Biology subjects

Brackley, C. A.

Publications and source records attributed to Brackley, C. A..

18 recordsLinked to original sources

bioRxiv2026

CpG islands act as topological sinks for transcription-induced DNA supercoiling

Strong evolutionary selection has maintained CpG-dense islands (CGIs) at the promoters of constitutively expressed genes throughout the vertebrate genome, suggesting an important role in regulating DNA topology. Here, using Twist-seq, a psoralen-based approach for quantitative genome-wide profiling of DNA supercoiling, we reveal distinct topological states across human gene promoters. We show that CGI promoters accumulate elevated levels of negative supercoiling relative to non-CGI promoters and define localised topological domains at highly transcribed genes. Integrating genome-wide analyses with reaction-diffusion modelling and coarse-grained molecular dynamics simulations, we find that this behaviour is encoded by the intrinsic physical properties of CGI DNA. The GC-rich sequence context promotes nucleosome depletion and focuses torsional stress onto embedded AT-rich pockets, driving localised DNA melting and plectoneme-tip bubble formation within promoter-proximal nucleosome-free regions. This provides an energetically favourable pathway for redistributing transcription-induced torsional stress through transient strand separation and writhe, consistent with increased ssDNA formation at CGI promoters observed by ssDNA-seq. We propose that CGIs function as sequence-encoded topological sinks that buffer supercoiling while maintaining a promoter architecture permissive for transcription initiation, thereby preserving promoter integrity and genome stability.

molecular biology↗

bioRxiv2026

Irregular nucleosome positioning governs a crystalline to liquid-like phase transition and tunes chromatin accessibility

Chromatin must fold tightly enough to protect the genome while being sufficiently accessible for DNA dependent processes such as transcription. The physical rules that balance these competing roles remain unclear, as DNA sequence encodes both biochemical information such as transcription factor binding sites, and biophysical cues that shape chromatin structure. Here, using synthetic chromatin fibres assembled from physiologically relevant DNA sequences, we show that nucleosome positioning dictates the material state of chromatin. Heterochromatin-like sequences produce compact fibres stabilised by nucleosome stacking, whereas euchromatin-like sequences generate irregular nucleosome positioning that yields disrupted, heterogeneous, and mechanically deformable fibres. Quantitative polymer modelling reveals that these irregular arrays are highly dynamic, continually sampling a broad ensemble of conformations as nucleosome stacking breaks down. We identify two previously unrecognised thresholds encoded by nucleosome positioning: minimal positional irregularity (2-3 bp) triggers a transition from an ordered paracrystalline state to a liquid-like phase, whereas an order of magnitude greater irregularity (~18 bp) is required to generate accessibility and mechanical fragility permissive for transcription factor binding. Euchromatin-like arrays reside at this accessibility threshold. These findings indicate that nucleosome positioning tunes chromatin toward or away from critical structural states that couple genome protection, chromatin dynamics, and transcriptional potential, providing a physical mechanism that helps connect DNA sequence to gene expression.

molecular biology↗

bioRxiv2026

Symmetry and force response of cohesin loop extrusion are determined by diffusion of its motor and anchor domains

Cohesin, a Structural Maintenance of Chromosomes (SMC) complex is thought to organize genomes by generating DNA loops, yet the molecular basis of loop extrusion remains incompletely understood. Here, we show that a single S. pombe cohesin complex extrudes DNA loops both symmetrically or asymmetrically, depending on the external force applied to DNA and that the mode and speed are controlled by stochastic switching of the cohesin motor between driving and diffusive states. We directly measured the weak forces generated during cohesin-mediated loop extrusion while simultaneously visualizing DNA loops and found that forces as low as 0.05 pN pull DNA out of cohesin loop without disrupting cohesin-DNA interaction. We further identified the Scc3 subunit as a diffusive DNA anchor required for loop extrusion and showed that its physical separation from the motor domain by unstructured regions of Scc1 promotes loop-extrusion initiation. Together, our results support a model in which cohesin loop extrusion is limited and tuned by diffusive motion of both anchor and motor modules, providing a mechanistic framework for how weak and diffusive cohesin-DNA interactions could modulate cohesin function in chromatin organization.

biophysics↗

bioRxiv2023

The discovery of an evolutionarily conserved enhancer within the MYEOV locus suggests an unexpected role for this non-coding region in cancer

The myeloma overexpressed gene (MYEOV) has been proposed to be a proto-oncogene due to high RNA transcript levels found in multiple cancers, including myeloma, breast, lung, pancreas and esophageal cancer. The presence of an open reading frame (ORF) in humans and other primates suggests protein-coding potential. Yet, we still lack evidence of a functional MYEOV protein. It remains undetermined how MYEOV overexpression affects cancerous tissues. In this work, we show that MYEOV has originated and may still function as an enhancer, possibly regulating CCND1. Firstly, MYEOV 3 end enhancer activity was confirmed in humans using publicly available ATAC-STARR-seq data, performed on B-cell-derived GM12878 cells. We detected enhancer histone marks H3K4me1 and H3K27ac overlapping MYEOV in multiple healthy human tissues, which include B cells, liver and lung tissue. The analysis of 3D genome datasets revealed chromatin interactions between the MYEOV-3-enhancer and the proto-oncogene CCND1. BLAST searches and multi-sequence alignments results showed that this human enhancer element is conserved from the amphibians/amniotes divergence, with a 273 bp conserved region also found in all mammals, and even in chickens, and it consistently located near the corresponding CCND1 orthologues. Furthermore, we observed conservation of active enhancer state in the MYEOV orthologues of four non-human primates, dog, rat and mouse. When studying this homologous region in mice, where the ORF of MYEOV is absent, we not only observed an enhancer chromatin state but also found interactions between the mouse enhancer homolog and Ccnd1 using 3D-genome interaction data. This is similar to the interaction observed in humans and, interestingly, coincides with CTCF binding sites in both species. Taken together, this suggests that MYEOV is a primate-specific gene with a de novo ORF that originated at an evolutionarily older enhancer region. This deeply conserved enhancer element is possibly regulating CCND1 in both humans and mice, opening the possibility of studying MYEOV regulatory functions in cancer using non-primate animal models.

genomics↗

bioRxiv2022

Urine production rate is critical in a model for catheter-associated urinary tract infection

Urinary catheters are used extensively in hospitals and long-term care and they are highly prone to infection. Understanding the pathways by which bacteria colonise a urinary catheter could guide strategies to mitigate infection, but quantitative models for this colonisation process are lacking. Here we present a mathematical model for bacterial colonisation of a urinary catheter, that integrates population dynamics and fluid dynamics. The model describes bacteria migrating up the outside surface of the catheter, spreading into the bladder and being swept through the catheter lumen. Computer simulations of the model reveal that clinical outcomes for long-term versus short-term catheterisation are controlled by different factors: the rate of urine production by the kidneys as opposed to urethral length, catheter surface properties and bacterial motility. Our work may help explain variable susceptibility to catheter-associated urinary tract infection (CAUTI) among individuals, and the mixed success of antimicrobial surface coatings. Our model suggests that for long-term catheterised patients, increasing fluid intake or reducing residual urine volume in the bladder may help prevent infection, while antimicrobial surface coatings are predicted to be effective only for short-term catheterised patients. Therefore, different catheter management strategies could be rationally targeted to long-term vs short-term catheterised patients.

biophysics↗

bioRxiv2022

Condensin reorganizes centromeric chromatin during mitotic entry into a bipartite structure stabilized by cohesin

The Structural Maintenance of Chromosomes (SMC) complexes cohesin and condensin establish the 3D organization of mitotic chromosomes1-3. Cohesin is essential to maintain sister chromatid pairing until anaphase onset4, while condensin is important for mitotic centromere structure and elastic resistance to spindle forces5-8. Both complexes are also important to form productive kinetochore-spindle attachments6, 8, 9. How condensin and cohesin work together to shape the mitotic centromere to ensure faithful chromosome segregation remains unclear. Here we show by super-resolution imaging, Capture-C analysis and polymer modeling that vertebrate centromeres are partitioned into two distinct condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres and also in human neocentromeres devoid of satellite repeats, and is therefore a fundamental feature of vertebrate centromere identity. Super-resolution imaging reveals that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to mitotic spindle forces. In its absence, separated bipartite kinetochores frequently engage in merotelic spindle attachments. Consistently, uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. The two-domain structure of vertebrate regional centromeres described here incorporates architectural roles for both condensin and cohesin and may have implications for avoiding chromosomal instability in cancer cells.

cell biology↗

bioRxiv2022

Gene structure heterogeneity drives transcription noise within human chromosomes

Classical observations have long suggested there is a link between 3D gene structure and transcription1-4. However, due to the many factors regulating gene expression, and to the challenge of visualizing DNA and chromatin dynamics at the same time in living cells, this hypothesis has been difficult to quantitatively test experimentally. Here we take an orthogonal approach and use computer simulations, based on the known biophysical principles of genome organisation5-7, to simultaneously predict 3D structure and transcriptional output of human chromatin genome wide. We validate our model by quantitative comparison with Hi-C contact maps, FISH, GRO-seq and single-cell RNA-seq data, and provide the 3DGene resource to visualise the panoply of structures adopted by any active human gene in a population of cells. We find transcription strongly correlates with the formation of protein-mediated microphase separated clusters of promoters and enhancers, associated with clouds of chromatin loops, and show that gene noise is a consequence of structural heterogeneity. Our results also indicate that loop extrusion by cohesin does not affect average transcriptional patterns, but instead impacts transcriptional noise. These findings provide a functional role for intranuclear microphase separation, and an evolutionary mechanism for loop extrusion halted at CTCF sites, to modulate transcriptional noise.

molecular biology↗

bioRxiv2022

A 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 analyse a model for quorum-sensing triggered biofilm initiation. In our model, individual, planktonic bacteria adhere to a surface, pro-liferate 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.

biophysics↗

bioRxiv2021

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 significant gap in our understanding. To overcome these limitations we applied the "highly predictive heteromorphic polymer" (HiP-HoP) model, validated by experiments, to determine chromatin fibre mobility at the Pax6 locus in three mouse cell lines with different transcription states. While transcriptional activity minimally affects the movement of 40 kbp regions, we observed that the motion of smaller 1 kbp regions depends strongly on local disruption to chromatin fibre structure marked by H3K27 acetylation. This also significantly influenced locus configuration dynamics by modulating promoter-enhancer loops associated with protein bridging. Importantly these simulations indicate that chromatin dynamics are sufficiently fast to sample all possible conformations of loci within minutes, generating wide dynamic variability of gene loci structure within single cells. Experiments inhibiting transcription change chromatin fibre structure subtly, yet we predict they should substantially affect mobility. This combination of simulation and experimental validation provide a novel insight and mechanistic model to explain how transcriptional activity influences chromatin structure and gene dynamics.

biophysics↗

bioRxiv2021

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

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to genome 3D 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 non-malignant 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 chain 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 remodelling 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 break-points.

genomics↗

bioRxiv2020

Complex small-world regulatory networks emerge from the 3D organisation of the human genome

The discovery that overexpressing one or a few critical transcription factors can switch cell state suggests that gene regulatory networks are relatively simple. In contrast, genome-wide association studies (GWAS) point to complex phenotypes being determined by hundreds of loci that rarely encode transcription factors and which individually have small effects. Here, we use computer simulations and a simple fitting-free polymer model of chromosomes to show that spatial correlations arising from 3D genome organisation naturally lead to stochastic and bursty transcription plus complex small-world regulatory networks (where the transcriptional activity of each genomic region subtly affects almost all others). These effects require factors to be present at sub-saturating levels; increasing levels dramatically simplifies networks as more transcription units are pressed into use. Consequently, results from GWAS can be reconciled with those involving overexpression. We apply this pan-genomic model to predict patterns of transcriptional activity in whole human chromosomes, and, as an example, the effects of the deletion causing the diGeorge syndrome.

biophysics↗

bioRxiv2018

Lamina and Heterochromatin Direct Chromosome Organisation in Senescence and Progeria

Lamina-associated domains (LADs) cover a large part of the human genome and are thought to play a major role in shaping the nuclear architectural landscape. Here, we use simulations based on concepts from polymer physics to dissect the roles played by heterochromatin- and lamina-mediated interactions in nuclear organisation. Our model explains the conventional organisation of heterochromatin and euchromatin in growing cells, as well as the pathological organisation found in oncogene-induced senescence and progeria. We show that the experimentally observed changes in the locality of contacts in senescent and progeroid cells can be explained naturally as arising due to phase transitions in the system. Our model predicts that LADs are highly stochastic, and that, once established, the senescent phenotype should be metastable even if lamina-mediated interactions were reinstated. Overall, our simulations uncover a universal physical mechanism that can regulate heterochromatin segregation and LAD formation in a wide range of mammalian nuclei.

biophysics↗

bioRxiv2018

capC-MAP: A Software Package for Analysis of Capture-C data

We present capC-MAP, a software package for the analysis of Capture-C data. Capture-C is a \"many-to-all\" chromosome-conformation-capture method. We summarise the method, then detail capC-MAP, the first software specifically designed and optimised for Capture-C data. capC-MAP has been developed with ease-of-use and flexibility in mind: the entire pipe-ine can be run with a single command, or the component programs can be run individually for custom data processing, in a strategy that will suit computational as well as experimental researchers. Finally, we compare and benchmark capC-MAP against another package which can perform (though is not optimised for) analysis of Capture-C data.

genomics↗

bioRxiv2018

Nucleosome positions alone determine micro-domains in yeast chromosomes.

We use molecular dynamics simulations based on publicly available MNase-seq data for nucleosome positions to predict the 3-D structure of chromatin in the yeast genome. Our main aim is to shed light on the mechanism underlying the formation of micro-domains, chromosome regions of around 0.5-10 kbp which show enriched self-interactions, which were experimentally observed in recent MicroC experiments. 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 characterise in more detail how irregular nucleosome spacing may affect local chromatin structure.

biophysics↗

bioRxiv2018

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

Chromatin folded into 3-D macromolecular structures is often analysed by 3C and FISH techniques, but frequently provide contradictory results. Instead, chromatin can be modelled as a simple polymer comprised of a connected chain of units. By embedding data for epigenetic marks (H3K27ac), genomic disruptions (ATAC-seq) and structural anchors (CTCF) we developed a highly predictive heteromorphic polymer (HiP-HoP) model, where the chromatin fibre varied along its length; combined with diffusing protein bridges and loop extrusion this model predicted the 3-D organisation 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 3-D and enable predictions of chromatin folding paths.

molecular biology↗

bioRxiv2018

Complementary chromosome folding by transcription factors and cohesin

The spatial organisation of interphase chromosomes is known to affect genomic function, yet the principles behind such organisation remain elusive. Here, we first compare and then combine two well-known biophysical models, the transcription factor (TF) and loop extrusion (LE) models, and dissect their respective roles in organising the genome. Our results suggest that extrusion and transcription factors play complementary roles in folding the genome: the former are necessary to compact gene deserts or \"inert chromatin\" regions, the latter are sufficient to explain most of the structure found in transcriptionally active or repressed domains. Finally, we find that to reproduce interaction patterns found in HiC experiments we do not need to postulate an explicit motor activity of cohesin (or other extruding factors): a model where co-hesin molecules behave as molecular slip-links sliding diffusively along chromatin works equally well.

biophysics↗

bioRxiv2018

Effect of chromatin flexibility on diffusive loop extrusion via molecular slip-links

We use Brownian dynamics simulations to study the formation of chromatin loops through diffusive sliding of molecular slip links, mimicking the behaviour of cohesin-like molecules. We recently proposed that diffusive sliding is sufficient to explain the extrusion of chromatin loops of hundreds of kilo-base-pairs (kbp), which may then be stabilised by interactions between cohesin and CTCF proteins. Here we show that the elasticity of the chromatin fibre strongly affects this dynamical process, and find that diffusive loop extrusion is more efficient on stiffer chromatin regions. Efficiency is also enhanced if cohesin loading sites are close to regions where CTCF is bound. In light of the heterogeneous physical properties of eukaryotic chromatin, we suggest that our results should be relevant to the looping and organisation of interphase chromosomes in vivo.

biophysics↗

bioRxiv2016

Non-equilibrium chromosome looping via molecular slip-links

We propose a model for the formation of chromatin loops based on the diffusive sliding of a DNA-bound factor which can dimerise to form a molecular slip-link. Our slip-links mimic the behaviour of cohesin-like molecules, which, along with the CTCF protein, stabilize loops which organize the genome. By combining 3D Brownian dynamics simulations and 1D exactly solvable non-equilibrium models, we show that diffusive sliding is sufficient to account for the strong bias in favour of convergent CTCF-mediated chromosome loops observed experimentally. Importantly, our model does not require any underlying, and energetically costly, motor activity of cohesin. We also find that the diffusive motion of multiple slip-links along chromatin may be rectified by an intriguing ratchet effect that arises if slip-links bind to the chromatin at a preferred \"loading site\". This emergent collective behaviour is driven by a 1D osmotic pressure which is set up near the loading point, and favours the extrusion of loops which are much larger than the ones formed by single slip-links.

biophysics↗