Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3’UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3’UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

The cancer epigenome has been studied in cells cultured in 2D monolayers on plastic surfaces, but recent studies highlight the impact of the extracellular matrix (ECM) and the 3D environment on multiple cellular functions. Here, we report the physical, biochemical and genomic differences between T47D breast cancer cells cultured in 2D monolayer and as 3D spheroids in Matrigel. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, and altered expression of over 2,000 genes, the majority of which become repressed. Hi-C analysis reveals that cells grown in 3D exhibit enrichment in regions belonging to the B compartment, decrease on chromatin bound CTCF and increased fusion of Topologically Associating Domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, likely responsible for the observed TAD fusions. Cells grown in 3D exhibit higher progesterone receptor (PR) binding to chromatin, leading to an increase in the number hormone-regulated genes. This effect is in part mediated by the LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

Chromatin remodeling is essential to allow full development of alternative gene expression programs in response to environmental changes. In fission yeast, oxidative stress triggers massive transcriptional changes including the activation of hundreds of genes, with the participation of histone modifying complexes and chromatin remodelers. DNA transcription is associated to alterations in DNA topology, and DNA topoisomerases facilitate elongation along gene bodies. Here, we test whether the DNA topoisomerase Top1 participates in the RNA polymerase II-dependent activation of the cellular response to oxidative stress. Cells lacking Top1 are resistant to H2O2 stress. The transcriptome of Δtop1 strain was not greatly affected in the absence of stress, but activation of the anti-stress gene expression program was more sustained than in wild-type cells. Top1 associated to stress open reading frames. While the nucleosomes of stress genes are partially and transiently evicted during stress, the chromatin configuration remains open for longer times in cells lacking Top1, facilitating RNA polymerase II progression. We propose that, by removing DNA tension arising from transcription, Top1 facilitates nucleosome reassembly and works in synergy with the chromatin remodeler Hrp1 as opposing forces to transcription and to Snf22 / Hrp3 opening remodelers.

Among the existing techniques for interrogating the genome structure, Hi-C assays have become the most performed experiments and constitute the majority of the publicly available datasets. As a result, there is a continuous demand to create and improve algorithms and methods to assist the scientific community in the interpretation of Hi-C experimental data. Here we introduce probabilistic TADbit (pTADbit), a new approach that combines Deep Learning and restraint-based modelling to infer the three-dimensional (3D) structure of genome and genomic domains interrogated by Hi-C experiments. pTADbit uses thousands of microscopy-based distances between genomic loci to train a neural network model that aims at predicting the population distribution of the spatial distance between two genomic loci based solely on their Hi-C interaction frequency. pTADbit produces more accurate chromatin models compared to the original TADbit as well as other available 3D modeling methods, while drastically reducing the required computation time. The resulting ensemble of models not only agree consistently with independent measures obtained by imaging experiments but also better capture the heterogeneity of the cell population. The development of pTADbit lays the basis for the integration of data produced from high-throughput imaging assays into the 3D modelling genomes and genomic domains.

BACKGROUND:In many organisms, aging is characterized by a loss of mitochondrial homeostasis. Multiple factors such as respiratory metabolism, mitochondrial fusion/fission, or mitophagy have been linked to cell longevity, but the exact impact of each one on the aging process is still unclear.RESULTS:Using the deletion mutant collection of the fission yeast Schizosaccharomyces pombe, we have developed a genome-wide screening for mutants with altered chronological lifespan. We have identified four mutants associated with proteolysis at the mitochondria that exhibit opposite effects on longevity. The analysis of the respiratory activity of these mutants revealed a positive correlation between increased respiration rate and prolonged lifespan. We also found that the phenotype of the long-lived protease mutants could not be explained by impaired mitochondrial fusion/fission activities, but it was dependent on mitophagy induction. The anti-aging role of mitophagy was supported by the effect of a mutant defective in degradation of mitochondria, which shortened lifespan of the long-lived mutants.CONCLUSIONS:Our characterization of the mitochondrial protease mutants demonstrates that mitophagy sustains the lifespan extension of long-lived mutants displaying a higher respiration potential.

Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer-promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2-3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription.

There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes. OligoFISSEQ combines Oligopaints with fluorescence in situ sequencing to enable the 3D mapping of many regions across the genome in human cells to interrogate genome organization at improved genomic resolution. OligoFISSEQ is compatible with immunochemistry and OligoSTORM for super-resolution imaging.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intraversus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression, We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Chromatin Conformation Capture techniques have unveiled several layers of chromosome organization such as the segregation in compartments, the folding in topologically associating domains (TADs), and site-specific looping interactions. The discovery of this genome hierarchical organization emerged from the computational analysis of chromatin capture data. With the increasing availability of such data, automatic pipelines for the robust comparison, grouping, and classification of multiple experiments are needed. Here we present a pipeline based on the TADbit framework that emphasizes reproducibility, automation, quality check, and statistical robustness. This comprehensive modular pipeline covers all the steps from the sequencing products to the visualization of reconstructed 3D models of the chromatin.

Chromosome conformation capture techniques, such as Hi-C, are fundamental in characterizing genome organization. These methods have revealed several genomic features, such as chromatin loops, whose disruption can have dramatic effects in gene regulation. Unfortunately, their detection is difficult; current methods require that the users choose the resolution of interaction maps based on dataset quality and sequencing depth. Here, we introduce Binless, a resolution-agnostic method that adapts to the quality and quantity of available data, to detect both interactions and differences. Binless relies on an alternate representation of Hi-C data, which leads to a more detailed classification of paired-end reads. Using a large-scale benchmark, we demonstrate that Binless is able to call interactions with higher reproducibility than other existing methods. Binless, which is freely available, can thus reliably be used to identify chromatin loops as well as for differential analysis of chromatin interaction maps.

ABSTRACT Multiscale Genomics (MuG) Virtual Research Environment (MuGVRE) is a cloud-based computational infrastructure created to support the deployment of software tools addressing the various levels of analysis in 3D/4D genomics. Integrated tools tackle needs ranging from high computationally demanding applications (e.g. molecular dynamics simulations) to high-throughput data analysis applications (like the processing of next generation sequencing). The MuG Infrastructure is based on openNebula cloud systems implemented at the Institute for research in Biomedicine, and the Barcelona Supercomputing Center, and has specific interfaces for users and developers. Interoperability of the tools included in MuGVRE is maintained through a rich set of metadata allowing the system to associate tools and data in a transparent manner. Execution scheduling is based in a traditional queueing system to handle demand peaks in applications of fixed needs, and an elastic and multi-scale programming model (pyCOMPSs, controlled by the PMES scheduler), for complex workflows requiring distributed or multi-scale executions schemes. MuGVRE is available at https://vre.multiscalegenomics.eu and documentation and general information at https://www.multiscalegenomics.eu . The infrastructure is open and freely accessible.

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome Conformation Capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a new tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We applied TADdyn on in-situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterized the structural rearrangements that take place upon changes in the transcriptional state of 11 genomic loci. TADdyn simulations show that structural cages form around the transcription starting site of active loci to stabilize their dynamics, by initiating ( hit ) and maintaining ( stick ) interactions with regulatory regions. Consistent findings with TADdyn for all loci under study suggest that this hit-and-stick mechanism may represent a general mechanism to trigger and stabilize transcription.

The sequence of a genome is insufficient to understand all genomic processes carried out in the cell nucleus. To achieve this, the knowledge of its three-dimensional architecture is necessary. Advances in genomic technologies and the development of new analytical methods, such as Chromosome Conformation Capture (3C) and its derivatives, provide unprecedented insights in the spatial organization of genomes. Here we present TADbit, a computational framework to analyze and model the chromatin fiber in three dimensions. Our package takes as input the sequencing reads of 3C-based experiments and performs the following main tasks: (i) pre-process the reads, (ii) map the reads to a reference genome, (iii) filter and normalize the interaction data, (iv) analyze the resulting interaction matrices, (v) build 3D models of selected genomic domains, and (vi) analyze the resulting models to characterize their structural properties. To illustrate the use of TADbit, we automatically modeled 50 genomic domains from the fly genome revealing differential structural features of the previously defined chromatin colors, establishing a link between the conformation of the genome and the local chromatin composition. TADbit provides three-dimensional models built from 3C-based experiments, which are ready for visualization and for characterizing their relation to gene expression and epigenetic states. TADbit is an open-source Python library available for download from https://github.com/3DGenomes/tadbit.

3C-like experiments, such as 4C or Hi-C, have been fundamental in understanding genome organization. Thanks to these technologies, it is now known, for example, that Topologically Associating Domains (TADs) and chromatin loops are implicated in the dynamic interplay of gene activation and repression, and their disruption can have dramatic effects on embryonic development. To make their detection easier, scientists have endeavored into deeper sequencing to mechanically increase the chances to detect weaker signals such as chromatin loops. Part of this mindset can be attributed to the limitations of existing software: the analysis of Hi-C experiments is both statistically and computationally demanding. Here, we devise a new way to represent Hi-C data, which leads to a more detailed classification of paired-end reads and, ultimately, to a new normalization and interaction detection method. Unlike any other, Binless is resolution-agnostic, and adapts to the quality and quantity of available data. We demonstrate its capacities to call interactions and differences and make the software freely available.

Light sheet fluorescence microscopy (LSFM) is used in many biological research experiments that require fast three-dimensional (3D) imaging up to a few volumes per second. Wavefront coding (WFC) microscopy provides LSFM with 3D real-time imaging capabilities introducing a controlled aberration that extends the depth of field (DOF) of the microscope objective. Resulting images, however, are blurred and need to be processed to recover the original sharpness in a CPU-intensive deconvolution which makes real-time visualization unattainable. We present computational tools based on GPU parallel computing to achieve realtime deconvolution and visualization of the images obtained with WFC light-sheet microscopy.