Author: "M. Chiariello" - Searchworks@Jio Institute Digital Library Search Results

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1. Multiscale modelling of chromatin 4D organization in SARS-CoV-2 infected cells

Author: Andrea M. Chiariello, Alex Abraham, Simona Bianco, Andrea Esposito, Andrea Fontana, Francesca Vercellone, Mattia Conte, and Mario Nicodemi
Subjects: Science
Abstract: Abstract SARS-CoV-2 can re-structure chromatin organization and alter the epigenomic landscape of the host genome, but the mechanisms that produce such changes remain unclear. Here, we use polymer physics to investigate how the chromatin of the host genome is re-organized upon infection with SARS-CoV-2. We show that re-structuring of A/B compartments can be explained by a re-modulation of intra-compartment homo-typic affinities, which leads to the weakening of A-A interactions and the enhancement of A-B mixing. At the TAD level, re-arrangements are physically described by a reduction in the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and a spread in space of the TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon response, such as DDX58 or IFIT, becomes more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysing the time trajectories of pairwise gene-enhancer and higher-order contacts reveals that this variability derives from increased fluctuations in the chromatin dynamics of infected cells. This suggests that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time.
Published: 2024
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2. Hmga2 protein loss alters nuclear envelope and 3D chromatin structure

Author: Giuseppina Divisato, Andrea M. Chiariello, Andrea Esposito, Pietro Zoppoli, Federico Zambelli, Maria Antonietta Elia, Graziano Pesole, Danny Incarnato, Fabiana Passaro, Silvia Piscitelli, Salvatore Oliviero, Mario Nicodemi, Silvia Parisi, and Tommaso Russo
Subjects: High mobility group proteins, Pluripotent stem cells, Topologically associated domains, Lamin, Nuclear envelope, Histone modifications, Biology (General), QH301-705.5
Abstract: Abstract Background The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. Results We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. Conclusions Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.
Published: 2022
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3. Loop-extrusion and polymer phase-separation can co-exist at the single-molecule level to shape chromatin folding

Author: Mattia Conte, Ehsan Irani, Andrea M. Chiariello, Alex Abraham, Simona Bianco, Andrea Esposito, and Mario Nicodemi
Subjects: Science
Abstract: Two main mechanisms have been proposed to shape 3D genome architecture - loop extrusion and phase separation. Here the authors combine these mechanisms in polymer models in a manner that best fits 3D genome, based on both Hi-C and super-resolution locus imaging data, proposing that these two physical processes can indeed coexist simultaneously within cells to define loops and TADs.
Published: 2022
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4. Polymer physics indicates chromatin folding variability across single-cells results from state degeneracy in phase separation

Author: Mattia Conte, Luca Fiorillo, Simona Bianco, Andrea M. Chiariello, Andrea Esposito, and Mario Nicodemi
Subjects: Science
Abstract: The molecular and physical mechanisms underlying chromatin folding at the single DNA molecule level remain poorly understood. Here, the authors use polymer modeling to investigate the conformations of two 2Mb-wide DNA loci in normal and cohesin depleted cells, and provide 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.
Published: 2020
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5. Polymer Models of Chromatin Imaging Data in Single Cells

Author: Mattia Conte, Andrea M. Chiariello, Alex Abraham, Simona Bianco, Andrea Esposito, Mario Nicodemi, Tommaso Matteuzzi, and Francesca Vercellone
Subjects: chromosome architecture, multiplexed FISH imaging, polymer physics, machine learning, computer simulations, Industrial engineering. Management engineering, T55.4-60.8, Electronic computers. Computer science, QA75.5-76.95
Abstract: Recent super-resolution imaging technologies enable tracing chromatin conformation with nanometer-scale precision at the single-cell level. They revealed, for example, that human chromosomes fold into a complex three-dimensional structure within the cell nucleus that is essential to establish biological activities, such as the regulation of the genes. Yet, to decode from imaging data the molecular mechanisms that shape the structure of the genome, quantitative methods are required. In this review, we consider models of polymer physics of chromosome folding that we benchmark against multiplexed FISH data available in human loci in IMR90 fibroblast cells. By combining polymer theory, numerical simulations and machine learning strategies, the predictions of the models are validated at the single-cell level, showing that chromosome structure is controlled by the interplay of distinct physical processes, such as active loop-extrusion and thermodynamic phase-separation.
Published: 2022
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6. Divergent Transcription of the Nkx2-5 Locus Generates Two Enhancer RNAs with Opposing Functions

Author: Irene Salamon, Simone Serio, Simona Bianco, Christina Pagiatakis, Silvia Crasto, Andrea M. Chiariello, Mattia Conte, Paola Cattaneo, Luca Fiorillo, Arianna Felicetta, Elisa di Pasquale, Paolo Kunderfranco, Mario Nicodemi, Roberto Papait, and Gianluigi Condorelli
Subjects: Biological Sciences, Molecular Biology, Molecular Mechanism of Gene Regulation, Science
Abstract: Summary: Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5—a gene specifying a master cardiomyogenic lineage transcription factor (TF)—which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription.
Published: 2020
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7. A Dynamic Folded Hairpin Conformation Is Associated with α-Globin Activation in Erythroid Cells

Author: Andrea M. Chiariello, Simona Bianco, A. Marieke Oudelaar, Andrea Esposito, Carlo Annunziatella, Luca Fiorillo, Mattia Conte, Alfonso Corrado, Antonella Prisco, Martin S.C. Larke, Jelena M. Telenius, Renato Sciarretta, Francesco Musella, Veronica J. Buckle, Douglas R. Higgs, Jim R. Hughes, and Mario Nicodemi
Subjects: Biology (General), QH301-705.5
Abstract: Summary: We investigate the three-dimensional (3D) conformations of the α-globin locus at the single-allele level in murine embryonic stem cells (ESCs) and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent fluorescence in situ hybridization (FISH) data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ESCs to erythroid cells, associated with the activation of the globin genes. We find that in ESCs, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, whereas in erythroid cells the α-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites, multiple molecular mechanisms cooperate to form an interacting domain. : Chiariello et al. use polymer physics models to infer the 3D conformations of the murine α-globin locus. In the transition from ESCs to erythroid cells, the locus rearranges from an inactive highly intermingled conformation to an active, hairpin-shaped structure, marked by cell-specific three-way contacts accurately predicted by the models. Keywords: globin loci, higher-order chromatin organization, polymer physics, gene regulation
Published: 2020
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8. The effect of configurational complexity in hetero-polymers on the coil-globule phase transition

Author: Fabrizio Tafuri and Andrea M. Chiariello
Subjects: Fluid Flow and Transfer Processes, General Physics and Astronomy
Abstract: The coil-globule transition of hetero-polymer chains is studied here. By means of extensive Molecular Dynamics simulations, we show that the transition is directly linked to the complexity of the chain, which depends on the number of chemical species defined in the environment and the location of the binding sites along the polymer. In addition, when the number of species increases, we find that the distribution of binding sites plays an important role in triggering the transition, beyond the standard control parameters of the polymer model, i.e. binders concentration and binding affinity. Overall, our results show that by increasing the system complexity new organizational layers emerge, thus allowing a more structured control on the polymer thermodynamic state. This can be potentially applied to the study of chromatin architecture, as such polymer models have been broadly used to understand the molecular mechanisms of genome folding.
Published: 2023
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9. Physics-Based Polymer Models to Probe Chromosome Structure in Single Molecules

Author: Mattia Conte, Andrea M. Chiariello, Simona Bianco, Andrea Esposito, Alex Abraham, and Mario Nicodemi
Published: 2023
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10. Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation

Author: James Fraser, Carmelo Ferrai, Andrea M Chiariello, Markus Schueler, Tiago Rito, Giovanni Laudanno, Mariano Barbieri, Benjamin L Moore, Dorothee CA Kraemer, Stuart Aitken, Sheila Q Xie, Kelly J Morris, Masayoshi Itoh, Hideya Kawaji, Ines Jaeger, Yoshihide Hayashizaki, Piero Carninci, Alistair RR Forrest, The FANTOM Consortium, Colin A Semple, Josée Dostie, Ana Pombo, and Mario Nicodemi
Subjects: chromatin contacts, chromosome architecture, epigenetics, gene expression, polymer modelling, Biology (General), QH301-705.5, Medicine (General), R5-920
Abstract: Abstract Mammalian chromosomes fold into arrays of megabase‐sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher‐order organization remains elusive. Here, we investigate TAD higher‐order interactions with Hi‐C through neuronal differentiation and show that they form a hierarchy of domains‐within‐domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree‐like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.
Published: 2015
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11. A Polymer Physics Investigation of the Architecture of the Murine Orthologue of the 7q11.23 Human Locus

Author: Andrea M. Chiariello, Andrea Esposito, Carlo Annunziatella, Simona Bianco, Luca Fiorillo, Antonella Prisco, and Mario Nicodemi
Subjects: polymer physics, chromatin, Neuroscience, congenital disease, 7q11.23 locus, Neurosciences. Biological psychiatry. Neuropsychiatry, RC321-571
Abstract: In the last decade, the developments of novel technologies, such as Hi-C or GAM methods, allowed to discover that chromosomes in the nucleus of mammalian cells have a complex spatial organization, encompassing the functional contacts between genes and regulators. In this work, we review recent progresses in chromosome modeling based on polymer physics to understand chromatin structure and folding mechanisms. As an example, we derive in mouse embryonic stem cells the full 3D structure of the Bmp7 locus, a genomic region that plays a key role in osteoblastic differentiation. Next, as an application to Neuroscience, we present the first 3D model for the mouse orthologoue of the Williams–Beuren syndrome 7q11.23 human locus. Deletions and duplications of the 7q11.23 region generate neurodevelopmental disorders with multi-system involvement and variable expressivity, and with autism. Understanding the impact of such mutations on the rewiring of the interactions of genes and regulators could be a new key to make sense of their related diseases, with potential applications in biomedicine.
Published: 2017
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12. Polymer models are a versatile tool to study chromatin 3D organization

Author: Andrea Esposito, Andrea M. Chiariello, Alex Abraham, Luca Fiorillo, Francesco Musella, Simona Bianco, Mattia Conte, Antonella Prisco, Mario Nicodemi, Esposito, A., Bianco, S., Fiorillo, L., Conte, M., Abraham, A., Musella, F., Nicodemi, M., Prisco, A., and Chiariello, A. M.
Subjects: Polymers, Computer science, DNA Folding, Inference, Molecular Dynamics Simulation, Models, Biological, Biochemistry, 03 medical and health sciences, 0302 clinical medicine, chromatin architecture, Human–computer interaction, computer simulation, 030304 developmental biology, Structure (mathematical logic), SBS model, 0303 health sciences, Genome, polymer physics, Chromatin, machine learning, Polymer physics, Single-Cell Analysis, phase separation, 030217 neurology & neurosurgery, Genome architecture
Abstract: The development of new experimental technologies is opening the way to a deeper investigation of the three-dimensional organization of chromosomes inside the cell nucleus. Genome architecture is linked to vital functional purposes, yet a full comprehension of the mechanisms behind DNA folding is still far from being accomplished. Theoretical approaches based on polymer physics have been employed to understand the complexity of chromatin architecture data and to unveil the basic mechanisms shaping its structure. Here, we review some recent advances in the field to discuss how Polymer Physics, combined with numerical Molecular Dynamics simulation and Machine Learning based inference, can capture important aspects of genome organization, including the description of tissue-specific structural rearrangements, the detection of novel, regulatory-linked architectural elements and the structural variability of chromatin at the single-cell level.
Published: 2021
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13. Structure of the human chromosome interaction network.

Author: Sergio Sarnataro, Andrea M Chiariello, Andrea Esposito, Antonella Prisco, and Mario Nicodemi
Subjects: Medicine, Science
Abstract: New Hi-C technologies have revealed that chromosomes have a complex network of spatial contacts in the cell nucleus of higher organisms, whose organisation is only partially understood. Here, we investigate the structure of such a network in human GM12878 cells, to derive a large scale picture of nuclear architecture. We find that the intensity of intra-chromosomal interactions is power-law distributed. Inter-chromosomal interactions are two orders of magnitude weaker and exponentially distributed, yet they are not randomly arranged along the genomic sequence. Intra-chromosomal contacts broadly occur between epigenomically homologous regions, whereas inter-chromosomal contacts are especially associated with regions rich in highly expressed genes. Overall, genomic contacts in the nucleus appear to be structured as a network of networks where a set of strongly individual chromosomal units, as envisaged in the 'chromosomal territory' scenario derived from microscopy, interact with each other via on average weaker, yet far from random and functionally important interactions.
Published: 2017
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14. Physical mechanisms of chromatin spatial organization

Author: Luca Fiorillo, Simona Bianco, Mattia Conte, Francesco Musella, Mario Nicodemi, Alex Abraham, Antonella Prisco, Andrea M. Chiariello, Ehsan Irani, Andrea Esposito, Chiariello, A. M., Bianco, S., Esposito, A., Fiorillo, L., Conte, M., Irani, E., Musella, F., Abraham, A., Prisco, A., and Nicodemi, M.
Subjects: 0301 basic medicine, Computational biology, Biology, Models, Biological, Biochemistry, 03 medical and health sciences, Biopolymers, 0302 clinical medicine, chromatin architecture, computer simulation, Molecular Biology, Gene, Spatial organization, Regulation of gene expression, Cell Biology, polymer physics, Chromatin, Folding (chemistry), machine learning, 030104 developmental biology, Gene Expression Regulation, 030220 oncology & carcinogenesis, Mutation, phase separation, active motor
Abstract: In higher eukaryotes, chromosomes have a complex three-dimensional (3D) conformation in the cell nucleus serving vital functional purposes, yet their folding principles remain poorly understood at the single-molecule level. Here, we summarize recent approaches from polymer physics to comprehend the physical mechanisms underlying chromatin architecture. In particular, we focus on two models that have been supported by recent, growing experimental evidence, the Loop Extrusion model and the Strings&Binders phase separation model. We discuss their key ingredients, how they compare to experimental data and some insight they provide on chromatin architecture and gene regulation. Progresses in that research field are opening the possibility to predict how genomic mutations alter the network of contacts between genes and their regulators and how that is linked to genetic diseases, such as congenital disorders and cancer.
Published: 2021
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15. Inference of chromosome 3D structures from GAM data by a physics computational approach

Author: Alfonso Corrado, Mattia Conte, Antonella Prisco, Andrea Esposito, Mario Nicodemi, Mariano Barbieri, Andrea M. Chiariello, Ana Pombo, Luca Fiorillo, Carlo Annunziatella, Simona Bianco, Fiorillo, L., Bianco, S., Chiariello, A. M., Barbieri, M., Esposito, A., Annunziatella, C., Conte, M., Corrado, A., Prisco, A., Pombo, A., and Nicodemi, M.
Subjects: Sprite (computer graphics), Polymers, Molecular Conformation, Inference, Whole chromosome, Chromosomes, General Biochemistry, Genetics and Molecular Biology, Mice, 03 medical and health sciences, Chromosome (genetic algorithm), Animals, Statistical Physic, Molecular Biology, 030304 developmental biology, 0303 health sciences, Genome, Physics, 030302 biochemistry & molecular biology, Chromosome Mapping, Experimental data, Mouse Embryonic Stem Cells, SOX9 Transcription Factor, GAM data, Models, Chemical, Genetic Loci, Polymer physics, Algorithm, Genome architecture
Abstract: The combination of modelling and experimental advances can provide deep insights for understanding chromatin 3D organization and ultimately its underlying mechanisms. In particular, models of polymer physics can help comprehend the complexity of genomic contact maps, as those emerging from technologies such as Hi-C, GAM or SPRITE. Here we discuss a method to reconstruct 3D structures from Genome Architecture Mapping (GAM) data, based on PRISMR, a computational approach introduced to find the minimal polymer model best describing Hi-C input data from only polymer physics. After recapitulating the PRISMR procedure, we describe how we extended it for treating GAM data. We successfully test the method on a 6 Mb region around the Sox9 gene and, at a lower resolution, on the whole chromosome 7 in mouse embryonic stem cells. The PRISMR derived 3D structures from GAM co-segregation data are finally validated against independent Hi-C contact maps. The method results to be versatile and robust, hinting that it can be similarly applied to different experimental data, such as SPRITE or microscopy distance data.
Published: 2020
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16. Polymer physics reveals a combinatorial code linking 3D chromatin architecture to 1D chromatin states

Author: Andrea Esposito, Simona Bianco, Andrea M. Chiariello, Alex Abraham, Luca Fiorillo, Mattia Conte, Raffaele Campanile, Mario Nicodemi, Esposito, A., Bianco, S., Chiariello, A. M., Abraham, A., Fiorillo, L., Conte, M., Campanile, R., and Nicodemi, M.
Subjects: Mammals, Genome, Animal, Polymers, Physics, Chromosome, biophysic, epigenomic, General Biochemistry, Genetics and Molecular Biology, Mammal, Chromatin, Chromosomes, machine learning, chromatin architecture, Cardiovascular and Metabolic Diseases, 3D genome organization, computer simulation, Physic, Animals, CP: Molecular biology, polymer-physic
Abstract: The mammalian genome has a complex, functional 3D organization. However, it remains largely unknown how DNA contacts are orchestrated by chromatin organizers. Here, we infer from only Hi-C the cell-type-specific arrangement of DNA binding sites sufficient to recapitulate, through polymer physics, contact patterns genome wide. Our model is validated by its predictions in a set of duplications at Sox9 against available independent data. The binding site types fall in classes that well match chromatin states from segmentation studies, yet they have an overlapping, combinatorial organization along chromosomes necessary to accurately explain contact specificity. The chromatin signatures of the binding site types return a code linking chromatin states to 3D architecture. The code is validated by extensive de novo predictions of Hi-C maps in an independent set of chromosomes. Overall, our results shed light on how 3D information is encrypted in 1D chromatin via the specific combinatorial arrangement of binding sites.
Published: 2022

17. Further Delineation of Duplications of ARX Locus Detected in Male Patients with Varying Degrees of Intellectual Disability

Author: Loredana Poeta, Michela Malacarne, Agnese Padula, Denise Drongitis, Lucia Verrillo, Maria Brigida Lioi, Andrea M. Chiariello, Simona Bianco, Mario Nicodemi, Maria Piccione, Emanuela Salzano, Domenico Coviello, Maria Giuseppina Miano, Poeta L., Malacarne M., Padula A., Drongitis D., Verrillo L., Lioi M.B., Chiariello A.M., Bianco S., Nicodemi M., Piccione M., Salzano E., Coviello D., Miano M.G., Poeta, L., Malacarne, M., Padula, A., Drongitis, D., Verrillo, L., Lioi, M. B., Chiariello, A. M., Bianco, S., Nicodemi, M., Piccione, M., Salzano, E., Coviello, D., and Miano, M. G.
Subjects: Male, Transcription Factor, Ultraconserved enhancers, Intellectual disability, 3D structure, Catalysis, Inorganic Chemistry, Mice, Animals, Humans, Physical and Theoretical Chemistry, Child, Molecular Biology, Spectroscopy, Homeodomain Proteins, Animal, KDM5C-SYN1 axi, Organic Chemistry, KDM5C-SYN1 axis, Genes, Homeobox, Homeodomain Protein, General Medicine, Xp21.3 duplication, Computer Science Applications, Ultraconserved enhancer, Settore MED/03 - Genetica Medica, Mutation, ARX, Human, Transcription Factors
Abstract: The X-linked gene encoding aristaless-related homeobox (ARX) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, paediatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telencephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that—depending on the involvement of tissue-specific enhancers—the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance.
Published: 2022

18. Connecting the Dots: PHF13 and cohesin promote polymer-polymer phase separation of chromatin into chromosomes

Author: Francesca Rossi, Rene Buschow, Laura V. Glaser, Tobias Schubert, Hannah Staege, Astrid Grimme, Hans Will, Thorsten Milke, Martin Vingron, Andrea M. Chiariello, and Sarah Kinkley
Subjects: History, Polymers and Plastics, Business and International Management, biological phenomena, cell phenomena, and immunity, Industrial and Manufacturing Engineering
Abstract: SummaryHow interphase chromatin compacts into mitotic chromosomes has eluded researchers for over a century. Here we show that PHF13, a tightly regulated H3K4me epigenetic reader, and cohesin, a mediator of chromatin architecture, cooperate to drive polymer-polymer-phase-separation (PPPS) and higher order compaction of chromatin into chromosomes. PHF13 interacts with cohesin, shows similar dynamics during mitosis, and their co-depletion dramatically impairs mitotic condensation. Mechanistically, we demonstrate that PHF13 stabilizes cohesin chromatin interactions and that itself oligomerizes, resulting in a polymer with increased chromatin avidity and the ability to bridge neighboring and distant nucleosomes. Consistently, molecular dynamic simulations modelling the ability of PHF13 and cohesin to drive chromatin phase separations recapitulated our in vivo observations and are in line with 2-step condensation models.
Published: 2022
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19. Further Delineation of Duplications of

Author: Loredana, Poeta, Michela, Malacarne, Agnese, Padula, Denise, Drongitis, Lucia, Verrillo, Maria Brigida, Lioi, Andrea M, Chiariello, Simona, Bianco, Mario, Nicodemi, Maria, Piccione, Emanuela, Salzano, Domenico, Coviello, and Maria Giuseppina, Miano
Subjects: Homeodomain Proteins, Male, Mice, Intellectual Disability, Mutation, Genes, Homeobox, Animals, Humans, Child, Transcription Factors
Abstract: The X-linked gene encoding aristaless-related homeobox (
Published: 2022

20. 8-oxodG accumulation within super-enhancers marks fragile CTCF-mediated chromatin loops

Author: Giovanni Scala, Francesca Gorini, Susanna Ambrosio, Andrea M Chiariello, Mario Nicodemi, Luigi Lania, Barbara Majello, Stefano Amente, Scala, Giovanni, Gorini, Francesca, Ambrosio, Susanna, Chiariello, Andrea M, Nicodemi, Mario, Lania, Luigi, Majello, Barbara, and Amente, Stefano
Subjects: CCCTC-Binding Factor, Enhancer Elements, Genetic, Transcription, Genetic, 8-Hydroxy-2'-Deoxyguanosine, Genetics, Humans, DNA, Promoter Regions, Genetic, Chromatin, Genomic Instability, Epigenesis, Genetic
Abstract: 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a major product of the DNA oxidization process, has been proposed to have an epigenetic function in gene regulation and has been associated with genome instability. NGS-based methodologies are contributing to the characterization of the 8-oxodG function in the genome. However, the 8-oxodG epigenetic role at a genomic level and the mechanisms controlling the genomic 8-oxodG accumulation/maintenance have not yet been fully characterized. In this study, we report the identification and characterization of a set of enhancer regions accumulating 8-oxodG in human epithelial cells. We found that these oxidized enhancers are mainly super-enhancers and are associated with bidirectional-transcribed enhancer RNAs and DNA Damage Response activation. Moreover, using ChIA-PET and HiC data, we identified specific CTCF-mediated chromatin loops in which the oxidized enhancer and promoter regions physically associate. Oxidized enhancers and their associated chromatin loops accumulate endogenous double-strand breaks which are in turn repaired by NHEJ pathway through a transcription-dependent mechanism. Our work suggests that 8-oxodG accumulation in enhancers–promoters pairs occurs in a transcription-dependent manner and provides novel mechanistic insights on the intrinsic fragility of chromatin loops containing oxidized enhancers-promoters interactions.
Published: 2022

21. Efficient computational implementation of polymer physics models to explore chromatin structure

Author: Raffaele Campanile, Alfonso Corrado, Luca Fiorillo, Andrea M. Chiariello, Andrea Esposito, Mattia Conte, Maria Gabriella Chiariello, Carlo Annunziatella, Simona Bianco, Conte, M., Esposito, A., Fiorillo, L., Campanile, R., Annunziatella, C., Corrado, A., Chiariello, M. G., Bianco, S., and Chiariello, A. M.
Subjects: Physics, Molecular dynamics, Molecular dynamic, Sprite (lightning), Computer Networks and Communications, Spatial structure, Polymer physics, Biological system, polymer physics, Software, Chromatin, chromatin organization
Abstract: The development of novel experimental technologies able to map genome-wide chromatin contacts, as Hi-C, GAM or SPRITE, allowed to derive detailed information about the spatial structure of chromosomes in the cell nucleus. They revealed that the genome has a complex spatial organisation, which is highly connected with its activity. In the last years, such an abundance of experimental data prompted the development of quantitative models based on Polymer Physics to describe the chromatin architecture, clarifying many aspects about the molecular mechanisms underlying genome folding. Efficient algorithms are thus fundamental to perform massive numerical simulations for testing the accuracy of these models and provide a good description for small genomic regions or for whole chromosomes. Here, we consider the performances of Molecular Dynamics (MD) implementation of commonly used polymer physics models. Such models can be combined with Machine Learning approaches informed with experimental data to produce more accurate descriptions of real genomic regions. However, the execution times increase as a power-law with the size of the input data, which ultimately reflects the complexity of the investigated system. The best strategy is therefore a convenient trade-off between the accuracy in the description and the availability of computational resources. The combination of innovative experimental data and polymer physics theories allow to reconstruct the 3D genome structure. This is achieved by the use of machine learning approaches and massive parallel computing. Efficient algorithms and computational resources are then fundamental to produce models of increasingly high accuracy.
Published: 2022

22. A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome

Author: Elena Manara, Luigia Cinque, Orazio Palumbo, Matteo Bertelli, Lucia Micale, Simona Bianco, Mario Nicodemi, Marco Castori, Angelantonio Notarangelo, Maria Grazia Giuffrida, Laura Bernardini, Andrea M. Chiariello, Giulia Guerri, Andrea Esposito, Cinque, L., Micale, L., Manara, E., Esposito, A., Palumbo, O., Chiariello, A. M., Bianco, S., Guerri, G., Bertelli, M., Giuffrida, M. G., Bernardini, L., Notarangelo, A., Nicodemi, M., and Castori, M.
Subjects: Genetics, Chromosome 17 (human), Cardiovascular and Metabolic Diseases, Regulatory sequence, Breakpoint, Locus (genetics), Chromosomal translocation, Biology, Enhancer, Gene, Genetics (clinical), Chromatin
Abstract: Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5' regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5' of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient's blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5' of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.
Published: 2022

23. A Polymer Physics Model to Dissect Genome Organization in Healthy and Pathological Phenotypes

Author: Luca Fiorillo, Andrea M. Chiariello, Alex Abraham, Simona Bianco, Francesco Flora, Andrea Esposito, Mattia Conte, Mario Nicodemi, Francesco Musella, Conte, M., Fiorillo, L., Bianco, S., Chiariello, A. M., Esposito, A., Musella, F., Flora, F., Abraham, A., and Nicodemi, M.
Subjects: Pathological phenotype, Computational biology, Biology, Compartmentalization (psychology), Phenotype, Genome organization, Chromatin, Cell nucleus, medicine.anatomical_structure, Architectural pattern, Regulatory sequence, Predictive model, medicine, Polymer physic, Structural variants, Gene, Genomic organization
Abstract: Novel technologies revealed a nontrivial spatial organization of the chromosomes within the cell nucleus, which includes different levels of compartmentalization and architectural patterns. Notably, such complex three-dimensional structure plays a crucial role in vital biological functions and its alterations can produce extensive rewiring of genomic regulatory regions, thus leading to gene misexpression and disease. Here, we show that theoretical and computational approaches, based on polymer physics, can be employed to dissect chromatin contacts in three-dimensional space and to predict the effects of pathogenic structural variants on the genome architecture. In particular, we discuss the folding of the human EPHA4 and the murine Pitx1 loci as case studies.
Published: 2022

24. Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes

Author: Alessa R. Ringel, Quentin Szabo, Andrea M. Chiariello, Konrad Chudzik, Robert Schöpflin, Patricia Rothe, Alexandra L. Mattei, Tobias Zehnder, Dermot Harnett, Verena Laupert, Simona Bianco, Sara Hetzel, Juliane Glaser, Mai H.Q. Phan, Magdalena Schindler, Daniel M. Ibrahim, Christina Paliou, Andrea Esposito, Cesar A. Prada-Medina, Stefan A. Haas, Peter Giere, Martin Vingron, Lars Wittler, Alexander Meissner, Mario Nicodemi, Giacomo Cavalli, Frédéric Bantignies, Stefan Mundlos, Michael I. Robson, Ringel, Alessa R, Szabo, Quentin, Chiariello, Andrea M, Chudzik, Konrad, Schöpflin, Robert, Rothe, Patricia, Mattei, Alexandra L, Zehnder, Tobia, Harnett, Dermot, Laupert, Verena, Bianco, Simona, Hetzel, Sara, Glaser, Juliane, Phan, Mai H Q, Schindler, Magdalena, Ibrahim, Daniel M, Paliou, Christina, Esposito, Andrea, Prada-Medina, Cesar A, Haas, Stefan A, Giere, Peter, Vingron, Martin, Wittler, Lar, Meissner, Alexander, Nicodemi, Mario, Cavalli, Giacomo, Bantignies, Frédéric, Mundlos, Stefan, Robson, Michael I, Centre National de la Recherche Scientifique (France), Université de Montpellier, CINECA, European Research Council, EMBO, Wellcome Trust, and German Research Foundation
Subjects: Cancer Research, CCCTC-Binding Factor, Transcription Factor, Placenta, cohesin, topologically associating domain, General Biochemistry, Genetics and Molecular Biology, Mammal, Evolution, Molecular, Pregnancy, 3D genome organization, evolution, Animals, Promoter Regions, Genetic, Mammals, DNA methylation, loop extrusion, Genome, Topologically associating domains, Animal, CTCF, Chromatin Assembly and Disassembly, Chromatin, enhancer-promoter specificity, Enhancer Elements, Genetic, developmental gene regulation, Cardiovascular and Metabolic Diseases, Lamina-associated domainenhancer-promoter specificityDNA methylationdevelopmental gene regulationevolutionloop extrusioncohesinCTCF3D genome organization, lamina-associated domain, Female, Transcription Factors
Abstract: Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1’s intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42’s unresponsiveness. Rather, Zfp42’s promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes., We thank the Montpellier Ressources Imagerie facility (BioCampus Montpellier, Centre National de la Recherche Scientifique [CNRS], INSERM, University of Montpellier) and for computer resources from CINECA (ISCRA grant thanks to computer resources from INFN and CINECA [ISCRA Grant HP10C8JWU7]). G.C., Q.S., and F.B. were supported by a grant from the European Research Council (Advanced Grant 3DEpi, 788972) and by the CNRS. This work was funded by EMBO and the Wellcome Trust (ALTF1554-2016 and 206475/Z/17/Z; to M.I.R.) as well as the Deutsche Forschungsgemeinschaft (KR3985/7-3 and MU 880/16-1 to S.M.).
Published: 2022

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

Author: Quentin Szabo, M. Phan, Giacomo Cavalli, Cesar Augusto Prada-Medina, Christina Paliou, Sara Hetzel, Michael I. Robson, K. Chudzik, Andrea M. Chiariello, Stephan Haas, Tobias Zehnder, Daniel M. Ibrahim, Alexander Meissner, Andrea Esposito, R. Schoepflin, P. Giere, P. Rothe, Simona Bianco, Martin Vingron, V. Laupert, Dermot Harnett, Stefan Mundlos, Frédéric Bantignies, Alessa R. Ringel, Lars Wittler, Alexandra L. Mattei, Mario Nicodemi, and Magdalena Schindler
Subjects: Regulation of gene expression, History, Polymers and Plastics, Cohesin, Rex1, Promoter, Biology, Industrial and Manufacturing Engineering, Cell biology, Gene expression, DNA methylation, Business and International Management, Enhancer, Psychological repression, Gene
Abstract: SUMMARYCohesin 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.HIGHLIGHTSNew genes can emerge in evolution without taking on the expression pattern of their surrounding pre-existing TAD.Compartmentalisation can restructure seemingly evolutionarily stable TADs to control a promoter’s access to enhancers.Lamina-associated domains neither prevent transcriptional activation nor enhancer-promoter communication.Repression rather than promoter-specificity refines when genes respond to promiscuous enhancer activities in specific tissues.
Published: 2021
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26. A Polymer Physics Model to Dissect Genome Organization in Healthy and Pathological Phenotypes

Author: Mattia, Conte, Luca, Fiorillo, Simona, Bianco, Andrea M, Chiariello, Andrea, Esposito, Francesco, Musella, Francesco, Flora, Alex, Abraham, and Mario, Nicodemi
Subjects: Mice, Phenotype, Polymers, Physics, Animals, Humans, Chromatin, Chromosomes
Abstract: Novel technologies revealed a nontrivial spatial organization of the chromosomes within the cell nucleus, which includes different levels of compartmentalization and architectural patterns. Notably, such complex three-dimensional structure plays a crucial role in vital biological functions and its alterations can produce extensive rewiring of genomic regulatory regions, thus leading to gene misexpression and disease. Here, we show that theoretical and computational approaches, based on polymer physics, can be employed to dissect chromatin contacts in three-dimensional space and to predict the effects of pathogenic structural variants on the genome architecture. In particular, we discuss the folding of the human EPHA4 and the murine Pitx1 loci as case studies.
Published: 2021

27. Dynamic and equilibrium properties of finite-size polymer models of chromosome folding

Author: Andrea M. Chiariello, Luca Fiorillo, Francesco Musella, Andrea Esposito, Carlo Annunziatella, Alex Abraham, Mattia Conte, Simona Bianco, Conte, M., Fiorillo, L., Annunziatella, C., Esposito, A., Musella, F., Abraham, A., Bianco, S., and Chiariello, A. M.
Subjects: chemistry.chemical_classification, Physics, Cell Nucleus, Scaling law, Polymers, Polymer, Chromatin, Chromosomes, Folding (chemistry), chemistry, Simple (abstract algebra), Polymer physics, Statistical physics, Scaling
Abstract: Novel technologies are revealing that chromosomes have a complex three-dimensional organization within the cell nucleus that serves functional purposes. Models from polymer physics have been developed to quantitively understand the molecular principles controlling their structure and folding mechanisms. Here, by using massive molecular-dynamics simulations we show that classical scaling laws combined with finite-size effects of a simple polymer model can effectively explain the scaling behavior that chromatin exhibits at the topologically associating domains level, as revealed by experimental observations. Model results are then validated against recently published high-resolution in situ Hi-C data.
Published: 2021

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

Author: Raffaele Campanile, Luca Fiorillo, Andrea M. Chiariello, Andrea Esposito, Alex Abraham, Mario Nicodemi, Mattia Conte, and Simona Bianco
Subjects: business.industry, Machine learning, computer.software_genre, Genome, Chromatin, DNA binding site, Epigenetic Profile, Epigenetics, Artificial intelligence, Binding site, Enhancer, business, Transcription factor, computer
Abstract: 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.
Published: 2021
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29. CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains

Author: Miao Yu, Bing Ren, Andrea M. Chiariello, Yuanyuan Han, Mattia Conte, Adam Jussila, Yanxiao Zhang, Quan Zhu, Ivan Juric, Xiaowei Zhuang, Hui Huang, Bogdan Bintu, Melodi Tastemel, Ming Hu, Mario Nicodemi, Simona Bianco, Rong Hu, Colin Kern, Huang, H., Zhu, Q., Jussila, A., Han, Y., Bintu, B., Kern, C., Conte, M., Zhang, Y., Bianco, S., Chiariello, A. M., Yu, M., Hu, R., Tastemel, M., Juric, I., Hu, M., Nicodemi, M., Zhuang, X., and Ren, B.
Subjects: CCCTC-Binding Factor, Transcription, Genetic, Context (language use), Plasma protein binding, Biology, Insulator Element, Article, 03 medical and health sciences, Mice, 0302 clinical medicine, Transcription (biology), Genetics, Animals, Humans, Binding site, Promoter Regions, Genetic, computational data analyses, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Binding Sites, Mouse Embryonic Stem Cells, Mouse Embryonic Stem Cell, Chromatin, Cell biology, Enhancer Elements, Genetic, Gene Expression Regulation, CTCF, Insulator Elements, 030217 neurology & neurosurgery, Function (biology), Human, Protein Binding
Abstract: Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer-promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.
Published: 2021

30. Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin

Author: Andrea M. Chiariello, Mattia Conte, Alexander Kukalev, Simona Bianco, Francesco Musella, Rieke Kempfer, Ana Pombo, Andrea Esposito, Luca Fiorillo, Alex Abraham, Mario Nicodemi, Antonella Prisco, Ibai Irastorza-Azcarate, Fiorillo, L., Musella, F., Conte, M., Kempfer, R., Chiariello, A. M., Bianco, S., Kukalev, A., Irastorza-Azcarate, I., Esposito, A., Abraham, A., Prisco, A., Pombo, A., and Nicodemi, M.
Subjects: Sprite (computer graphics), Computer science, In silico, Computational biology, Biochemistry, Genome, 03 medical and health sciences, Mice, Computer Simulation, Inverse power law, Polymer, Molecular Biology, 030304 developmental biology, 0303 health sciences, Animal, Chromosome Mapping, Cell Biology, Replicate, Chromatin, Single Molecule Imaging, Single-Cell Analysi, Models, Chemical, Cardiovascular and Metabolic Diseases, Benchmark (computing), Genome architecture, Biotechnology, Human
Abstract: Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb. This Analysis reports a computational approach to implement Hi-C, SPRITE and GAM, which allows researchers to assess the performances of the three technologies to capture DNA contacts in chromatin three-dimensional models.
Published: 2021

31. Analysis of Genome Architecture Mapping Data with a Machine Learning and Polymer-Physics-Based Tool

Author: Andrea Esposito, Andrea M. Chiariello, Luca Fiorillo, Francesco Flora, Mattia Conte, Francesco Musella, Simona Bianco, Fiorillo, L., Conte, M., Esposito, A., Musella, F., Flora, F., Chiariello, A. M., and Bianco, S.
Subjects: Structure (mathematical logic), 0303 health sciences, Sprite (computer graphics), Computer science, business.industry, Folding (DSP implementation), Machine learning, computer.software_genre, Genome, GAM, Data mapping, 03 medical and health sciences, 0302 clinical medicine, Chromatin organization, Polymer physics, Artificial intelligence, Experimental methods, business, computer, 030217 neurology & neurosurgery, Genome architecture, 030304 developmental biology
Abstract: Understanding the mechanisms driving the folding of chromosomes in nuclei is a major goal of modern Molecular Biology. Recent technological advances in microscopy (FISH, STORM) and sequencing approaches (Hi-C, GAM, SPRITE) enabled to collect quantitative data about chromatin 3D architecture, revealing a non-random and highly specific organization. To transform such tremendous amount of data into valuable insights on genome folding, heavy computational analyses are required. Here, we study the performances of PRISMR, a computational tool based on Machine Learning strategies and Polymer Physics principles, to explore genome 3D structure from Genome Architecture Mapping (GAM) data. Using such data, we show that PRISMR can successfully reconstruct the 3D structure of real genomic regions at various length scales, from mega-base sized loci to whole chromosomes. Importantly, the inferred structures are validated against independent Hi-C data. Finally, we show how PRISMR can be effectively employed to explore differences between experimental methods.
Published: 2021
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32. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Author: D. KLIONSKY, A. ABDEL-AZIZ, S. ABDELFATAH, M. ABDELLATIF, A. ABDOLI, S. ABEL, H. ABELIOVICH, M. ABILDGAARD, Y. ABUDU, A. ACEVEDO-AROZENA, I. ADAMOPOULOS, K. ADELI, T. ADOLPH, A. ADORNETTO, E. AFLAKI, G. AGAM, A. AGARWAL, B. AGGARWAL, M. AGNELLO, P. AGOSTINIS, J. AGREWALA, A. AGROTIS, P. AGUILAR, S. AHMAD, Z. AHMED, U. AHUMADA-CASTRO, S. AITS, S. AIZAWA, Y. AKKOC, T. AKOUMIANAKI, H. AKPINAR, A. AL-ABD, L. AL-AKRA, A. AL-GHARAIBEH, M. ALAOUI-JAMALI, S. ALBERTI, E. ALCOCER-GOMEZ, C. ALESSANDRI, M. ALI, M. AL-BARI, S. ALIWAINI, J. ALIZADEH, E. ALMACELLAS, A. ALMASAN, A. ALONSO, G. ALONSO, N. ALTAN-BONNET, D. ALTIERI, S. ALVES, C. DA COSTA, M. ALZAHARNA, M. AMADIO, C. AMANTINI, C. AMARAL, S. AMBROSIO, A. AMER, V. AMMANATHAN, Z. AN, S. ANDERSEN, S. ANDRABI, M. ANDRADE-SILVA, A. ANDRES, S. ANGELINI, D. ANN, U. ANOZIE, M. ANSARI, P. ANTAS, A. ANTEBI, Z. ANTON, T. ANWAR, L. APETOH, N. APOSTOLOVA, T. ARAKI, Y. ARAKI, K. ARASAKI, W. ARAUJO, J. ARAYA, C. ARDEN, M. AREVALO, S. ARGUELLES, E. ARIAS, J. ARIKKATH, H. ARIMOTO, A. ARIOSA, D. ARMSTRONG-JAMES, L. ARNAUNE-PELLOQUIN, A. AROCA, D. ARROYO, I. ARSOV, R. ARTERO, D. ASARO, M. ASCHNER, M. ASHRAFIZADEH, O. ASHUR-FABIAN, A. ATANASOV, A. AU, P. AUBERGER, H. AUNER, L. AURELIAN, R. AUTELLI, L. AVAGLIANO, Y. AVALOS, S. AVEIC, C. AVELEIRA, T. AVINWITTENBERG, Y. AYDIN, S. AYTON, S. AYYADEVARA, M. AZZOPARDI, M. BABA, J. BACKER, S. BACKUES, D. BAE, O. BAE, S. BAE, E. BAEHRECKE, A. BAEK, S. BAEK, G. BAGETTA, A. BAGNIEWSKA-ZADWORNA, H. BAI, J. BAI, X. BAI, Y. BAI, N. BAIRAGI, S. BAKSI, T. BALBI, C. BALDARI, W. BALDUINI, A. BALLABIO, M. BALLESTER, S. BALAZADEH, R. BALZAN, R. BANDOPADHYAY, S. BANERJEE, Y. BAO, M. BAPTISTA, A. BARACCA, C. BARBATI, A. BARGIELA, D. BARILA, P. BARLOW, S. BARMADA, E. BARREIRO, G. BARRETO, J. BARTEK, B. BARTEL, A. BARTOLOME, G. BARVE, S. BASAGOUDANAVAR, D. BASSHAM, R. JR, A. BASU, H. BATOKO, I. BATTEN, E. BAULIEU, B. BAUMGARNER, J. BAYRY, R. BEALE, I. BEAU, F. BEAUMATIN, L. BECHARA, G. BECK, M. BEERS, J. BEGUN, C. BEHRENDS, G. BEHRENS, R. BEI, E. BEJARANO, S. BEL, C. BEHL, A. BELAID, N. BELGAREH-TOUZE, C. BELLAROSA, F. BELLEUDI, M. PEREZ, R. BELLO-MORALES, J. BELTRAN, S. BELTRAN, D. BENBROOK, M. BENDORIUS, B. BENITEZ, I. BENITO-CUESTA, J. BENSALEM, M. BERCHTOLD, S. BEREZOWSKA, D. BERGAMASCHI, M. BERGAMI, A. BERGMANN, L. BERLIOCCHI, C. BERLIOZ-TORRENT, A. BERNARD, L. BERTHOUX, C. BESIRLI, S. BESTEIRO, V. BETIN, R. BEYAERT, J. BEZBRADICA, K. BHASKAR, I. BHATIA-KISSOVA, R. BHATTACHARYA, S. BHATTACHARYA, S. BHATTACHARYYA, M. BHUIYAN, S. BHUTIA, L. BI, X. BI, T. BIDEN, K. BIJIAN, V. BILLES, N. BINART, C. BINCOLETTO, A. BIRGISDOTTIR, G. BJORKOY, G. BLANCO, A. BLAS-GARCIA, J. BLASIAK, R. BLOMGRAN, K. BLOMGREN, J. BLUM, E. BOADA-ROMERO, M. BOBAN, K. BOESZEBATTAGLIA, P. BOEUF, B. BOLAND, P. BOMONT, P. BONALDO, S. BONAM, L. BONFILI, J. BONIFACINO, B. BOONE, M. BOOTMAN, M. BORDI, C. BORNER, B. BORNHAUSER, G. BORTHAKUR, J. BOSCH, S. BOSE, L. BOTANA, J. BOTAS, C. BOULANGER, M. 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TUXWORTH, J. TYLER, E. TYUTEREVA, Y. UCHIYAMA, A. UGUNKLUSEK, H. UHLIG, I. ULASOV, M. UMEKAWA, C. UNGERMANN, R. UNNO, S. URBE, E. URIBE-CARRETERO, S. USTUN, V. UVERSKY, T. VACCARI, M. VACCARO, B. VAHSEN, H. VAKIFAHMETOGLU-NORBERG, R. VALDOR, M. VALENTE, A. VALKO, R. VALLEE, A. VALVERDE, G. VAN DEN BERGHE, S. VAN DER VEEN, L. VAN KAER, J. VAN LOOSDREGT, S. VAN WIJK, W. VANDENBERGHE, I. VANHOREBEEK, M. VANNIER-SANTOS, N. VANNINI, M. VANRELL, C. VANTAGGIATO, G. VARANO, I. VARELA-NIETO, M. VARGA, M. VASCONCELOS, S. VATS, D. VAVVAS, I. VEGANAREDO, S. VEGA-RUBIN-DE-CELIS, G. VELASCO, A. VELAZQUEZ, T. VELLAI, E. VELLENGA, F. VELOTTI, M. VERDIER, P. VERGINIS, I. VERGNE, P. VERKADE, M. VERMA, P. VERSTREKEN, T. VERVLIET, J. VERVOORTS, A. VESSONI, V. VICTOR, M. VIDAL, C. VIDONI, O. VIEIRA, R. VIERSTRA, S. VIGANO, H. VIHINEN, V. VIJAYAN, M. VILA, M. VILAR, J. VILLALBA, A. VILLALOBO, B. VILLAREJO-ZORI, F. VILLARROYA, J. VILLARROYA, O. VINCENT, C. VINDIS, C. VIRET, M. VISCOMI, D. VISNJIC, I. VITALE, D. VOCADLO, O. VOITSEKHOVSKAJA, C. VOLONTE, M. VOLTA, M. VOMERO, C. VON HAEFEN, M. VOOIJS, W. VOOS, L. VUCICEVIC, R. WADE-MARTINS, S. WAGURI, K. WAITE, S. WAKATSUKI, D. WALKER, M. WALKER, S. WALKER, J. WALTER, F. WANDOSELL, B. WANG, C. WANG, D. WANG, F. WANG, G. WANG, H. WANG, J. WANG, K. WANG, L. WANG, M. WANG, N. WANG, P. WANG, Q. WANG, W. WANG, X. WANG, Y. WANG, Z. WANG, G. WARNES, V. WARNSMANN, H. WATADA, E. WATANABE, M. WATCHON, T. WEAVER, G. WEGRZYN, A. WEHMAN, H. WEI, L. WEI, T. WEI, Y. WEI, O. WEIERGRABER, C. WEIHL, G. WEINDL, R. WEISKIRCHEN, A. WELLS, R. WEN, X. WEN, A. WERNER, B. WEYKOPF, S. WHEATLEY, J. WHITTON, A. WHITWORTH, K. WIKTORSKA, M. WILDENBERG, T. WILEMAN, S. WILKINSON, D. WILLBOLD, B. WILLIAMS, R. WILLIAMS, P. WILLIAMSON, R. WILSON, B. WINNER, N. WINSOR, S. WITKIN, H. WODRICH, U. WOEHLBIER, T. WOLLERT, E. WONG, J. WONG, R. WONG, V. WONG, W. WONG, A. WU, C. WU, J. WU, K. WU, M. WU, S. WU, W. WU, X. WU, Y. WU, R. XAVIER, H. XIA, L. XIA, Z. XIA, G. XIANG, J. XIANG, M. XIANG, W. XIANG, B. XIAO, G. XIAO, H. XIAO, J. XIAO, L. XIAO, S. XIAO, Y. XIAO, B. XIE, C. XIE, M. XIE, Y. XIE, Z. XIE, M. XILOURI, C. XU, E. XU, H. XU, J. XU, L. XU, W. XU, X. XU, Y. XUE, S. YAKHINE-DIOP, M. YAMAGUCHI, O. YAMAGUCHI, A. YAMAMOTO, S. YAMASHINA, S. YAN, Z. YAN, Y. YANAGI, C. YANG, D. YANG, H. YANG, J. YANG, L. YANG, M. YANG, P. YANG, Q. YANG, S. YANG, W. YANG, X. YANG, Y. YANG, H. YAO, S. YAO, X. YAO, Y. YAO, T. YASUI, M. YAZDANKHAH, P. YEN, C. YI, X. YIN, Y. YIN, Z. YIN, M. YING, Z. YING, C. YIP, S. YIU, Y. YOO, K. YOSHIDA, S. YOSHII, T. YOSHIMORI, B. YOUSEFI, B. YU, H. YU, J. YU, L. YU, M. YU, S. YU, V. YU, W. YU, Z. YU, J. YUAN, L. YUAN, S. YUAN, Y. YUAN, Z. YUAN, J. YUE, Z. YUE, J. YUN, R. YUNG, D. ZACKS, G. ZAFFAGNINI, V. ZAMBELLI, I. ZANELLA, Q. ZANG, S. ZANIVAN, S. ZAPPAVIGNA, P. ZARAGOZA, K. ZARBALIS, A. ZAREBKOHAN, A. ZARROUK, S. ZEITLIN, J. ZENG, E. ZEROVNIK, L. ZHAN, B. ZHANG, D. ZHANG, H. ZHANG, J. ZHANG, K. ZHANG, L. ZHANG, M. ZHANG, P. ZHANG, S. ZHANG, W. ZHANG, X. ZHANG, Y. ZHANG, Z. ZHANG, H. ZHAO, L. ZHAO, S. ZHAO, T. ZHAO, X. ZHAO, Y. ZHAO, G. ZHENG, K. ZHENG, L. ZHENG, S. ZHENG, X. ZHENG, Y. ZHENG, Z. ZHENG, B. ZHIVOTOVSKY, Q. ZHONG, A. ZHOU, B. ZHOU, C. ZHOU, G. ZHOU, H. ZHOU, J. ZHOU, K. ZHOU, R. ZHOU, X. ZHOU, Y. ZHOU, Z. ZHOU, B. ZHU, C. ZHU, G. ZHU, H. ZHU, W. ZHU, Y. ZHU, H. ZHUANG, X. ZHUANG, K. ZIENTARA-RYTTER, C. ZIMMERMANN, E. ZIVIANI, T. ZOLADEK, W. ZONG, D. ZOROV, A. ZORZANO, W. ZOU, Z. ZOU, S. ZURYN, W. ZWERSCHKE, B. BRAND-SABERI, C. KENCHAPPA, S. OSHIMA, Y. RONG, J. SLUIMER, and C. STALLINGS
Subjects: flux, macroautophagy, phagophore, stress, vacuole, Autophagosome, LC3, lysosome, neurodegeneration, cancer
Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Published: 2021

33. Author response for 'Physical mechanisms of chromatin spatial organization'

Author: Ehsan Irani, Francesco Musella, Andrea Esposito, Simona Bianco, Luca Fiorillo, Alex Abraham, Andrea M. Chiariello, Mattia Conte, Antonella Prisco, and Mario Nicodemi
Subjects: Biology, Neuroscience, Spatial organization, Chromatin
Published: 2020
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34. Multiplex-GAM: genome-wide identification of chromatin contacts yields insights not captured by Hi-C

Author: D.C.A. Kramer, Yingnan Zhang, Markus Schueler, Antonio Scialdone, Rieke Kempfer, Andrea M. Chiariello, Robert A. Beagrie, Christoph J. Thieme, C. Baugher, Lonnie R. Welch, Ana Pombo, Alexander Kukalev, S. Bianco, Yichao Li, Mario Nicodemi, and Carlo Annunziatella
Subjects: Regulation of gene expression, Sequence assembly, Identification (biology), Multiplex, Computational biology, Biology, Genome, Gene, Genome architecture, Chromatin
Abstract: SummaryTechnologies 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.
Published: 2020
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35. CTCF Mediates Dosage and Sequence-context-dependent Transcriptional Insulation through Formation of Local Chromatin Domains

Author: Andrea M. Chiariello, Mario Nicodemi, Ivan Juric, Mattia Conte, Yanxiao Zhang, Rong Hu, Ming Hu, Adam Jussila, Bing Ren, Bogdan Bintu, Colin Kern, Yuanyuan Han, Hui Huang, Miao Yu, Simona Bianco, Quan Zhu, and Xiaowei Zhuang
Subjects: Reporter gene, Spatiotemporal gene expression, Chemistry, CTCF, Mammalian genome, Binding site, Embryonic stem cell, DNA sequencing, Cell biology, Chromatin
Abstract: Insulators play a critical role in spatiotemporal gene expression in metazoans by separating active and repressive chromatin domains and preventing inappropriate enhancer-promoter contacts. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here, we explore the sequence requirements of CTCF-mediated transcriptional insulation with the use of a sensitive insulator reporter assay in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on DNA sequences flanking its core binding motifs, and CTCF binding sites at topologically associating domain(TAD) boundaries are more likely to function as insulators than those outside TAD boundaries, independent of binding strength. Using chromosomal conformation capture assays and high-resolution chromatin imaging techniques, we demonstrate that insulators form local chromatin domain boundaries and reduce enhancer-promoter contacts. Taken together, our results provide strong genetic, molecular, and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.
Published: 2020
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36. Chromatin folding variability across single-cells results from state degeneracy in phase-separation

Author: Mario Nicodemi, Mattia Conte, Simona Bianco, Andrea Esposito, Luca Fiorillo, and Andrea M. Chiariello
Subjects: chemistry.chemical_compound, Histone, biology, Cohesin, CTCF, Chemistry, biology.protein, Biophysics, Degeneracy (biology), Binding site, Affinities, DNA, Chromatin
Abstract: 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.
Published: 2020
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37. Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Author: Mark A. Ungless, Izabela Harabula, Sarah A. Teichmann, Georg Dechant, Leonid Serebreni, Mandy Meijer, Vedran Franke, Luna Zea Redondo, Warren Winick-Ng, Andreas Abentung, Aleksandra A. Kolodziejczyk, Gonçalo Castelo-Branco, Rieke Kempfer, Christoph J. Thieme, Francesco Musella, Mario Nicodemi, Alexander Kukalev, Simona Bianco, Ana Pombo, Elena Torlai Triglia, Galina Apostolova, Luca Fiorillo, Eleanor J. Paul, Dominik Szabo, Ehsan Irani, Ibai Irastorza Azcarate, Andrea M. Chiariello, and Altuna Akalin
Subjects: Regulation of gene expression, 0303 health sciences, Cell type, Cellular differentiation, Biology, Genome, Oligodendrocyte, Chromatin, Cell biology, 03 medical and health sciences, 0302 clinical medicine, medicine.anatomical_structure, medicine, Compartment (development), Gene, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: Neurons and oligodendrocytes are terminally differentiated cells that perform highly specialized functions, which depend on cascades of gene activation and repression to retain homeostatic control over a lifespan. Gene expression is regulated by three-dimensional (3D) genome organisation, from local levels of chromatin compaction to the organisation of topological domains and chromosome compartments. Whereas our understanding of 3D genome architecture has vastly increased in the past decade, it remains difficult to study specialized cells in their native environment without disturbing their activity. To develop the application of Genome Architecture Mapping (GAM) in small numbers of specialized cells in complex tissues, we combined GAM with immunoselection. We applied immunoGAM to map the genome architecture of specific cell populations in the juvenile/adult mouse brain: dopaminergic neurons (DNs) from the midbrain, pyramidal glutamatergic neurons (PGNs) from the hippocampus, and oligodendrocyte lineage cells (OLGs) from the cortex. We integrate 3D genome organisation with single-cell transcriptomics data, and find specific chromatin structures that relate with cell-type specific patterns of gene expression. We discover abundant changes in compartment organisation, especially a strengthening of heterochromatin compartments which establish strong contacts spanning tens of megabases, especially in brain cells. These compartments contain olfactory and taste receptor genes, which are de-repressed in a subpopulation of PGNs with molecular signatures of long-term potentiation (LTP). We also show extensive reorganisation of topological domains where activation of neuronal or oligodendrocyte genes coincides with formation of new TAD borders. Finally, we discover loss of TAD organisation, or ‘TAD melting’, at long (>1Mb) neuronal genes when they are most highly expressed. Our work shows that the 3D organisation of the genome is highly cell-type specific in terminally differentiated cells of the brain, and essential to better understand brain-specific mechanisms of gene regulation.
Published: 2020
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38. The Physics of DNA Folding: Polymer Models and Phase-Separation

Author: Andrea Esposito, Alex Abraham, Mattia Conte, Francesca Vercellone, Antonella Prisco, Simona Bianco, and Andrea M. Chiariello
Subjects: Polymers and Plastics, General Chemistry
Abstract: Within cell nuclei, several biophysical processes occur in order to allow the correct activities of the genome such as transcription and gene regulation. To quantitatively investigate such processes, polymer physics models have been developed to unveil the molecular mechanisms underlying genome functions. Among these, phase-separation plays a key role since it controls gene activity and shapes chromatin spatial structure. In this paper, we review some recent experimental and theoretical progress in the field and show that polymer physics in synergy with numerical simulations can be helpful for several purposes, including the study of molecular condensates, gene-enhancer dynamics, and the three-dimensional reconstruction of real genomic regions.
Published: 2022
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39. Publisher Correction: Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin

Author: Ana Pombo, Mattia Conte, Alex Abraham, Antonella Prisco, Rieke Kempfer, Ibai Irastorza-Azcarate, Luca Fiorillo, Francesco Musella, Alexander Kukalev, Simona Bianco, Mario Nicodemi, Andrea Esposito, and Andrea M. Chiariello
Subjects: Physics, Computational model, Sprite (computer graphics), Computational biophysics, Cell Biology, Computational biology, Molecular Biology, Biochemistry, Biotechnology, Chromatin
Published: 2021
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40. Divergent Transcription of the Nkx2-5 Locus Generates Two Enhancer RNAs with Opposing Functions

Author: Andrea M. Chiariello, Simone Serio, Paolo Kunderfranco, Mario Nicodemi, Mattia Conte, Christina Pagiatakis, Arianna Felicetta, Silvia Crasto, Gianluigi Condorelli, Roberto Papait, Elisa Di Pasquale, Paola Cattaneo, Luca Fiorillo, Simona Bianco, Irene Salamon, Salamon, I., Serio, S., Bianco, S., Pagiatakis, C., Crasto, S., Chiariello, A. M., Conte, M., Cattaneo, P., Fiorillo, L., Felicetta, A., di Pasquale, E., Kunderfranco, P., Nicodemi, M., Papait, R., Condorelli, G., Salamon I., Serio S., Bianco S., Pagiatakis C., Crasto S., Chiariello A.M., Conte M., Cattaneo P., Fiorillo L., Felicetta A., di Pasquale E., Kunderfranco P., Nicodemi M., Papait R., and Condorelli G.
Subjects: 0301 basic medicine, Polymer Physic, 02 engineering and technology, Article, Statistical Mechanics, 03 medical and health sciences, Transcription (biology), Molecular Mechanism of Gene Regulation, Computer Simulation, Enhancer, lcsh:Science, Gene, Transcription factor, Molecular Biology, Multidisciplinary, biology, Sirtuin 1, Promoter, Biological Science, Biological Sciences, 021001 nanoscience & nanotechnology, Long non-coding RNA, Cell biology, 030104 developmental biology, biology.protein, lcsh:Q, Histone deacetylase, 0210 nano-technology
Abstract: Summary Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5—a gene specifying a master cardiomyogenic lineage transcription factor (TF)—which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription., Graphical Abstract, Highlights • Two eRNAs (IRENE-SS, IRENE-div) with opposing functions are found upstream of Nkx2-5 • IRENE-SS works as a classical eRNA, acting as a transcriptional activator • IRENE-div acts unconventionally, functioning as a transcriptional repressor • IRENEs epigenetically control enhancer status and, subsequently, locus architecture, Biological Sciences; Molecular Biology; Molecular Mechanism of Gene Regulation
Published: 2020

41. Hybrid Machine Learning and Polymer Physics Approach to Investigate 3D Chromatin Structure

Author: Alfonso Corrado, Andrea Esposito, Renato Sciarretta, Carlo Annunziatella, Andrea M. Chiariello, Mattia Conte, Francesco Musella, Simona Bianco, Luca Fiorillo, Conte, M., Esposito, A., Fiorillo, L., Annunziatella, C., Corrado, A., Musella, F., Sciarretta, R., Chiariello, A. M., and Bianco, S.
Subjects: Polymer Physics, 0303 health sciences, Hybrid machine, Computer science, In silico, Chromosome, Locus (genetics), Computational biology, 01 natural sciences, Genome, Chromatin, Machine Learning, 03 medical and health sciences, Cell nucleus, medicine.anatomical_structure, 0103 physical sciences, Chromatin organization, medicine, Polymer physics, 010306 general physics, Genome architecture, 030304 developmental biology
Abstract: Innovative experimental protocols from Molecular Biology provided in recent years quantitative data about the structure of the cell nucleus. These technologies, such as Hi-C, GAM or SPRITE, revealed that the genome has a non-random three-dimensional (3D) spatial organization, which serves functional purposes. In order to dissect the complexity of chromosome folding, models from Polymer Physics have been employed, highlighting many key aspects of large-scale chromatin organization. A deep understanding of the molecular mechanisms underlying the genome architecture is currently a crucial problem in Biology, since chromatin misfolding or structural variants can reconfigure chromatin domains, thereby resulting in pathogenic phenotypes and disease. Here, we discuss a numerical Polymer-Physics-based approach (PRISMR), able to model 3D chromatin folding by using Machine Learning strategies informed with experimental data. Using as a case study the Pitx1 locus, a genomic region critically involved in hindlimb development, we show that the PRISMR algorithm reproduces in silico with high accuracy the experimental contact data, thus providing a powerful computational tool for analyzing and predicting the 3D chromatin structure.
Published: 2020

42. Higher-order Chromosome Structures Investigated by Polymer Physics in Cellular Morphogenesis and Differentiation

Author: Luca Fiorillo, Francesco Musella, Mattia Conte, Simona Bianco, Andrea M. Chiariello, Andrea Esposito, Renato Sciarretta, Esposito, A., Chiariello, A. M., Conte, M., Fiorillo, L., Musella, F., Sciarretta, R., and Bianco, S.
Subjects: Macromolecular Substances, Molecular Conformation, Locus (genetics), Computational biology, Biology, Pitx1, Hierarchical folding, 03 medical and health sciences, Mice, 0302 clinical medicine, Structural Biology, Gene expression, Genome architecture, medicine, Animals, Hierarchical organization, Macromolecular Substance, Molecular Biology, Cell Shape, 030304 developmental biology, Epigenomics, Neurons, Regulation of gene expression, Spatial Analysis, 0303 health sciences, Animal, Cell Differentiation, Neuron, Spatial Analysi, Chromatin, medicine.anatomical_structure, Principled approach, Structural variant, Ectopic expression, Forelimb, 030217 neurology & neurosurgery
Abstract: Experimental advances in Molecular Biology demonstrated that chromatin architecture and gene regulation are deeply related. Hi-C data, for instance, returned a scenario where chromosomes form a complex pattern of interactions, including TADs, metaTADs, and compartments, correlated with genomic and epigenomic features. Here, we discuss the emerging hierarchical organization of chromatin and show how it remains partially conserved during mouse neuronal differentiation with changes highly related to modifications in gene expression. In this scenario, models of polymer physics, such as the Strings & Binders (SBS) model, can be a crucial instrument to understand the molecular mechanisms underlying the formation of such a higher order 3D structure. In particular, we focus on the case study of the murine Pitx1 genomic region. At this locus, two alternative spatial conformations take place in the hindlimb and forelimb tissues, corresponding to two different transcriptional states of Pitx1. We finally show how the structural variants can affect the locus 3D organization leading to ectopic gene expression and limb malformations.
Published: 2020

43. Computational approaches from polymer physics to investigate chromatin folding

Author: Mario Nicodemi, Mattia Conte, Luca Fiorillo, Andrea M. Chiariello, Francesco Musella, Andrea Esposito, Simona Bianco, Bianco, S., Chiariello, A. M., Conte, M., Esposito, A., Fiorillo, L., Musella, F., and Nicodemi, M.
Subjects: Loop extrusion model, Polymers, Computational biology, Biology, Pitx1, Models, Biological, Chromosomes, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Molecular level, Chromatin organization, Humans, Statistical Physic, 030304 developmental biology, 0303 health sciences, SBS model, bioinformatic, Physics, Computational Biology, Chromosome, Statistical mechanic, systems biology, Cell Biology, Computer simulation, Key features, Cell function, Chromatin, Folding (chemistry), chemistry, Polymer model, Polymer physics, EPHA4, Structural variants, 030217 neurology & neurosurgery, DNA
Abstract: Microscopy and sequencing-based technologies are providing increasing insights into chromatin architecture. Nevertheless, a full comprehension of chromosome folding and its link with vital cell functions is far from accomplished at the molecular level. Recent theoretical and computational approaches are providing important support to experiments to dissect the three-dimensional structure of chromosomes and its organizational mechanisms. Here, we review, in particular, the String&Binders polymer model of chromatin that describes the textbook scenario where contacts between distal DNA sites are established by cognate binders. It has been shown to recapitulate key features of chromosome folding and to be able at predicting how phenotypes causing structural variants rewire the interactions between genes and regulators.
Published: 2020

44. A modern challenge of polymer physics: Novel ways to study, interpret, and reconstruct chromatin structure

Author: Andrea M. Chiariello, Francesco Musella, Simona Bianco, Renato Sciarretta, Luca Fiorillo, Andrea Esposito, Mattia Conte, Fiorillo, L., Bianco, S., Esposito, A., Conte, M., Sciarretta, R., Musella, F., and Chiariello, A. M.
Subjects: Physics, chromatin structure, molecular dynamic, Nanotechnology, polymer physics, Biochemistry, Computer Science Applications, Chromatin, Computational Mathematics, Molecular dynamics, Materials Chemistry, Polymer physics, Physical and Theoretical Chemistry, genetic
Abstract: The constant development of sophisticated technologies is allowing to dissect three-dimensional chromatin structure at high resolution level. The tremendous amount of quantitative experimental data available today requires a conceptual framework able to make sense of them. In this perspective, polymer physics offers a key tool to interpret chromatin architecture data and to unveil the basic mechanisms shaping its structure. In the very last years, several polymer models have been proposed and have allowed to capture complex features emerging from the data. The major peculiarity distinguishing the different models is represented by the more or less complicated physical mechanism used to explain chromatin folding. Here, we review very popular models which have been recently developed and which represent brilliant examples from this interdisciplinary research field. In order to highlight the wide range of practical applications they have, we discuss the cases of the murine Pitx1 and the human EPHA4 loci, showing that polymer physics allows to effectively study chromatin structure in different cell lines and to predict the impact of pathogenic structural variants on the genome three-dimensional architecture. This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Molecular Dynamics and Monte-Carlo Methods Theoretical and Physical Chemistry > Statistical Mechanics.
Published: 2020

45. The Strings and Binders Switch Model of Chromatin

Author: Carlo Annunziatella, Simona Bianco, Luca Fiorillo, Andrea Esposito, Mario Nicodemi, Andrea M. Chiariello, Bianco, Simona, Chiariello, Andrea M., Annunziatella, Carlo, Esposito, Andrea, Fiorillo, Luca, and Nicodemi, Mario
Subjects: Chemistry, Chromatin, Cell biology
Published: 2019
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46. Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations

Author: Laura Furia, Nicola Crosetto, Simona Bianco, Mario Nicodemi, Andrea M. Chiariello, Gaetano Ivan Dellino, Giulia De Conti, Giorgio E. M. Melloni, Britta A. M. Bouwman, Luciano Giacò, Davide Guido, Pier Giuseppe Pelicci, Mario Faretta, Lucilla Luzi, Fernando Palluzzi, Davide Cittaro, Rossana Piccioni, Dellino, G. I., Palluzzi, F., Chiariello, A. M., Piccioni, R., Bianco, S., Furia, L., De Conti, G., Bouwman, B. A. M., Melloni, G., Guido, D., Giaco, L., Luzi, L., Cittaro, D., Faretta, M., Nicodemi, M., Crosetto, N., and Pelicci, P. G.
Subjects: DNA Repair, DNA repair, Intron, Fluorescent Antibody Technique, RNA polymerase II, Topoisomerase Inhibitor, Biology, Statistical models: physic, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Cell Line, Tumor, Genetics, DNA Breaks, Double-Stranded, Promoter Regions, Genetic, Gene, 030304 developmental biology, Settore MED/04 - Patologia Generale, 0303 health sciences, Promoter, DNA repair protein XRCC4, Flow Cytometry, Chromatin, Cell biology, Gene Expression Regulation, Neoplastic, Enhancer Elements, Genetic, chemistry, RNA Splice Site, Genetic Loci, biology.protein, Genomic, Neoplasm, RNA Polymerase II, Transcription Initiation Site, 030217 neurology & neurosurgery, DNA
Abstract: It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.
Published: 2019

47. Understanding chromatin structure: Efficient computational implementation of polymer physics models

Author: Raffaele Campanile, Mattia Conte, Luca Fiorillo, Carlo Annunziatella, Andrea Esposito, Simona Bianco, Andrea M. Chiariello, Bianco, S., Annunziatella, C., Esposito, A., Fiorillo, L., Conte, M., Campanile, R., and Chiariello, A. M.
Subjects: 0301 basic medicine, chemistry.chemical_classification, Sequence, Molecular dynamic, Theoretical computer science, Computer science, Folding (DSP implementation), Polymer, Genome, Chromatin, 03 medical and health sciences, Cell nucleus, Molecular dynamics, 030104 developmental biology, 0302 clinical medicine, medicine.anatomical_structure, chemistry, medicine, Polymer physics, Focus (optics), 030217 neurology & neurosurgery
Abstract: In recent years the development of novel technologies, as Hi-C or GAM, allowed to investigate the spatial structure of chromatin in the cell nucleus with a constantly increasing level of accuracy. Polymer physics models have been developed and improved to better interpret the wealth of complex information coming from the experimental data, providing highly accurate understandings on chromatin architecture and on the mechanisms regulating genome folding. To investigate the capability of the models to explain the experiments and to test their agreement with the data, massive parallel simulations are needed and efficient algorithms are fundamental. In this work, we consider general computational Molecular Dynamics (MD) techniques commonly used to implement such models, with a special focus on the Strings & Binders Switch polymer model. By combining this model with machine learning computational approaches, it is possible to give an accurate description of real genomic loci. In addition, it is also possible to make predictions about the impact of structural variants of the genomic sequence, which are known to be linked to severe congenital diseases.
Published: 2019

48. Models of polymer physics for the architecture of the cell nucleus

Author: Mario Nicodemi, Andrea Esposito, Luca Fiorillo, Simona Bianco, Andrea M. Chiariello, Carlo Annunziatella, Esposito, Andrea, Annunziatella, Carlo, Bianco, Simona, Chiariello, Andrea M., Fiorillo, Luca, and Nicodemi, Mario
Subjects: Theoretical computer science, statistical physic, Polymers, Computer science, Medicine (miscellaneous), Models, Biological, Biochemistry, Genetics and Molecular Biology (miscellaneous), Article, polymer physic, 03 medical and health sciences, 0302 clinical medicine, Development (topology), computational biology, Chromosome (genetic algorithm), Humans, Architecture, 030304 developmental biology, Cell Nucleus, 0303 health sciences, LOOP (programming language), DNA, Folding (DSP implementation), Chromatin Assembly and Disassembly, Thermodynamics, Polymer physics, chromatin, Focus (optics), 030217 neurology & neurosurgery
Abstract: The depth and complexity of data now available on chromosome 3D architecture, derived by new technologies such as Hi-C, have triggered the development of models based on polymer physics to explain the observed patterns and the underlying molecular folding mechanisms. Here, we give an overview of some of the ideas and models from physics introduced to date, along with their progresses and limitations in the description of experimental data. In particular, we focus on the Strings&Binders and the Loop Extrusion model of chromatin architecture. This article is categorized under: Analytical and Computational Methods > Computational Methods.
Published: 2019

49. Preformed chromatin topology assists transcriptional robustness of Shh during limb development

Author: Guillaume Andrey, Verena Heinrich, Martin Vingron, Carlo Annunziatella, Christina Paliou, Lars Wittler, Norbert Brieske, Ivana Jerković, Johannes Helmuth, Andrea M. Chiariello, Robert Schöpflin, Stefan A. Haas, Stefan Mundlos, Mario Nicodemi, Bernd Timmermann, Andrea Esposito, Philine Guckelberger, Simona Bianco, Paliou, C., Guckelberger, P., Schopflin, R., Heinrich, V., Esposito, A., Chiariello, A. M., Bianco, S., Annunziatella, C., Helmuth, J., Haas, S., Jerkovic, I., Brieske, N., Wittler, L., Timmermann, B., Nicodemi, M., Vingron, M., Mundlos, S., and Andrey, G.
Subjects: CCCTC-Binding Factor, Transcription, Genetic, statistical physic, Chromosomal Proteins, Non-Histone, Polymer modelling, Down-Regulation, Cell Cycle Proteins, Development, Limb, Mice, 03 medical and health sciences, 0302 clinical medicine, Genetic, Transcription (biology), Gene expression, Animals, Limb development, Hedgehog Proteins, Promoter Regions, Genetic, Enhancer, Loss function, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Binding Sites, Multidisciplinary, Chemistry, Membrane Proteins, Extremities, Promoter, respiratory system, Chromatin, 3D genome, Cell biology, Gene regulation, Enhancer Elements, Genetic, PNAS Plus, 030217 neurology & neurosurgery
Abstract: 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 the 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.
Published: 2019

50. Loxosceles gaucho venom gland proteome: A new perspective on Loxosceles venom biochemical composition

Author: Fan Hui Wen, Thiago M. Chiariello, Rodrigo Valladão, Emidio Beraldo Neto, Adriana Rios Lopes, Daniel C. Pimenta, Daiane Laise da Silva, Aline Vivian Vatti Auada, and Guilherme Rabelo Coelho
Subjects: Loxosceles gaucho, Proteome, Biochemical composition, Venom gland, Biology, Toxicology, Loxosceles venom, Microbiology
Published: 2020
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