Comparative assessment of cytometry by time-of-flight and full spectral flow cytometry based on a 33-color antibody panel.

Mass cytometry and full spectrum flow cytometry have recently emerged as new promising single cell proteomic analysis tools that can be exploited to decipher the extensive diversity of immune cell repertoires and their implication in human diseases. In this study, we evaluated the performance of mass cytometry against full spectrum flow cytometry using an identical 33-color antibody panel on four healthy individuals. Our data revealed an overall high concordance in the quantification of major immune cell populations between the two platforms using a semi-automated clustering approach. We further showed a strong correlation of cluster assignment when comparing manual and automated clustering. Both comparisons revealed minor disagreements in the quantification and assignment of rare cell subpopulations. Our study showed that both single cell proteomic technologies generate highly overlapping results and substantiate that the choice of technology is not a primary factor for successful biological assessment of cell profiles but must be considered in a broader design framework of clinical studies.

Chromosome-level organization of the regulatory genome in the Drosophila nervous system.

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.

Essential role of Cp190 in physical and regulatory boundary formation.

Boundaries in animal genomes delimit contact domains with enhanced internal contact frequencies and have debated functions in limiting regulatory cross-talk between domains and guiding enhancers to target promoters. Most mammalian boundaries form by stalling of chromosomal loop-extruding cohesin by CTCF, but most Drosophila boundaries form CTCF independently. However, how CTCF-independent boundaries form and function remains largely unexplored. Here, we assess genome folding and developmental gene expression in fly embryos lacking the ubiquitous boundary-associated factor Cp190. We find that sequence-specific DNA binding proteins such as CTCF and Su(Hw) directly interact with and recruit Cp190 to form most promoter-distal boundaries. Cp190 is essential for early development and prevents regulatory cross-talk between specific gene loci that pattern the embryo. Cp190 was, in contrast, dispensable for long-range enhancer-promoter communication at tested loci. Cp190 is thus currently the major player in fly boundary formation and function, revealing that diverse mechanisms evolved to partition genomes into independent regulatory domains.

A single-cell morpho-transcriptomic map of brassinosteroid action in the Arabidopsis root.

The effects of brassinosteroid signaling on shoot and root development have been characterized in great detail but a simple consistent positive or negative impact on a basic cellular parameter was not identified. In this study, we combined digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize root meristems and mature root segments of brassinosteroid-blind mutants and wild type. The resultant datasets demonstrate that brassinosteroid signaling affects neither cell volume nor cell proliferation capacity. Instead, brassinosteroid signaling is essential for the precise orientation of cell division planes and the extent and timing of anisotropic cell expansion. Moreover, we found that the cell-aligning effects of brassinosteroid signaling can propagate to normalize the anatomy of both adjacent and distant brassinosteroid-blind cells through non-cell-autonomous functions, which are sufficient to restore growth vigor. Finally, single-cell transcriptome data discern directly brassinosteroid-responsive genes from genes that can react non-cell-autonomously and highlight arabinogalactans as sentinels of brassinosteroid-dependent anisotropic cell expansion.

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.

CTCF loss has limited effects on global genome architecture in Drosophila despite critical regulatory functions.

Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. We genetically ablate CTCF in Drosophila and examine impacts on genome folding and transcriptional regulation in the central nervous system. We find that CTCF is required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. We also find that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution.

Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes.

Due to helical structure of DNA, massive amounts of positive supercoils are constantly introduced ahead of each replication fork. Positive supercoiling inhibits progression of replication forks but various mechanisms evolved that permit very efficient relaxation of that positive supercoiling. Some of these mechanisms lead to interesting topological situations where DNA supercoiling, catenation and knotting coexist and influence each other in DNA molecules being replicated. Here, we first review fundamental aspects of DNA supercoiling, catenation and knotting when these qualitatively different topological states do not coexist in the same circular DNA but also when they are present at the same time in replicating DNA molecules. We also review differences between eukaryotic and prokaryotic cellular strategies that permit relaxation of positive supercoiling arising ahead of the replication forks. We end our review by discussing very recent studies giving a long-sought answer to the question of how slow DNA topoisomerases capable of relaxing just a few positive supercoils per second can counteract the introduction of hundreds of positive supercoils per second ahead of advancing replication forks.

Are TADs supercoiled?

Topologically associating domains (TADs) are megabase-sized building blocks of interphase chromosomes in higher eukaryotes. TADs are chromosomal regions with increased frequency of internal interactions. On average a pair of loci separated by a given genomic distance contact each other 2-3 times more frequently when they are in the same TAD as compared to a pair of loci located in two neighbouring TADs. TADs are also functional blocks of chromosomes as enhancers and their cognate promoters are normally located in the same TAD, even if their genomic distance from each other can be as large as a megabase. The internal structure of TADs, causing their increased frequency of internal interactions, is not established yet. We survey here experimental studies investigating presence of supercoiling in interphase chromosomes. We also review numerical simulation studies testing whether transcription-induced supercoiling of chromatin fibres can explain how TADs are formed and how they can assure very efficient interactions between enhancers and their cognate promoters located in the same TAD.

KnotProt 2.0: a database of proteins with knots and other entangled structures.

The KnotProt 2.0 database (the updated version of the KnotProt database) collects information about proteins which form knots and other entangled structures. New features in KnotProt 2.0 include the characterization of both probabilistic and deterministic entanglements which can be formed by disulfide bonds and interactions via ions, a refined characterization of entanglement in terms of knotoids, the identification of the so-called cysteine knots, the possibility to analyze all or a non-redundant set of proteins, and various technical updates. The KnotProt 2.0 database classifies all entangled proteins, represents their complexity in the form of a knotting fingerprint, and presents many biological and geometrical statistics based on these results. Currently the database contains >2000 entangled structures, and it regularly self-updates based on proteins deposited in the Protein Data Bank (PDB).

Knoto-ID: a tool to study the entanglement of open protein chains using the concept of knotoids.

Summary: The backbone of most proteins forms an open curve. To study their entanglement, a common strategy consists in searching for the presence of knots in their backbones using topological invariants. However, this approach requires to close the curve into a loop, which alters the geometry of curve. Knoto-ID allows evaluating the entanglement of open curves without the need to close them, using the recent concept of knotoids which is a generalization of the classical knot theory to open curves. Knoto-ID can analyse the global topology of the full chain as well as the local topology by exhaustively studying all subchains or only determining the knotted core. Knoto-ID permits to localize topologically non-trivial protein folds that are not detected by informatics tools detecting knotted protein folds. Availability and implementation: Knoto-ID is written in C++ and includes R (www.R-project.org) scripts to generate plots of projections maps, fingerprint matrices and disk matrices. Knoto-ID is distributed under the GNU General Public License (GPL), version 2 or any later version and is available at https://github.com/sib-swiss/Knoto-ID. A binary distribution for Mac OS X, Linux and Windows with detailed user guide and examples can be obtained from https://www.vital-it.ch/software/Knoto-ID.

Expert curation for building network-based dynamical models: a case study on atherosclerotic plaque formation.

Database URL: http://biomodels.caltech.edu.

Transcription-induced supercoiling as the driving force of chromatin loop extrusion during formation of TADs in interphase chromosomes.

Using molecular dynamics simulations, we show here that growing plectonemes resulting from transcription-induced supercoiling have the ability to actively push cohesin rings along chromatin fibres. The pushing direction is such that within each topologically associating domain (TAD) cohesin rings forming handcuffs move from the source of supercoiling, constituted by RNA polymerase with associated DNA topoisomerase TOP1, towards borders of TADs, where supercoiling is released by topoisomerase TOPIIB. Cohesin handcuffs are pushed by continuous flux of supercoiling that is generated by transcription and is then progressively released by action of TOPIIB located at TADs borders. Our model explains what can be the driving force of chromatin loop extrusion and how it can be ensured that loops grow quickly and in a good direction. In addition, the supercoiling-driven loop extrusion mechanism is consistent with earlier explanations proposing why TADs flanked by convergent CTCF binding sites form more stable chromatin loops than TADs flanked by divergent CTCF binding sites. We discuss the role of supercoiling in stimulating enhancer promoter contacts and propose that transcription of eRNA sends the first wave of supercoiling that can activate mRNA transcription in a given TAD.

Transcription-induced supercoiling explains formation of self-interacting chromatin domains in S. pombe.

The question of how self-interacting chromatin domains in interphase chromosomes are structured and generated dominates current discussions on eukaryotic chromosomes. Numerical simulations using standard polymer models have been helpful in testing the validity of various models of chromosome organization. Experimental contact maps can be compared with simulated contact maps and thus verify how good is the model. With increasing resolution of experimental contact maps, it became apparent though that active processes need to be introduced into models to recapitulate the experimental data. Since transcribing RNA polymerases are very strong molecular motors that induce axial rotation of transcribed DNA, we present here models that include such rotational motors. We also include into our models swivels and sites for intersegmental passages that account for action of DNA topoisomerases releasing torsional stress. Using these elements in our models, we show that transcription-induced supercoiling generated in the regions with divergent-transcription and supercoiling relaxation occurring between these regions are sufficient to explain formation of self-interacting chromatin domains in chromosomes of fission yeast (S. pombe).

Molecular Dynamics Simulation of Supercoiled, Knotted, and Catenated DNA Molecules, Including Modeling of Action of DNA Gyrase.

A detailed protocol of molecular dynamics simulations of supercoiled DNA molecules that can be in addition knotted or catenated is described. We also describe how to model ongoing action of DNA gyrase that introduces negative supercoing into DNA molecules. The protocols provide detailed instructions about model parameters, equations of used potentials, simulation, and visualization. Implementation of the model into a frequently used molecular dynamics simulation environment, ESPResSo, is shown step by step.

Studies of global and local entanglements of individual protein chains using the concept of knotoids.

We study here global and local entanglements of open protein chains by implementing the concept of knotoids. Knotoids have been introduced in 2012 by Vladimir Turaev as a generalization of knots in 3-dimensional space. More precisely, knotoids are diagrams representing projections of open curves in 3D space, in contrast to knot diagrams which represent projections of closed curves in 3D space. The intrinsic difference with classical knot theory is that the generalization provided by knotoids admits non-trivial topological entanglement of the open curves provided that their geometry is frozen as it is the case for crystallized proteins. Consequently, our approach doesn't require the closure of chains into loops which implies that the geometry of analysed chains does not need to be changed by closure in order to characterize their topology. Our study revealed that the knotoid approach detects protein regions that were classified earlier as knotted and also new, topologically interesting regions that we classify as pre-knotted.

Topological Models for Open-Knotted Protein Chains Using the Concepts of Knotoids and Bonded Knotoids.

In this paper we introduce a method that offers a detailed overview of the entanglement of an open protein chain. Further, we present a purely topological model for classifying open protein chains by also taking into account any bridge involving the backbone. To this end, we implemented the concepts of planar knotoids and bonded knotoids. We show that the planar knotoids technique provides more refined information regarding the knottedness of a protein when compared to established methods in the literature. Moreover, we demonstrate that our topological model for bonded proteins is robust enough to distinguish all types of lassos in proteins.

Boolean regulatory network reconstruction using literature based knowledge with a genetic algorithm optimization method.

BACKGROUND: Prior knowledge networks (PKNs) provide a framework for the development of computational biological models, including Boolean models of regulatory networks which are the focus of this work. PKNs are created by a painstaking process of literature curation, and generally describe all relevant regulatory interactions identified using a variety of experimental conditions and systems, such as specific cell types or tissues. Certain of these regulatory interactions may not occur in all biological contexts of interest, and their presence may dramatically change the dynamical behaviour of the resulting computational model, hindering the elucidation of the underlying mechanisms and reducing the usefulness of model predictions. Methods are therefore required to generate optimized contextual network models from generic PKNs. RESULTS: We developed a new approach to generate and optimize Boolean networks, based on a given PKN. Using a genetic algorithm, a model network is built as a sub-network of the PKN and trained against experimental data to reproduce the experimentally observed behaviour in terms of attractors and the transitions that occur between them under specific perturbations. The resulting model network is therefore contextualized to the experimental conditions and constitutes a dynamical Boolean model closer to the observed biological process used to train the model than the original PKN. Such a model can then be interrogated to simulate response under perturbation, to detect stable states and their properties, to get insights into the underlying mechanisms and to generate new testable hypotheses. CONCLUSIONS: Generic PKNs attempt to synthesize knowledge of all interactions occurring in a biological process of interest, irrespective of the specific biological context. This limits their usefulness as a basis for the development of context-specific, predictive dynamical Boolean models. The optimization method presented in this article produces specific, contextualized models from generic PKNs. These contextualized models have improved utility for hypothesis generation and experimental design. The general applicability of this methodological approach makes it suitable for a variety of biological systems and of general interest for biological and medical research. Our method was implemented in the software optimusqual, available online at http://www.vital-it.ch/software/optimusqual/ .

How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation.

Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation.

Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation.

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA-DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling.

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA-DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.