Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins.

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.

In silico design of DNA sequences for in vivo nucleosome positioning.

The computational design of synthetic DNA sequences with designer in vivo properties is gaining traction in the field of synthetic genomics. We propose here a computational method which combines a kinetic Monte Carlo framework with a deep mutational screening based on deep learning predictions. We apply our method to build regular nucleosome arrays with tailored nucleosomal repeat lengths (NRL) in yeast. Our design was validated in vivo by successfully engineering and integrating thousands of kilobases long tandem arrays of computationally optimized sequences which could accommodate NRLs much larger than the yeast natural NRL (namely 197 and 237 bp, compared to the natural NRL of ∼165 bp). RNA-seq results show that transcription of the arrays can occur but is not driven by the NRL. The computational method proposed here delineates the key sequence rules for nucleosome positioning in yeast and should be easily applicable to other sequence properties and other genomes.

P-Body Purification Reveals the Condensation of Repressed mRNA Regulons.

Within cells, soluble RNPs can switch states to coassemble and condense into liquid or solid bodies. Although these phase transitions have been reconstituted in vitro, for endogenous bodies the diversity of the components, the specificity of the interaction networks, and the function of the coassemblies remain to be characterized. Here, by developing a fluorescence-activated particle sorting (FAPS) method to purify cytosolic processing bodies (P-bodies) from human epithelial cells, we identified hundreds of proteins and thousands of mRNAs that structure a dense network of interactions, separating P-body from non-P-body RNPs. mRNAs segregating into P-bodies are translationally repressed, but not decayed, and this repression explains part of the poor genome-wide correlation between RNA and protein abundance. P-bodies condense thousands of mRNAs that strikingly encode regulatory processes. Thus, we uncovered how P-bodies, by condensing and segregating repressed mRNAs, provide a physical substrate for the coordinated regulation of posttranscriptional mRNA regulons.

Genome-wide prediction of DNA mutation effect on nucleosome positions for yeast synthetic genomics.

Genetically modified genomes are often used today in many areas of fundamental and applied research. In many studies, coding or noncoding regions are modified in order to change protein sequences or gene expression levels. Modifying one or several nucleotides in a genome can also lead to unexpected changes in the epigenetic regulation of genes. When designing a synthetic genome with many mutations, it would thus be very informative to be able to predict the effect of these mutations on chromatin. We develop here a deep learning approach that quantifies the effect of every possible single mutation on nucleosome positions on the full Saccharomyces cerevisiae genome. This type of annotation track can be used when designing a modified S. cerevisiae genome. We further highlight how this track can provide new insights on the sequence-dependent mechanisms that drive nucleosomes' positions in vivo.

Improving distance measures between genomic tracks with mutual proximity.

An increasing number of genomic tracks such as DNA methylation, histone modifications or transcriptomes are being produced to annotate genomes with functional states. The comparison of such high dimensional vectors obtained under various experimental conditions requires the use of a distance or dissimilarity measure. Pearson, Cosine and $L_{p}$-norm distances are commonly used for both count and binary vectors. In this article, we highlight how enhancement methods such as the contrast increasing mutual proximity' (MP) or local scaling' improve common distance measures. We present a systematic approach to evaluate the performance of such enhanced distance measures in terms of separability of groups of experimental replicates to outline their effect. We show that the MP' applied on the various distance measures drastically increases performance. Depending on the type of epigenetic experiment, MP' coupled together with Pearson, Cosine, $L_1$, Yule or Jaccard distances proves to be highly efficient in discriminating epigenomic profiles.

Contribution of 3D genome topological domains to genetic risk of cancers: a genome-wide computational study.

BACKGROUND: Genome-wide association studies have identified statistical associations between various diseases, including cancers, and a large number of single-nucleotide polymorphisms (SNPs). However, they provide no direct explanation of the mechanisms underlying the association. Based on the recent discovery that changes in three-dimensional genome organization may have functional consequences on gene regulation favoring diseases, we investigated systematically the genome-wide distribution of disease-associated SNPs with respect to a specific feature of 3D genome organization: topologically associating domains (TADs) and their borders. RESULTS: For each of 449 diseases, we tested whether the associated SNPs are present in TAD borders more often than observed by chance, where chance (i.e., the null model in statistical terms) corresponds to the same number of pointwise loci drawn at random either in the entire genome, or in the entire set of disease-associated SNPs listed in the GWAS catalog. Our analysis shows that a fraction of diseases displays such a preferential localization of their risk loci. Moreover, cancers are relatively more frequent among these diseases, and this predominance is generally enhanced when considering only intergenic SNPs. The structure of SNP-based diseasome networks confirms that localization of risk loci in TAD borders differs between cancers and non-cancer diseases. Furthermore, different TAD border enrichments are observed in embryonic stem cells and differentiated cells, consistent with changes in topological domains along embryogenesis and delineating their contribution to disease risk. CONCLUSIONS: Our results suggest that, for certain diseases, part of the genetic risk lies in a local genetic variation affecting the genome partitioning in topologically insulated domains. Investigating this possible contribution to genetic risk is particularly relevant in cancers. This study thus opens a way of interpreting genome-wide association studies, by distinguishing two types of disease-associated SNPs: one with an effect on an individual gene, the other acting in interplay with 3D genome organization.

Condensin- and Replication-Mediated Bacterial Chromosome Folding and Origin Condensation Revealed by Hi-C and Super-resolution Imaging.

Chromosomes of a broad range of species, from bacteria to mammals, are structured by large topological domains whose precise functional roles and regulatory mechanisms remain elusive. Here, we combine super-resolution microscopies and chromosome-capture technologies to unravel the higher-order organization of the Bacillus subtilis chromosome and its dynamic rearrangements during the cell cycle. We decipher the fine 3D architecture of the origin domain, revealing folding motifs regulated by condensin-like complexes. This organization, along with global folding throughout the genome, is present before replication, disrupted by active DNA replication, and re-established thereafter. Single-cell analysis revealed a strict correspondence between sub-cellular localization of origin domains and their condensation state. Our results suggest that the precise 3D folding pattern of the origin domain plays a role in the regulation of replication initiation, chromosome organization, and DNA segregation.

MNHN-Tree-Tools: a toolbox for tree inference using multi-scale clustering of a set of sequences.

SUMMARY: Genomic sequences are widely used to infer the evolutionary history of a given group of individuals. Many methods have been developed for sequence clustering and tree building. In the early days of genome sequencing, these were often limited to hundreds of sequences but due to the surge of high throughput sequencing, it is now common to have millions of sampled sequences at hand. We introduce MNHN-Tree-Tools, a high performance set of algorithms that builds multi-scale, nested clusters of sequences found in a FASTA file. MNHN-Tree-Tools does not rely on multiple sequence alignment and can thus be used on large datasets to infer a sequence tree. Herein, we outline two applications: a human alpha-satellite repeats classification and a tree of life derivation from 16S/18S rDNA sequences. AVAILABILITY AND IMPLEMENTATION: Open source with a Zlib License via the Git protocol: https://gitlab.in2p3.fr/mnhn-tools/mnhn-tree-tools. MANUAL: A detailed users guide and tutorial: https://gitlab.in2p3.fr/mnhn-tools/mnhn-tree-tools-manual/-/raw/master/manual.pdf. WEBSITE AND FAQ: http://treetools.haschka.net. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

keras_dna: a wrapper for fast implementation of deep learning models in genomics.

SUMMARY: Prediction of genomic annotations from DNA sequences using deep learning is today becoming a flourishing field with many applications. Nevertheless, there are still difficulties in handling data in order to conveniently build and train models dedicated for specific end-user's tasks. keras_dna is designed for an easy implementation of Keras models (TensorFlow high level API) for genomics. It can handle standard bioinformatic files formats as inputs such as bigwig, gff, bed, wig, bedGraph or fasta and returns standardized inputs for model training. keras_dna is designed to implement existing models but also to facilitate the development of news models that can have single or multiple targets or inputs. AVAILABILITY AND IMPLEMENTATION: Freely available with a MIT License using pip install keras_dna or cloning the github repo at https://github.com/etirouthier/keras_dna.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

High-salt-recovered sequences are associated with the active chromosomal compartment and with large ribonucleoprotein complexes including nuclear bodies.

The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt-recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to cis-regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type-specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.

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.

The 3D folding of metazoan genomes correlates with the association of similar repetitive elements.

The potential roles of the numerous repetitive elements found in the genomes of multi-cellular organisms remain speculative. Several studies have suggested a role in stabilizing specific 3D genomic contacts. To test this hypothesis, we exploited inter-chromosomal contacts frequencies obtained from Hi-C experiments and show that the folding of the human, mouse and Drosophila genomes is associated with a significant co-localization of several specific repetitive elements, notably many elements of the SINE family. These repeats tend to be the oldest ones and are enriched in transcription factor binding sites. We propose that the co-localization of these repetitive elements may explain the global conservation of genome folding observed between homologous regions of the human and mouse genome. Taken together, these results support a contribution of specific repetitive elements in maintaining and/or reshaping genome architecture over evolutionary times.

3D genome reconstruction from chromosomal contacts.

A computational challenge raised by chromosome conformation capture (3C) experiments is to reconstruct spatial distances and three-dimensional genome structures from observed contacts between genomic loci. We propose a two-step algorithm, ShRec3D, and assess its accuracy using both in silico data and human genome-wide 3C (Hi-C) data. This algorithm avoids convergence issues, accommodates sparse and noisy contact maps, and is orders of magnitude faster than existing methods.

High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome.

Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10(-2)-10(3) sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to <30 nm. Finally, we show that the Rouse model is also relevant to analyze chromosome motion in mutant cells depleted of proteins that bind to or assemble chromatin, and suggest that it provides a consistent framework to study chromatin dynamics. We discuss the implications of our findings for yeast genome architecture and for target search mechanisms in the nucleus.

DNA topology in chromosomes: a quantitative survey and its physiological implications.

Using a simple geometric model, we propose a general method for computing the linking number of the DNA embedded in chromatin fibers. The relevance of the method is reviewed through the single molecule experiments that have been performed in vitro with magnetic tweezers. We compute the linking number of the DNA in the manifold conformational states of the nucleosome which have been evidenced in these experiments and discuss the functional dynamics of chromosomes in the light of these manifold states.

Transcription-induced domains form the elementary constraining building blocks of bacterial chromosomes.

Transcription generates local topological and mechanical constraints on the DNA fiber, leading to the generation of supercoiled chromosome domains in bacteria. However, the global impact of transcription on chromosome organization remains elusive, as the scale of genes and operons in bacteria remains well below the resolution of chromosomal contact maps generated using Hi-C (~5-10 kb). Here we combined sub-kb Hi-C contact maps and chromosome engineering to visualize individual transcriptional units. We show that transcriptional units form discrete three-dimensional transcription-induced domains that impose mechanical and topological constraints on their neighboring sequences at larger scales, modifying their localization and dynamics. These results show that transcriptional domains constitute primary building blocks of bacterial chromosome folding and locally impose structural and dynamic constraints.

Condensin I folds the Caenorhabditis elegans genome.

The structural maintenance of chromosome (SMC) complexes-cohesin and condensins-are crucial for chromosome separation and compaction during cell division. During the interphase, mammalian cohesins additionally fold the genome into loops and domains. Here we show that, in Caenorhabditis elegans, a species with holocentric chromosomes, condensin I is the primary, long-range loop extruder. The loss of condensin I and its X-specific variant, condensin IDC, leads to genome-wide decompaction, chromosome mixing and disappearance of X-specific topologically associating domains, while reinforcing fine-scale epigenomic compartments. In addition, condensin I/IDC inactivation led to the upregulation of X-linked genes and unveiled nuclear bodies grouping together binding sites for the X-targeting loading complex of condensin IDC. C. elegans condensin I/IDC thus uniquely organizes holocentric interphase chromosomes, akin to cohesin in mammals, as well as regulates X-chromosome gene expression.