causing oncogene reactivation in several types of cancer, including GBM. Importantly, it is shown that mutations affecting the isocitrate dehydrogenase (IDH) 1 and 2 genes, one of the most frequent genetic alterations in gliomas, lead to genome-wide DNA hypermethylation and the consequent IE dysfunction. The relevance of IEs has also been observed in a small population of cancer stem cells known as glioma stem cells (GSCs), which are thought to participate in GBM tumor initiation and drug resistance. Recent studies revealed that epigenomic alterations, specifically chromatin insulation and DNA loop formation, play a crucial role in establishing and maintaining the GSC transcriptional program. This review focuses on the relevance of IEs in GBM biology and their implementation as a potential theranostic target to stratify GBM patients and develop novel therapeutic approaches. We will also discuss the state-of-the-art emerging technologies using big data analysis and how they will settle the bases on future diagnosis and treatment strategies in GBM patients.