Competitive binding of MatP and topoisomerase IV to the MukB dimerization hinge

ABSTRACTSMC complexes have ubiquitous roles in chromosome organisation. In Escherichia coli, the interplay between the SMC complex, MukBEF, and matS-bound MatP in the replication termination region, ter, results in depletion of MukBEF from ter, thus promoting chromosome individualisation by directing replichores to separate cell halves. MukBEF also interacts with topoisomerase IV ParC2E2 heterotetramers, to direct its chromosomal distribution to mirror that of MukBEF, thereby facilitating coordination between chromosome organisation and decatenation by topoisomerase IV. Here we demonstrate that the MukB dimerization hinge binds ParC and MatP with the same dimer to dimer stoichiometry. MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding. Furthermore, the MukB hinge fails to stably associate with matS-bound MatP, while MatP mutants deficient in matS binding are impaired in MukB hinge binding, demonstrating that mats competes with the hinge for MatP binding. Cells expressing MukBEF complexes containing a mutation in the MukB hinge interface for ParC/MatP binding are deficient in ParC binding in vivo, despite having a Muk+ topoisomerase IV+ phenotype. This mutant protein is also impaired in MatP binding in vitro, and cells expressing this variant exhibit a MukBEF cellular localisation consistent with impaired MatP binding.

Principles of meiotic chromosome assembly revealed in S. cerevisiae

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

FACT mediates cohesin function on chromatin

Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion 1,2 and the organisation of higher-order structures during interphase 3 and mitosis 4,5. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes 6-8. Here we show that cohesin role in chromosome organisation requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin-dependent compartmentalization of chromosomes into loop domains.

Self-organised segregation of bacterial chromosomal origins

The chromosomal replication origin region (ori) of characterised bacteria is dynamically positioned throughout the cell cycle. In slowly growing Escherichia coli, ori is maintained at mid-cell from birth until its replication, after which newly replicated sister oris move to opposite quarter positions. Here, we provide an explanation for ori positioning based on the self-organisation of the Structural Maintenance of Chromosomes complex, MukBEF, which forms dynamically positioned clusters on the chromosome. We propose that a non-trivial feedback between the self-organising gradient of MukBEF complexes and the oris leads to accurate ori positioning. We find excellent agreement with quantitative experimental measurements and confirm key predictions. Specifically, we show that oris exhibit biased motion towards MukBEF clusters, rather than mid-cell. Our findings suggest that MukBEF and oris act together as a self-organising system in chromosome organisation-segregation and introduces protein self-organisation as an important consideration for future studies of chromosome dynamics.

Cohesin disrupts polycomb-dependent chromosome interactions

How chromosome organisation is related to genome function remains poorly understood. Cohesin, loop-extrusion, and CTCF have been proposed to create structures called topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find as in other cell types that cohesin is required to create TADs and regulate A/B compartmentalisation. However, in the absence of cohesin we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system, depend on PRC1, and we discover that cohesin counteracts these interactions. This disruptive activity is independent of CTCF and TADs, and regulates gene repression by the polycomb system. Therefore, in contrast to the proposal that cohesin creates structure in chromosomes, we discover a new role for cohesin in disrupting polycomb-dependent chromosome interactions to regulate gene expression.

Bayesian parameter estimation in non-stationary semiflexible polymers from ensembles of trajectories

During the cell-cycle and meiosis, during development, or in response to stress, chromosomes undertake dramatic programs of reorganisation, which can result in major changes to genomic architecture, as well as local changes to chromatin structure via chromatin remodelling and epigenetic modification. The biophysical properties of the genome may therefore vary significantly over time, from region to region, and from cell to cell. Semifleixble polymer models are frequently used to decipher the spatial and temporal aspects of chromosome organisation. Such models allow for parameter estimation from experimental observations (Bystricky et al., 2004, Ding et al., 2006, Koszul et al., 2008, Arbona et al., 2017), and so provide a concise quantification of the state of the system in terms of meaningful biophysical parameters, such as the compaction factor and bending-modulus. Simulation studies using appropriately parameterised models may also provide novel insights, and allow for predictions without confounding pleiotropic effects (Penfold et al., 2012), thus guiding future studies. Most semifleixble polymer models do not explicitly consider the spatial non-stationarity of chromosomes and chromatin. Furthermore, recent advances in chromosome conformation capture (3C)-based allow chromosome organisation to be (indirectly) measured in single cells (Belton et al., 2012, Nagano et al., 2013, 2016). The increasing availability of ensembles of trajectories sampled from potentially heterogeneous populations of cells means it is of interest to develop polymer statistic models that can capture both the spatial nonstationarity of the biophysical parameters, and the statistical relationships that exist within the population. Here we outline a statistical framework for non-stationary semiflexible polymers, and demonstrate how inference can be performed using ensembles of trajectories. For cells belonging to a homogenous population where the biophysical parameters are approximately identical in all cells, a (transformed) Gaussian process prior is assigned to the bending-modulus, and Markov chain Monte Carlo (MCMC) used to infer the posterior distribution of free parameters. For heterogeneous populations of cells, a transformed hierarchical GP (HGP) prior is assigned to the biophysical parameters, which naturally captures the statistical dependency of the parameters that exist across the population. Simulation studies demonstrate the accuracy of the model for homogenous and heterogeneous populations, while applications to yeast chromosome data demonstrates an improved ability to recapitulate trajectories of held out loci compared to related stationary models.

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.

Principles of Meiotic Chromosome Assembly

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we elucidate how this elaborate three-dimensional chromosome organisation is underpinned by genomic sequence in Saccharomyces cerevisiae. Entering meiosis, strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion limited by barriers, yet are patterned by convergent transcription rather than binding of the mammalian interphase factor, CTCF, which is absent in S. cerevisiae—thereby both challenging and extending current paradigms of local chromosome organisation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

Self-organised segregation of bacterial chromosomal origins

In spite of much effort, many aspects of chromosome organisation and segregation in bacteria remain unclear. Even for Escherichia coli, the most widely studied bacterial model organism, we still do not know the underlying mechanisms. Like many other bacteria, the chromosomal origin of replication in E. coli is dynamically positioned throughout the cell cycle. Initially maintained at mid-cell, where replication occurs, origins are subsequently partitioned to opposite quarter positions. The Structural Maintenance of Chromosomes (SMC) complex, MukBEF, which is required for correct chromosome compaction and organisation, has been implicated in this behaviour but the mode of action is unknown. Here, we build on a recent self-organising model for the positioning of E. coli MukBEF, to propose an explanation for the positioning and partitioning of origins. We propose that a specific association of MukBEF with the origin region, results in a non-trivial feedback between the self-organising MukBEF gradient and the origins, leading to accurate positioning and partitioning as an emergent property. We compare the model to quantitative experimental data of origin dynamics and their colocalisation with MukBEF clusters and find excellent agreement. Overall, the model suggests that MukBEF and origins act together as a self-organising system for chromosome segregation and introduces protein self-organisation as an important consideration for future studies of chromosome dynamics.