Biology subjects

Esposito, A.

Publications and source records attributed to Esposito, A..

32 records · Page 2Linked to original sources

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

The biochemical resolving power of fluorescence lifetime imaging

A deeper understanding of spatial resolution has led to innovations in microscopy and the disruption of biomedical research, as with super-resolution microscopy. To foster similar advances in time-resolved and spectral imaging, we have previously introduced the concept of biochemical resolving power in fluorescence microscopy. Here, we apply those concepts to investigate how the instrument response function (IRF), sampling conditions, and photon-statistics limit the biochemical resolution of fluorescence lifetime microscopy. Using Fisher information analysis and Monte Carlo simulations, we reveal the complex dependencies between photon-statistics and the IRF, permitting us to quantify resolution limits that have been poorly understood (e.g., the minimum resolvable decay time for a given width of the IRF and photon-statistics) or previously underappreciated (e.g., optimization of the IRF for biochemical detection). With this work, we unravel common misunderstandings on the role of the IRF and provide theoretical insights with significant practical implications on the design and use of time-resolved instrumentation.

biophysics↗

bioRxiv2020

TGS1 controls snRNA 3' end processing, prevents neurodegeneration and ameliorates SMN-dependent neurological phenotypes in vivo

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5 mono-methylguanosine cap of snRNAs to a trimethylguanosine cap. Here, we show that loss of TGS1 in C. elegans, D. melanogaster and D. rerio results in neurological phenotypes similar to those caused by Survival Motor Neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3 tails that are often uridylated. snRNAs with defective 3 terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

genetics↗

bioRxiv2020

A new model of chronic Mycobacterium abscessus lung infection in immunocompetent mice

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of neutrophils, lymphocytes and aggregates of vacuolated foamy cells, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.

microbiology↗

bioRxiv2020

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted -methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

cancer 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↗

bioRxiv2019

Photon-statistics in sensitized emission FRET and FLIM: a comparative theoretical analysis

FRET imaging is an essential analytical method in biomedical research. The limited photon-budget experimentally available, however, imposes compromises between spatiotemporal and biochemical resolutions, photodamage and phototoxicity. The study of photon-statistics in biochemical imaging is thus important in guiding the efficient design of instrumentation and assays. Here, we show a comparative analysis of photon-statistics in FRET imaging demonstrating how the precision of FRET imaging varies vastly with imaging parameters. Therefore, we provide analytical and numerical tools for assay optimization. FLIM is a very robust technique with excellent photon-efficiencies but also intensity-based FRET imaging can reach very high precision by utilizing also information within acceptor fluorescence.

biophysics↗

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↗

bioRxiv2018

Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

cancer biology↗

bioRxiv2018

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

Two decades of high-paced innovation have improved the spatial resolution of fluorescence microscopy to enable molecular resolution combined with the low-invasiveness and specificity characteristic of optical microscopy. However, fluorescence microscopy also enables scientists and clinicians to map and quantitate the physico-chemical properties (e.g., analyte concentration, enzymatic activities and protein-protein interactions) of biological samples. But the optimization of the biochemical resolving power in fluorescence microscopy is not as well-developed compared to its spatial resolution. Typical techniques rely on the observation of individual properties of fluorescence thus limiting the opportunities for sensing and multiplexing. Aiming to overcome existing limitations, we demonstrate a new imaging paradigm -- Hyper Dimensional Imaging Microscopy (HDIM) -- that enables the orthogonal properties of fluorescence emission (excited state lifetime, polarization and spectra) in biological samples to be quantified simultaneously and efficiently. Therefore, akin to how multi-dimensional separation in mass-spectroscopy and multi-dimensional spectra in NMR impacted proteomics and structural biology, we envisage that HDIM spectra of unprecedented dimensionality will impact the fields of systems biology and medical diagnostics by maximizing the biochemical resolving power of fluorescence microscopy.

biophysics↗

bioRxiv2018

Cooperation of partially-transformed clones: the invisible force behind the early stages of carcinogenesis

Most tumours exhibit significant heterogeneity and are best described as communities of cellular populations competing for resources. Growing experimental evidence also suggests, however, that cooperation between cancer clones is important as well for the maintenance of tumour heterogeneity and tumour progression. However, a role for cell communication during the earliest steps in oncogenesis is not well characterised despite its vital importance in normal tissue and clinically manifest tumours. By modelling the interaction between the mutational process and cell-to-cell communication in three-dimensional tissue architecture, we show that non-cell-autonomous mechanisms of carcinogenesis could support and accelerate pre-cancerous clonal expansion through the cooperation of different, non- or partially- transformed mutants. We predict the existence of a cell-autonomous time-horizon, a time before which cooperation between cell-to-cell communication and DNA mutations might be one of the most fundamental forces shaping the early stages of oncogenesis. The understanding of this process could shed new light on the mechanisms leading to clinically manifest cancers.

cancer biology↗

bioRxiv2018

Checkpoint non-fidelity induces a complex landscape of lineage fitness after DNA damage

DNA damage in proliferating mammalian cells causes death1, senescence2 or continued survival, via checkpoints that monitor damage and regulate cell cycle progression, DNA repair and fate determination3. Cell cycle checkpoints facilitate tumour suppression by preventing the generation of proliferating mutated cells4, particularly by blocking passage of DNA lesions into replication and mitosis5. While checkpoint non-fidelity permits cells to carry genomic aberrations into subsequent cell cycle phases6, its long-term consequences on lineages descendant from damaged cells remains poorly characterised. Devising methods for microscopy-based lineage tracing, we unexpectedly demonstrate that transient DNA damage to single living cells bearing a negligent checkpoint induces heterogenous cell-fate outcomes in their descendant generations removed from the initial insult. After transiently damaged cells undergo an initial arrest, pairs of descendant cells without obvious cell-cycle abnormalities either divide or die in a seemingly stochastic way. Progeny of transiently damaged cells may die generations afterwards, creating considerable variability of lineage fitness that promotes overall persistence in a mutagenic environment. Descendants of damaged cells frequently form micronuclei, activating immunogenic signalling. Our findings reveal previously unrecognized, heterogenous effects of cellular DNA damage that manifest long afterwards in descendant cells. We suggest that these heterogenous descendant cell-fate responses may function physiologically to ensure the elimination and immune clearance of damaged cell lineages, but pathologically, may enable the prolonged survival of cells bearing mutagenic damage.

cancer biology↗

bioRxiv2018

mTOR-dependent phosphorylation controls TFEB nuclear export

The transcriptional activation of catabolic processes during starvation is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient re-feeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data reveal that modulation of TFEB nuclear export via phosphorylation plays a major role in the modulation of TFEB localization and activity.

cell biology↗