Sir3 mediates long-range chromosome interactions in budding yeast.

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here, we investigate the well-conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing SIR complex composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long-range contacts between distant regions, including the rDNA, telomeres, and internal Sir3-bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach, we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, Sir4), Sir2 activity, or chromosome context. Altogether, these data suggest that Sir3 acts as a molecular bridge that stabilizes long-range interactions.

A mean-field theory for predicting single polymer collapse induced by neutral crowders.

Macromolecular crowding can induce the collapse of a single long polymer into a globular form due to depletion forces of entropic nature. This phenomenon has been shown to play a significant role in compacting the genome within the bacterium Escherichia coli into a well-defined region of the cell known as the nucleoid. Motivated by the biological significance of this process, numerous theoretical and computational studies have searched for the primary determinants of the behavior of polymer-crowder phases. However, our understanding of this process remains incomplete and there is debate on a quantitatively unified description. In particular, different simulation studies with explicit crowders have proposed different order parameters as potential predictors for the collapse transition. In this work, we present a comprehensive analysis of published simulation data obtained from different sources. Based on the common behavior we find in this data, we develop a unified phenomenological model that we show to be predictive. Finally, to further validate the accuracy of the model, we conduct new simulations on polymers of various sizes, and investigate the role of jamming of the crowders.

Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle.

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome-wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher-order organization of the Saccharomyces cerevisiae genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long-range intra-chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin-dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin-mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle-dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.

Serpentine: a flexible 2D binning method for differential Hi-C analysis.

MOTIVATION: Hi-C contact maps reflect the relative contact frequencies between pairs of genomic loci, quantified through deep sequencing. Differential analyses of these maps enable downstream biological interpretations. However, the multi-fractal nature of the chromatin polymer inside the cellular envelope results in contact frequency values spanning several orders of magnitude: contacts between loci pairs separated by large genomic distances are much sparser than closer pairs. The same is true for poorly covered regions, such as repeated sequences. Both distant and poorly covered regions translate into low signal-to-noise ratios. There is no clear consensus to address this limitation. RESULTS: We present Serpentine, a fast, flexible procedure operating on raw data, which considers the contacts in each region of a contact map. Binning is performed only when necessary on noisy regions, preserving informative ones. This results in high-quality, low-noise contact maps that can be conveniently visualized for rigorous comparative analyses. AVAILABILITY AND IMPLEMENTATION: Serpentine is available on the PyPI repository and https://github.com/koszullab/serpentine; documentation and tutorials are provided at https://serpentine.readthedocs.io/en/latest/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C.

In chromosome conformation capture experiments (Hi-C), the accuracy with which contacts are detected varies due to the uneven distribution of restriction sites along genomes. In addition, repeated sequences or homologous regions remain indistinguishable because of the ambiguities they introduce during the alignment of the sequencing reads. We addressed both limitations by designing and engineering 144 kb of a yeast chromosome with regularly spaced restriction sites (Syn-HiC design). In the Syn-HiC region, Hi-C signal-to-noise ratio is enhanced and can be used to measure the shape of an unbiased distribution of contact frequencies, allowing to propose a robust definition of a Hi-C experiment resolution. The redesigned region is also distinguishable from its native homologous counterpart in an otherwise isogenic diploid strain. As a proof of principle, we tracked homologous chromosomes during meiotic prophase in synchronized and pachytene-arrested cells and captured important features of their spatial reorganization, such as chromatin restructuration into arrays of Rec8-delimited loops, centromere declustering, individualization, and pairing. Overall, we illustrate the promises held by redesigning genomic regions to explore complex biological questions.

Kinetic Signature of Cooperativity in the Irreversible Collapse of a Polymer.

We investigate the kinetics of a polymer collapse due to the formation of irreversible cross-links between its monomers. Using the contact probability P(s) as a scale-dependent order parameter depending on the chemical distance s, our simulations show the emergence of a cooperative pearling instability. Namely, the polymer undergoes a sharp conformational transition to a set of absorbing states characterized by a length scale ξ corresponding to the mean pearl size. This length and the transition time depend on the polymer equilibrium dynamics and the cross-linking rate. We confirm experimentally this transition using a DNA conformation capture experiment in yeast.

Combined collapse by bridging and self-adhesion in a prototypical polymer model inspired by the bacterial nucleoid.

Recent experimental results suggest that the E. coli chromosome feels a self-attracting interaction of osmotic origin, and is condensed in foci by bridging interactions. Motivated by these findings, we explore a generic modeling framework combining solely these two ingredients, in order to characterize their joint effects. Specifically, we study a simple polymer physics computational model with weak ubiquitous short-ranged self attraction and stronger sparse bridging interactions. Combining theoretical arguments and simulations, we study the general phenomenology of polymer collapse induced by these dual contributions, in the case of regularly spaced bridging. Our results distinguish a regime of classical Flory-like coil-globule collapse dictated by the interplay of excluded volume and attractive energy and a switch-like collapse where bridging interactions compete with entropy loss terms from the looped arms of a star-like rosette. Additionally, we show that bridging can induce stable compartmentalized domains. In these configurations, different "cores" of bridging proteins are kept separated by star-like polymer loops in an entropically favorable multi-domain configuration, with a mechanism that parallels micellar polysoaps. Such compartmentalized domains are stable, and do not need any intra-specific interactions driving their segregation. Domains can be stable also in the presence of uniform attraction, as long as the uniform collapse is above its theta point.

NuST: analysis of the interplay between nucleoid organization and gene expression.

UNLABELLED: Different experimental results suggest the presence of an interplay between global transcriptional regulation and chromosome spatial organization in bacteria. The identification and clear visualization of spatial clusters of contiguous genes targeted by specific DNA-binding proteins or sensitive to nucleoid perturbations can elucidate links between nucleoid structure and gene expression patterns. Similarly, statistical analysis to assess correlations between results from independent experiments can provide the integrated analysis needed in this line of research. NuST (Nucleoid Survey tools), based on the Escherichia coli genome, gives the non-expert the possibility to analyze the aggregation of genes or loci sets along the genome coordinate, at different scales of observation. It is useful to discover correlations between different sources of data (e.g. expression, binding or genomic data) and genome organization. A user can use it on datasets in the form of gene lists coming from his/her own experiments or bioinformatic analyses, but also make use of the internal database, which collects data from many published studies. AVAILABILITY AND IMPLEMENTATION: NuST is a web server (available at http://www.lgm.upmc.fr/nust/). The website is implemented in PHP, SQLite and Ajax, with all major browsers supported, while the core algorithms are optimized and implemented in C. NuST has an extensive help page and provides a direct visualization of results as well as different downloadable file formats. A template Perl code for automated access to the web server can be downloaded at http://www.lgm.upmc.fr/nust/downloads/, in order to allow the users to use NuST in systematic bioinformatic analyses.

Physical descriptions of the bacterial nucleoid at large scales, and their biological implications.

Recent experimental and theoretical approaches have attempted to quantify the physical organization (compaction and geometry) of the bacterial chromosome with its complement of proteins (the nucleoid). The genomic DNA exists in a complex and dynamic protein-rich state, which is highly organized at various length scales. This has implications for modulating (when not directly enabling) the core biological processes of replication, transcription and segregation. We overview the progress in this area, driven in the last few years by new scientific ideas and new interdisciplinary experimental techniques, ranging from high space- and time-resolution microscopy to high-throughput genomics employing sequencing to map different aspects of the nucleoid-related interactome. The aim of this review is to present the wide spectrum of experimental and theoretical findings coherently, from a physics viewpoint. In particular, we highlight the role that statistical and soft condensed matter physics play in describing this system of fundamental biological importance, specifically reviewing classic and more modern tools from the theory of polymers. We also discuss some attempts toward unifying interpretations of the current results, pointing to possible directions for future investigation.

Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics.

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.

3D organization of synthetic and scrambled chromosomes.

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) strains.

Genomic analysis reveals epistatic silencing of "expensive" genes in Escherichia coli K-12.

A barrier for horizontal gene transfer is high gene expression, which is metabolically expensive. Silencing of horizontally-acquired genes in the bacterium Escherichia coli is caused by the global transcriptional repressor H-NS. The activity of H-NS is enhanced or diminished by other proteins including its homologue StpA, and Hha and YdgT. The interconnections of H-NS with these regulators and their role in silencing gene expression in E. coli are not well understood on a genomic scale. In this study, we use transcriptome sequencing to show that there is a bi-layered gene silencing system - involving the homologous H-NS and StpA - operating on horizontally-acquired genes among others. We show that H-NS-repressed genes belong to two types, termed "epistatic" and "unilateral". In the absence of H-NS, the expression of "epistatically controlled genes" is repressed by StpA, whereas that of "unilaterally controlled genes" is not. Epistatic genes show a higher tendency to be non-essential and recently acquired, when compared to unilateral genes. Epistatic genes reach much higher expression levels than unilateral genes in the absence of the silencing system. Finally, epistatic genes contain more high affinity H-NS binding motifs than unilateral genes. Therefore, both the DNA binding sites of H-NS as well as the function of StpA as a backup system might be selected for silencing highly transcribable genes.

Gene clusters reflecting macrodomain structure respond to nucleoid perturbations.

Focusing on the DNA-bridging nucleoid proteins Fis and H-NS, and integrating several independent experimental and bioinformatic data sources, we investigate the links between chromosomal spatial organization and global transcriptional regulation. By means of a novel multi-scale spatial aggregation analysis, we uncover the existence of contiguous clusters of nucleoid-perturbation sensitive genes along the genome, whose expression is affected by a combination of topological DNA state and nucleoid-shaping protein occupancy. The clusters correlate well with the macrodomain structure of the genome. The most significant of them lay symmetrically at the edges of the Ter macrodomain and involve all of the flagellar and chemotaxis machinery, in addition to key regulators of biofilm formation, suggesting that the regulation of the physical state of the chromosome by the nucleoid proteins plays an important role in coordinating the transcriptional response leading to the switch between a motile and a biofilm lifestyle.