Biology subjects

Bianco, S.

Publications and source records attributed to Bianco, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Promoter repression and 3D-restructuring resolves divergent developmental gene expression in TADs

Cohesin loop extrusion facilitates precise gene expression by continuously driving promoters to sample all enhancers located within the same topologically-associated domain (TAD). However, many TADs contain multiple genes with divergent expression patterns, thereby indicating additional forces further refine how enhancer activities are utilised. Here, we unravel the mechanisms enabling a new gene, Rex1, to emerge with divergent expression within the ancient Fat1 TAD in placental mammals. We show that such divergent expression is not determined by a strict enhancer-promoter compatibility code, intra-TAD position or nuclear envelope-attachment. Instead, TAD-restructuring in embryonic stem cells (ESCs) separates Rex1 and Fat1 with distinct proximal enhancers that independently drive their expression. By contrast, in later embryonic tissues, DNA methylation renders the inactive Rex1 promoter profoundly unresponsive to Fat1 enhancers within the intact TAD. Combined, these features adapted an ancient regulatory landscape during evolution to support two entirely independent Rex1 and Fat1 expression programs. Thus, rather than operating only as rigid blocks of co-regulated genes, TAD-regulatory landscapes can orchestrate complex divergent expression patterns in evolution. HIGHLIGHTSO_LINew genes can emerge in evolution without taking on the expression pattern of their surrounding pre-existing TAD. C_LIO_LICompartmentalisation can restructure seemingly evolutionarily stable TADs to control a promoters access to enhancers. C_LIO_LILamina-associated domains neither prevent transcriptional activation nor enhancer-promoter communication. C_LIO_LIRepression rather than promoter-specificity refines when genes respond to promiscuous enhancer activities in specific tissues. C_LI

genetics↗

bioRxiv2021

Semi-supervised identification of SARS-CoV-2 molecular targets

SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. In this work, we analyzed a corpus of 66,000 SARS-CoV-2 genome sequences. We developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on use of a single reference genome and by overcoming atypical genome traits. Using this method, we identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction compared to proteome references including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools such as Prokka (base) and VAPiD, we yielded an 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 molecular target sequences-- some conserved across time and geography while others represent emerging variants. We observed 3,362 non-redundant sequences per protein on average within this corpus and describe key D614G and N501Y variants spatiotemporally. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized Receptor Binding Domain variants. Here, we comprehensively present the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable high-accuracy method to analyze newly sequenced infections.

bioinformatics↗

bioRxiv2021

AI-driven prediction of SARS-CoV-2 variant binding trends from atomistic simulations

We present a novel technique to predict binding affinity trends between two molecules from atomistic molecular dynamics simulations. The technique uses a neural network algorithm applied to a series of images encoding the distance between two molecules in time. We demonstrate that our algorithm is capable of separating with high accuracy non-hydrophobic mutations with low binding affinity from those with high binding affinity. Moreover, we show high accuracy in prediction using a small subset of the simulation, therefore requiring a much shorter simulation time. We apply our algorithm to the binding between several variants of the SARS-CoV-2 spike protein and the human receptor ACE2.

biophysics↗

bioRxiv2021

Polymer physics and machine learning reveal a combinatorial code linking chromatin 3D architecture to 1D epigenetics

The mammalian genome has a complex 3D organization, serving vital functional purposes, yet it remains largely unknown how the multitude of specific DNA contacts, e.g., between transcribed and regulatory regions, is orchestrated by chromatin organizers, such as Transcription Factors. Here, we implement a method combining machine learning and polymer physics to infer from only Hi-C data the genomic 1D arrangement of the minimal set of binding sites sufficient to recapitulate, through only physics, 3D contact patterns genome-wide in human and mouse cells. The inferred binding sites are validated by their predictions on how chromatin refolds in a set of duplications at the Sox9 locus against available independent cHi-C data, showing that their different phenotypes originate from distinct enhancer hijackings in their 3D structure. Albeit derived from only Hi-C, our binding sites fall in epigenetic classes that well match chromatin states from epigenetic segmentation studies, such as active, poised and repressed states. However, the inferred binding domains have an overlapping, combinatorial organization along chromosomes, missing in epigenetic segmentations, which is required to explain Hi-C contact specificity with high accuracy. In a reverse approach, the epigenetic profile of binding domains provides a code to derive from only epigenetic marks the DNA binding sites and, hence, the 3D architecture, as validated by successful predictions of Hi-C matrices in an independent set of chromosomes. Overall, our results shed light on how complex 3D architectural information is encrypted in 1D epigenetics via the related, combinatorial arrangement of specific binding sites along the genome.

molecular biology↗

bioRxiv2021

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome

1During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model. We experimentally determined virus and DI replication during co-infection, and use these data to infer model parameters. Our model predicts, and our experimental measurements confirm, that DI replication and encapsidation are the most important determinants for the outcome of the competition. WT replication inversely correlates with DI replication. Our model predicts that genome replication and effective DI genome encapsidation are critical to effectively inhibit WT production, but an equilibrium can be established which enables WT to replicate, albeit to reduce levels.

microbiology↗

bioRxiv2020

Endoderm Nitric Oxide Signals to Regulate Nascent Development of Cardiac Progenitors in Chicken Embryos

Heart development in the chicken embryo is regulated by a concert of cardiogenic morphogens and signaling molecules, but the physiological signal molecule nitric oxide(NO) has not been studied in the context of heart formation. A dynamic investigation of endoderm NO formation demonstrates for the first time a correlation with the established development events of the cardiac heart fields and heart tube. Manipulation of endoderm NO signaling demonstrate a role of NO signaling in the differentiation and proliferation of cardiac progenitors for heart tube formation and cardiac heart field development. To investigate NO in the proliferation of myocardial cells in the heart tube embryos, a computer vision based artificial intelligence approach is followed to automate the long and tedious job of counting cells in a large image dataset. We document NO as an important signaling molecule in the regulation of nascent embryonic cardiogenesis whose effects on other early cardiogenic morphogens is unknown.

developmental biology↗

bioRxiv2020

Multiplex-GAM: genome-wide identification of chromatin contacts yields insights not captured by Hi-C

Technologies for measuring 3D genome topology are increasingly important for studying mechanisms of gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of Genome Architecture Mapping (GAM), a ligation-free technique to map chromatin contacts genome-wide. We perform a detailed comparison of contacts obtained by multiplex-GAM and Hi-C using mouse embryonic stem (mES) cells. We find that both methods detect similar topologically associating domains (TADs). However, when examining the strongest contacts detected by either method, we find that only one third of these are shared. The strongest contacts specifically found in GAM often involve "active" regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain "inactive" regions. Our work shows that active genomic regions are involved in extensive complex contacts that currently go under-estimated in genome-wide ligation-based approaches, and highlights the need for orthogonal advances in genome-wide contact mapping technologies.

molecular biology↗

bioRxiv2020

Chromatin folding variability across single-cells results from state degeneracy in phase-separation

Chromosome spatial organization controls functional interactions between genes and regulators, yet the molecular and physical mechanisms underlying folding at the single DNA molecule level remain to be understood. Here we employ models of polymer physics to investigate the conformations of two 2Mb-wide DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on the 3D structure of single-molecules are consistently validated against super-resolution single-cell imaging data, providing evidence that the architecture of the studied loci is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules. The process is driven by interactions between distinct types of cognate binding sites, correlating each with a different combination of chromatin factors, including CTCF, cohesin and histone marks. The intrinsic thermodynamics degeneracy of conformations results in a broad structural and time variability of single-molecules, reflected in their varying TAD-like contact patterns. Globules breathe in time, inducing stochastic unspecific interactions, yet they produce stable, compact environments where specific contacts become highly favored between regions enriched for cognate binding sites, albeit characterized by weak biochemical affinities. Cohesin depletion tends to reverse globule phase separation into a coil, randomly folded state, resulting in much more variable contacts across single-molecules, hence erasing population-averaged patterns. Overall, globule phase separation appears to be a robust, reversible mechanism of chromatin organization, where stochasticity and specificity coexist.

molecular biology↗

bioRxiv2020

Comparison of the Hi-C, GAM and SPRITE methods by use of polymer models of chromatin

Powerful technologies have been developed to probe chromatin 3D physical interactions genome-wide, such as Hi-C, GAM and SPRITE. Due to their intrinsic differences and without a benchmarking reference, it is currently difficult to assess how well each method represents the genome 3D structure and their relative performance. Here, we develop a computational approach to implement Hi-C, GAM and SPRITE in-silico to compare the three methods in a simplified, yet controlled framework against known polymer 3D structures. We test our approach on models of three 6-Mb genomic regions, around the Sox9 and the HoxD genes in mouse ES cells, and around the Epha4 gene in mouse CHLX-12 cells. The model-derived contact matrices consistently match Hi-C, GAM and SPRITE experiments. We show that in-silico Hi-C, GAM and SPRITE average data are overall faithful to the 3D structures of the polymer models. We find that the inherent variability of model single-molecule 3D conformations and experimental efficiency differently affect the contact data of the different methods. Similarly, the noise-to-signal levels vary with genomic distance differently in in-silico Hi-C, SPRITE and GAM. We benchmark the performance of each technology in bulk and in single-cell experiments, and identify the minimal number of cells required for replicates to return statistically consistent chromatin contact measures. Under the same experimental conditions, SPRITE requires the lowest number of cells, Hi-C is close to SPRITE, while GAM is the most reproducible method to capture interactions at large genomic distances.

biophysics↗

bioRxiv2020

Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Neurons and oligodendrocytes are terminally differentiated cells that sustain cascades of gene activation and repression to execute highly specialized functions, while retaining homeostatic control. To study long-range chromatin folding without disturbing the native tissue environment, we developed Genome Architecture Mapping in combination with immunoselection (immunoGAM), and applied it to three cell types from the adult murine brain: dopaminergic neurons (DNs) from the midbrain, pyramidal glutamatergic neurons (PGNs) from the hippocampus, and oligodendroglia (OLGs) from the cortex. We find cell-type specific 3D chromatin structures that relate with patterns of gene expression at multiple genomic scales, including extensive reorganization of topological domains (TADs) and chromatin compartments. We discover the loss of TAD insulation, or TAD melting, at long genes (>400 kb) when they are highly transcribed. We find many neuron-specific contacts which contain accessible chromatin regions enriched for putative binding sites for multiple neuronal transcription factors, and which connect cell-type specific genes that are associated with neurodegenerative disorders such as Parkinsons disease, or specialized functions such as synaptic plasticity and memory. Lastly, sensory receptor genes exhibit increased membership in heterochromatic compartments that establish strong contacts in brain cells. However, their silencing is compromised in a subpopulation of PGNs with molecular signatures of long-term potentiation. Overall, our work shows that the 3D organization of the genome is highly cell-type specific, and essential to better understand mechanisms of gene regulation in highly specialized tissues such as the brain.

molecular biology↗

bioRxiv2020

CTCF Promotes Long-range Enhancer-promoter Interactions and Lineage-specific Gene Expression in Mammalian Cells

Topologically associating domains (TAD) and insulated neighborhoods (INs) have been proposed to constrain enhancer-promoter communications to enable cell-type specific transcription programs, but recent studies show that disruption of TADs and INs resulted in relatively mild changes in gene expression profiles. To better understand the role of chromatin architecture in dynamic enhancer-promoter contacts and lineage-specific gene expression, we have utilized the auxin-inducible degron system to acutely deplete CTCF, a key factor involved in TADs and IN formation, in mouse embryonic stem cells (mESCs) and examined chromatin architecture and gene regulation during neural differentiation. We find that while CTCF depletion leads to global weakening of TAD boundaries and loss of INs, only a minor fraction of enhancer-promoter contacts are lost, affecting a small subset of genes. The CTCF-dependent enhancer-promoter contacts tend to be long-range, spanning hundreds of kilobases, and are established directly by CTCF binding to promoters. Disruption of CTCF binding at the promoter reduces enhancer-promoter contacts and transcription, while artificial tethering of CTCF to the promoter restores the enhancer-promoter contacts and gene activation. Genome-wide analysis of CTCF binding and gene expression across multiple mouse tissues suggests that CTCF-dependent promoter-enhancer contacts may regulate expression of additional mouse genes, particularly those expressed in the brain. Our results uncover both CTCF-dependent and independent enhancer-promoter contacts, and highlight a distinct role for CTCF in promoting enhancer-promoter contacts and gene activation in addition to its insulator function.

genomics↗

bioRxiv2020

Genetic interactions shaping evolutionary trajectories in an RNA virus population

A quantitative understanding of evolution rests on the analysis of the mutation accumulation process in biological populations, but is largely limited to high-frequency mutations due to the resolution of conventional sequencing technologies. Here, we examine the mutation composition of a poliovirus population over multiple passages using a highly-accurate sequencing strategy, that enables detection of up to 99% of all possible mutations, most of which are present at low-frequency. This data informs a mathematical model describing trajectory patterns of individual mutations to understand the type of interactions shaping population dynamics. We identify mutations consistent with a locus-independent behavior, and others deviating from that simple model by interactions. Clonal interference, followed by hitchhiking, appear to be the most prevalent interactions in the virus population. Epistasis, while presents, but does not significantly affect the distribution of mutational fitness on the short time scale examined in our study. Our study provides a comprehensive analysis of the allelic composition and how mutation rate, fitness, epistasis, clonal interference and hitchhiking influence population dynamics and evolution.

evolutionary biology↗

bioRxiv2019

Establishing the baseline for using plankton as biosensor

Plankton is at the bottom of the food chain. Microscopic phytoplankton account for about 50% of all photosynthesis on Earth, corresponding to 50 billion tons of carbon each year, or about 125 billion tonnes of sugar[1]. Plankton is also the food for most species of fish, and therefore it represents the backbone of the aquatic environment. Thus, monitoring plankton is paramount to infer potential dangerous changes to the ecosystem. In this work we use a collection of plankton species extracted from a large dataset of images from the Woods Hole Oceanographic Institution (WHOI), to establish a basic set of morphological features for supporting the use of plankton as a biosensor. Using a perturbation detection approach, we show that it is possible to detect deviation from the average space of features for each species of plankton microorganisms, that we propose could be related to environmental threat or perturbations. Such an approach can open the way for the development of an automatic Artificial Intelligence (AI) based system for using plankton as biosensor.

bioengineering↗

bioRxiv2019

Stereo In-Line Holographic Digital Microscope

Biologists use optical microscopes to study plankton in the lab, but their size, complexity and cost makes widespread deployment of microscopes in lakes and oceans challenging. Monitoring the morphology, behavior and distribution of plankton in situ is essential as they are excellent indicators of marine environment health and provide a majority of Earths oxygen and carbon sequestration. Direct in-line holographic microscopy (DIHM) eliminates many of these obstacles, but image reconstruction is computationally intensive and produces monochromatic images. By using one laser and one white LED, it is possible to obtain the 3D location plankton by triangulation, limiting holographic reconstruction to only the voxels occupied by the plankton, reducing computation by several orders of magnitude. The color information from the white LED assists in the classification of plankton, as phytoplankton contains green-colored chlorophyll. The reconstructed plankton images are rendered in a 3D interactive environment, viewable from a browser, providing the user the experience of observing plankton from inside a drop of water.

cell biology↗

bioRxiv2019

Preformed Chromatin Topology Assists TranscriptionalRobustness of Shh during Limb Development

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases proximity coincides with gene activation, whereas in others preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in a loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele resulting in severe Shh loss-of-function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

developmental biology↗

bioRxiv2019

Experimental and mathematical insights on the competition between poliovirus and a defective interfering genome

During replication, RNA virus populations accumulate genome alterations, such as mutations and deletions. The interactions between individual variants within the population can determine the fitness of the virus and, thus, the outcome of infection. We developed an ordinary differential equation model to infer the effect of the interaction between defective interfering (DI) replicons and wild-type (WT) poliovirus. We measure production of RNA and viral particles during a single infection cycle, and use these data to infer model parameters. We find that DI replicates faster than WT, but an equilibrium is established when both WT and DI compete for resources needed for RNA replication and genome encapsidation. In the presence of DI, the concentration of WT virions at cell lysis is suppressed by the factor of 5. Multiple generations within a single cell infection provide opportunities for significant inhibition of WT replication by competition with the faster replicating DI genomes.

microbiology↗

bioRxiv2018

Single-cell chromatin interactions reveal regulatory hubs in dynamic compartmentalized domains

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact within single cells is not understood. To address this we developed Tri-C, a new Chromosome Conformation Capture (3C) approach to identify concurrent chromatin interactions at individual alleles within single cells. The heterogeneity of interactions observed between such cells shows that CTCF-mediated formation of chromatin domains and interactions within them are dynamic processes. Importantly, our analyses reveal higher-order structures involving simultaneous interactions between multiple enhancers and promoters within individual cells. This provides a structural basis for understanding how multiple cis-elements act together to establish robust regulation of gene expression.

genetics↗

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↗