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Chromatin is an essential component of nuclear mechanical response and shape that maintains nuclear compartmentalization and function. The biophysical properties of chromatin alter nuclear shape and stability, but little is known about whether or how major genomic functions can impact the integrity of the nucleus. We hypothesized that transcription might affect cell nuclear shape and rupture through its effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with the RNA polymerase II inhibitor alpha-amanitin in wild type cells and perturbed cells that present increased nuclear blebbing. Transcription inhibition suppresses nuclear blebbing for several cell types, nuclear perturbations, and transcription inhibitors. Furthermore, transcription is necessary for robust nuclear bleb formation, bleb stabilization, and bleb-based nuclear ruptures. These morphological effects appear to occur through a novel biophysical pathway, since transcription does not alter either chromatin histone modification state or nuclear rigidity, which typically control nuclear blebbing. We find that active/phosphorylated RNA pol II Ser5, marking transcription initiation, is enriched in nuclear blebs relative to DNA. Thus, transcription initiation is a hallmark of nuclear blebs. Polymer simulations suggest that motor activity within chromatin, such as that of RNA pol II, can generate active forces that deform the nuclear periphery, and that nuclear deformations depend on motor dynamics. Our data provide evidence that the genomic function of transcription impacts nuclear shape stability, and suggests a novel mechanism, separate and distinct from chromatin rigidity, for regulating large-scale nuclear shape and function.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging of CTCF-cohesin collisions, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-electron microscopy structure of the intact cohesin-CTCF complex reveals that this CTCF motif ahead of zinc-fingers can only reach its binding site on the STAG1 cohesin subunit when the N-terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling is intrinsic to cohesin itself, and other proteins can substitute for CTCF in fruit flies and other eukaryotes. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries. Our results provide direct evidence that CTCF orientation and R-loops shape the 3D genome by directly regulating cohesin.

Objective. To develop a metaphase chromosome model representing the complete genome of a human lymphocyte cell to support microscopic Monte Carlo (MMC) simulation-based radiation-induced DNA damage studies. Approach. We first employed coarse-grained polymer physics simulation to obtain a rod-shaped chromatid segment of 730 nm in diameter and 460 nm in height to match Hi–C data. We then voxelized the segment with a voxel size of 11 nm per side and connected the chromatid with 30 types of pre-constructed nucleosomes and 6 types of linker DNAs in base pair (bp) resolutions. Afterward, we piled different numbers of voxelized chromatid segments to create 23 pairs of chromosomes of 1–5 μm long. Finally, we arranged the chromosomes at the cell metaphase plate of 5.5 μm in radius to create the complete set of metaphase chromosomes. We implemented the model in gMicroMC simulation by denoting the DNA structure in a four-level hierarchical tree: nucleotide pairs, nucleosomes and linker DNAs, chromatid segments, and chromosomes. We applied the model to compute DNA damage under different radiation conditions and compared the results to those obtained with G0/G1 model and experimental measurements. We also performed uncertainty analysis for relevant simulation parameters. Main results. The chromatid segment was successfully voxelized and connected in bps resolution, containing 26.8 mega bps (Mbps) of DNA. With 466 segments, we obtained the metaphase chromosome containing 12.5 Gbps of DNA. Applying it to compute the radiation-induced DNA damage, the obtained results were self-consistent and agreed with experimental measurements. Through the parameter uncertainty study, we found that the DNA damage ratio between metaphase and G0/G1 phase models was not sensitive to the chemical simulation time. The damage was also not sensitive to the specific parameter settings in the polymer physics simulation, as long as the produced metaphase model followed a similar contact map distribution. Significance. Experimental data reveal that ionizing radiation induced DNA damage is cell cycle dependent. Yet, DNA chromosome models, except for the G0/G1 phase, are not available in the state-of-the-art MMC simulation. For the first time, we successfully built a metaphase chromosome model and implemented it into MMC simulation for radiation-induced DNA damage computation.

International audience; Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing minor roles for cross-links and topological effects and challenging the view that interphase chromatin is a gel-like material. Our technology opens avenues for future research in areas from chromosome mechanics to genome functions.

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 a new type of 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.Significance StatementLoop extrusion by cohesin is critical to folding the mammalian genome into loops. Extrusion can be halted by CTCF proteins bound at specific genomic loci, which generates chromosomal domains and can regulate gene expression. However, the process of transcription itself can modulate cohesin, thus refolding chromosomes near active genes. Through experiments and simulations, we show that transcribing RNA polymerases (RNAPs) act as “moving barriers” to loop-extruding cohesins. Unlike stationary CTCF barriers, RNAPs actively relocalize cohesins, which generates characteristic patterns of spatial organization around active genes. Our model predicts that the barrier function of RNAP can explain why cohesin accumulates at active promoters and provides a mechanism for clustering active promoters. Through transcription-extrusion interactions, cells might dynamically regulate functional genomic contacts.