Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin.

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.

Polymer Simulations of Heteromorphic Chromatin Predict the 3D Folding of Complex Genomic Loci.

Chromatin folded into 3D macromolecular structures is often analyzed by chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) techniques, but these frequently provide contradictory results. Chromatin can be modeled as a simple polymer composed of a connected chain of units. By embedding data for epigenetic marks (H3K27ac), chromatin accessibility (assay for transposase-accessible chromatin using sequencing [ATAC-seq]), and structural anchors (CCCTC-binding factor [CTCF]), we developed a highly predictive heteromorphic polymer (HiP-HoP) model, where the chromatin fiber varied along its length; combined with diffusing protein bridges and loop extrusion, this model predicted the 3D organization of genomic loci at a population and single-cell level. The model was validated at several gene loci, including the complex Pax6 gene, and was able to determine locus conformations across cell types with varying levels of transcriptional activity and explain different mechanisms of enhancer use. Minimal a priori knowledge of epigenetic marks is sufficient to recapitulate complex genomic loci in 3D and enable predictions of chromatin folding paths.

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types.

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

Predicting genome organisation and function with mechanistic modelling.

Fitting-free mechanistic models based on polymer simulations predict chromatin folding in 3D by focussing on the underlying biophysical mechanisms. This class of models has been increasingly used in conjunction with experiments to study the spatial organisation of eukaryotic chromosomes. Feedback from experiments to models leads to successive model refinement and has previously led to the discovery of new principles for genome organisation. Here, we review the basis of mechanistic polymer simulations, explain some of the more recent approaches and the contexts in which they have been useful to explain chromosome biology, and speculate on how they might be used in the future.

High-resolution simulations of chromatin folding at genomic rearrangements in malignant B cells provide mechanistic insights into proto-oncogene deregulation.

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to 3D genome structure remains poorly understood. Here, we used the highly predictive heteromorphic polymer (HiP-HoP) model to predict chromatin conformations at the proto-oncogene CCND1 in healthy and malignant B cells. After confirming that the model gives good predictions of Hi-C data for the nonmalignant human B cell-derived cell line GM12878, we generated predictions for two cancer cell lines, U266 and Z-138. These possess genome rearrangements involving CCND1 and the immunoglobulin heavy locus (IGH), which we mapped using targeted genome sequencing. Our simulations showed that a rearrangement in U266 cells where a single IGH super-enhancer is inserted next to CCND1 leaves the local topologically associated domain (TAD) structure intact. We also observed extensive changes in enhancer-promoter interactions within the TAD, suggesting that it is the downstream chromatin remodeling which gives rise to the oncogene activation, rather than the presence of the inserted super-enhancer DNA sequence per se. Simulations of the IGH-CCND1 reciprocal translocation in Z-138 cells revealed that an oncogenic fusion TAD is created, encompassing CCND1 and the IGH super-enhancers. We predicted how the structure and expression of CCND1 changes in these different cell lines, validating this using qPCR and fluorescence in situ hybridization microscopy. Our work demonstrates the power of polymer simulations to predict differences in chromatin interactions and gene expression for different translocation breakpoints.

Epigenomic translocation of H3K4me3 broad domains over oncogenes following hijacking of super-enhancers.

Chromosomal translocations are important drivers of haematological malignancies whereby proto-oncogenes are activated by juxtaposition with enhancers, often called enhancer hijacking We analyzed the epigenomic consequences of rearrangements between the super-enhancers of the immunoglobulin heavy locus (IGH) and proto-oncogene CCND1 that are common in B cell malignancies. By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we characterized the normal chromatin landscape of the human IGH locus and its dynamics after pathological genomic rearrangement. We detected an H3K4me3 broad domain (BD) within the IGH locus of healthy B cells that was absent in samples with IGH-CCND1 translocations. The appearance of H3K4me3-BD over CCND1 in the latter was associated with overexpression and extensive chromatin accessibility of its gene body. We observed similar cancer-specific H3K4me3-BDs associated with hijacking of super-enhancers of other common oncogenes in B cell (MAF, MYC, and FGFR3/NSD2) and T cell malignancies (LMO2, TLX3, and TAL1). Our analysis suggests that H3K4me3-BDs can be created by super-enhancers and supports the new concept of epigenomic translocation, in which the relocation of H3K4me3-BDs from cell identity genes to oncogenes accompanies the translocation of super-enhancers.

Nonequilibrium dynamics and action at a distance in transcriptionally driven DNA supercoiling.

We study the effect of transcription on the kinetics of DNA supercoiling in three dimensions by means of Brownian dynamics simulations of a single-nucleotide-resolution coarse-grained model for double-stranded DNA. By explicitly accounting for the action of a transcribing RNA polymerase (RNAP), we characterize the geometry and nonequilibrium dynamics of the ensuing twin supercoiling domains. Contrary to the typical textbook picture, we find that the generation of twist by RNAP results in the formation of plectonemes (writhed DNA) some distance away. We further demonstrate that this translates into an "action at a distance" on DNA-binding proteins; for instance, positive supercoils downstream of an elongating RNAP destabilize nucleosomes long before the transcriptional machinery reaches the histone octamer. We also analyze the relaxation dynamics of supercoiled double-stranded DNA, and characterize the widely different timescales of twist diffusion, which is a simple and fast process, and writhe relaxation, which is much slower and entails multiple steps.

Computational design of probes to detect bacterial genomes by multivalent binding.

Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target-probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.

Simulating the chromatin-mediated phase separation of model proteins with multiple domains.

We perform simulations of a system containing simple model proteins and a polymer representing chromatin. We study the interplay between protein-protein and protein-chromatin interactions, and the resulting condensates that arise due to liquid-liquid phase separation, or a via a "bridging-induced attraction" mechanism. For proteins that interact multivalently, we obtain a phase diagram which includes liquid-like droplets, droplets with absorbed polymer, and coated polymer regimes. Of particular interest is a regime where protein droplets only form due to interaction with the polymer; here, unlike a standard phase separating system, droplet density rather than size varies with the overall protein concentration. We also observe that protein dynamics within droplets slow down as chromatin is absorbed. If the protein-protein interactions have a strictly limited valence, fractal or gel-like condensates are instead observed. A specific example that inspired our model is heterochromatin protein 1, or HP1. Recent in vivo experiments have shown that HP1 exhibits similar droplet size buffering behavior as our simulations. Overall, our results provide biologically relevant insights into the general nature of protein-chromatin condensates in living cells.

Model for Quorum-Sensing Mediated Stochastic Biofilm Nucleation.

Surface-attached bacterial biofilms cause disease and industrial biofouling, as well as being widespread in the natural environment. Density-dependent quorum sensing is one of the mechanisms implicated in biofilm initiation. Here we present and analyze a model for quorum-sensing triggered biofilm initiation. In our model, individual, planktonic bacteria adhere to a surface, proliferate, and undergo a collective transition to a biofilm phenotype. This model predicts a stochastic transition between a loosely attached, finite layer of bacteria near the surface and a growing biofilm. The transition is governed by two key parameters: the collective transition density relative to the carrying capacity and the immigration rate relative to the detachment rate. Biofilm initiation is complex, but our model suggests that stochastic nucleation phenomena may be relevant.

Nucleosome positions alone can be used to predict domains in yeast chromosomes.

We use molecular dynamics simulations based on publicly available micrococcal nuclease sequencing data for nucleosome positions to predict the 3D structure of chromatin in the yeast genome. Our main aim is to shed light on the mechanism underlying the formation of chromosomal interaction domains, chromosome regions of around 0.5 to 10 kbp which show enriched self-interactions, which were experimentally observed in recent MicroC experiments (importantly these are at a different length scale from the 100- to 1,000-kbp-sized domains observed in higher eukaryotes). We show that the sole input of nucleosome positioning data is already sufficient to determine the patterns of chromatin interactions and domain boundaries seen experimentally to a high degree of accuracy. Since the nucleosome spacing so strongly affects the larger-scale domain structure, we next examine the genome-wide linker-length distribution in more detail, finding that it is highly irregular and varies in different genomic regions such as gene bodies, promoters, and active and inactive genes. Finally we use our simple simulation model to characterize in more detail how irregular nucleosome spacing may affect local chromatin structure.

capC-MAP: software for analysis of Capture-C data.

SUMMARY: Capture-C is a member of the chromosome-conformation-capture family of experimental methods which probes the 3D organization of chromosomes within the cell nucleus. It provides high-resolution information on the genome-wide chromatin interactions from a set of 'target' genomic locations, and is growing in popularity as a tool for improving our understanding of cis-regulation and gene function. Yet, analysis of the data is complicated, and to date there has been no dedicated or easy-to-use software to automate the process. We present capC-MAP, a software package for the analysis of Capture-C data. AVAILABILITY AND IMPLEMENTATION: Implemented with both ease of use and flexibility in mind, capC-MAP is a suit of programs written in C++ and Python, where each program can be run separately, or an entire analysis can be performed with a single command line. It is available under an open-source licence at https://github.com/cbrackley/capC-MAP, as well as via the conda package manager, and should run on any standard Unix-style system. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptional Bursts in a Nonequilibrium Model for Gene Regulation by Supercoiling.

We analyze transcriptional bursting within a stochastic nonequilibrium model, which accounts for the coupling between the dynamics of DNA supercoiling and gene transcription. We find a clear signature of bursty transcription when there is a separation between the timescales of transcription initiation and supercoiling dissipation (the latter may either be diffusive or mediated by topological enzymes, such as type I or type II topoisomerases). In multigenic DNA domains, we observe either bursty transcription or transcription waves; the type of behavior can be selected for by controlling gene activity and orientation. In the bursty phase, the statistics of supercoiling fluctuations at the promoter are markedly non-Gaussian.

Simulated binding of transcription factors to active and inactive regions folds human chromosomes into loops, rosettes and topological domains.

Biophysicists are modeling conformations of interphase chromosomes, often basing the strengths of interactions between segments distant on the genetic map on contact frequencies determined experimentally. Here, instead, we develop a fitting-free, minimal model: bivalent or multivalent red and green 'transcription factors' bind to cognate sites in strings of beads ('chromatin') to form molecular bridges stabilizing loops. In the absence of additional explicit forces, molecular dynamic simulations reveal that bound factors spontaneously cluster-red with red, green with green, but rarely red with green-to give structures reminiscent of transcription factories. Binding of just two transcription factors (or proteins) to active and inactive regions of human chromosomes yields rosettes, topological domains and contact maps much like those seen experimentally. This emergent 'bridging-induced attraction' proves to be a robust, simple and generic force able to organize interphase chromosomes at all scales.

Ephemeral Protein Binding to DNA Shapes Stable Nuclear Bodies and Chromatin Domains.

Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear bodies exchange rapidly with the soluble pool while the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins able to switch between an "on" (binding) and an "off" (nonbinding) state. This system provides a model for any DNA-binding protein that can be posttranslationally modified to change its affinity for DNA (e.g., through phosphorylation). Protein switching is a nonequilibrium process, and it leads to the formation of clusters of self-limiting size, where individual proteins in a cluster exchange with the soluble pool with kinetics similar to those seen in photobleaching experiments. This behavior contrasts sharply with that exhibited by nonswitching proteins, which are permanently in the on-state; when these bind to DNA nonspecifically, they form clusters that grow indefinitely in size. To explain these findings, we propose a mean-field theory from which we obtain a scaling relation between the typical cluster size and the protein switching rate. Protein switching also reshapes intrachromatin contacts to give networks resembling those seen in topologically associating domains, as switching markedly favors local (short-range) contacts over distant ones. Our results point to posttranslational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, nonequilibrium, protein clusters with the properties of nuclear bodies.

Exploiting native forces to capture chromosome conformation in mammalian cell nuclei.

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure-to-function relationship. However, all 3C-based methods rely on chemical cross-linking to stabilize spatial interactions. This step remains a "black box" as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed "i3C", a novel approach for capturing spatial interactions without a need for cross-linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation-based orthogonal validation method, "TALE-iD", we show that native interactions resemble cross-linked ones, but display improved signal-to-noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.

Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization.

Molecular dynamics simulations are used to model proteins that diffuse to DNA, bind, and dissociate; in the absence of any explicit interaction between proteins, or between templates, binding spontaneously induces local DNA compaction and protein aggregation. Small bivalent proteins form into rows [as on binding of the bacterial histone-like nucleoid-structuring protein (H-NS)], large proteins into quasi-spherical aggregates (as on nanoparticle binding), and cylinders with eight binding sites (representing octameric nucleosomal cores) into irregularly folded clusters (like those seen in nucleosomal strings). Binding of RNA polymerase II and a transcription factor (NFκB) to the appropriate sites on four human chromosomes generates protein clusters analogous to transcription factories, multiscale loops, and intrachromosomal contacts that mimic those found in vivo. We suggest that this emergent behavior of clustering is driven by an entropic bridging-induced attraction that minimizes bending and looping penalties in the template.

Effect of DNA conformation on facilitated diffusion.

Within a living cell, site-specific DNA-binding proteins need to search the whole genome to find a target of ~10-20 bp. That they find the target, and do so quickly, is vital for the correct functioning of the DNA, and of the cell as a whole. The current understanding is that this search is performed via facilitated diffusion, i.e. by combining three-dimensional bulk diffusion within the cytoplasm or nucleoplasm, with one-dimensional diffusion along the DNA backbone, to which the protein binds non-specifically. After reviewing the standard theory of facilitated diffusion, we discuss in the present article the still rather rare direct computer simulations of this process, focusing on the three-dimensional part of the search, and the effect of DNA looping and the general DNA conformation on its efficiency. We close by highlighting some open questions in this field.

Transcription modulates chromatin dynamics and locus configuration sampling.

In living cells, the 3D structure of gene loci is dynamic, but this is not revealed by 3C and FISH experiments in fixed samples, leaving a notable gap in our understanding. To overcome these limitations, we applied the highly predictive heteromorphic polymer (HiP-HoP) model to determine chromatin fiber mobility at the Pax6 locus in three mouse cell lines with different transcription states. While transcriptional activity minimally affects movement of 40-kbp regions, we observed that motion of smaller 1-kbp regions depends strongly on local disruption to chromatin fiber structure marked by H3K27 acetylation. This also substantially influenced locus configuration dynamics by modulating protein-mediated promoter-enhancer loops. Importantly, these simulations indicate that chromatin dynamics are sufficiently fast to sample all possible locus conformations within minutes, generating wide dynamic variability within single cells. This combination of simulation and experimental validation provides insight into how transcriptional activity influences chromatin structure and gene dynamics.

A max-plus model of ribosome dynamics during mRNA translation.

We examine the dynamics of the translation stage of cellular protein production, in which ribosomes move uni-directionally along an mRNA strand, building amino acid chains as they go. We describe the system using a timed event graph-a class of Petri net useful for studying discrete events, which have to satisfy constraints. We use max-plus algebra to describe a deterministic version of the model, where the constraints represent steric effects which prevent more than one ribosome reading a given codon at a given time and delays associated with the availability of the different tRNAs. We calculate the protein production rate and density of ribosomes on the mRNA and find exact agreement between these analytical results and numerical simulations of the deterministic model, even in the case of heterogeneous mRNAs.