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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.
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COVID-19 , Cromatina , SARS-CoV-2 , Cromatina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , Humanos , COVID-19/virología , COVID-19/genética , COVID-19/metabolismoRESUMEN
Midbrain dopamine neurons (DNs) respond to a first exposure to addictive drugs and play key roles in chronic drug usage1-3. As the synaptic and transcriptional changes that follow an acute cocaine exposure are mostly resolved within a few days4,5, the molecular changes that encode the long-term cellular memory of the exposure within DNs remain unknown. To investigate whether a single cocaine exposure induces long-term changes in the 3D genome structure of DNs, we applied Genome Architecture Mapping and single nucleus transcriptomic analyses in the mouse midbrain. We found extensive rewiring of 3D genome architecture at 24 hours past exposure which remains or worsens by 14 days, outlasting transcriptional responses. The cocaine-induced chromatin rewiring occurs at all genomic scales and affects genes with major roles in cocaine-induced synaptic changes. A single cocaine exposure triggers extensive long-lasting changes in chromatin condensation in post-synaptic and post-transcriptional regulatory genes, for example the unfolding of Rbfox1 which becomes most prominent 14 days post exposure. Finally, structurally remodeled genes are most expressed in a specific DN sub-type characterized by low expression of the dopamine auto-receptor Drd2, a key feature of highly cocaine-sensitive cells. These results reveal an important role for long-lasting 3D genome remodelling in the cellular memory of a single cocaine exposure, providing new hypotheses for understanding the inception of drug addiction and 3D genome plasticity.
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Olfactory receptor (OR) choice provides an extreme example of allelic competition for transcriptional dominance, where every olfactory neuron stably transcribes one of approximately 2,000 or more OR alleles1,2. OR gene choice is mediated by a multichromosomal enhancer hub that activates transcription at a single OR3,4, followed by OR-translation-dependent feedback that stabilizes this choice5,6. Here, using single-cell genomics, we show formation of many competing hubs with variable enhancer composition, only one of which retains euchromatic features and transcriptional competence. Furthermore, we provide evidence that OR transcription recruits enhancers and reinforces enhancer hub activity locally, whereas OR RNA inhibits transcription of competing ORs over distance, promoting transition to transcriptional singularity. Whereas OR transcription is sufficient to break the symmetry between equipotent enhancer hubs, OR translation stabilizes transcription at the prevailing hub, indicating that there may be sequential non-coding and coding mechanisms that are implemented by OR alleles for transcriptional prevalence. We propose that coding OR mRNAs possess non-coding functions that influence nuclear architecture, enhance their own transcription and inhibit transcription from their competitors, with generalizable implications for probabilistic cell fate decisions.
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Neuronas Receptoras Olfatorias , ARN , Receptores Odorantes , Alelos , Linaje de la Célula , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , ARN/genética , Transcripción Genética , Genómica , Análisis de la Célula IndividualRESUMEN
SARS-CoV-2 is able to re-structure chromatin organization and alters the epigenomic landscape of the host genome, though the mechanisms that produce such changes are still poorly understood. Here, we investigate with polymer physics chromatin re-organization of the host genome, in space and time upon SARS-CoV-2 viral infection. We show that re-structuring of A/B compartments is well explained by a re-modulation of intra-compartment homotypic affinities, which leads to the weakening of A-A interactions and enhances A-B mixing. At TAD level, re-arrangements are physically described by a general reduction of the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and spread in space of TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon (IFN) response, such as DDX58 or IFIT, results more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysis of time trajectories of pairwise gene-enhancer and higher-order contacts reveals that such variability derives from a more fluctuating dynamics in infected case, suggesting that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time. Overall, our study provides the first polymer-physics based 4D reconstruction of SARS-CoV-2 infected genome with mechanistic insights on the consequent gene mis-regulation.
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Extrachromosomal DNAs (ecDNAs) are found in the nucleus of an array of human cancer cells where they can form clusters that were associated to oncogene overexpression, as they carry genes and cis-regulatory elements. Yet, the mechanisms of aggregation and gene amplification beyond copy-number effects remain mostly unclear. Here, we investigate, at the single molecule level, MYC-harboring ecDNAs of COLO320-DM colorectal cancer cells by use of a minimal polymer model of the interactions of ecDNA BRD4 binding sites and BRD4 molecules. We find that BRD4 induces ecDNAs phase separation, resulting in the self-assembly of clusters whose predicted structure is validated against HiChIP data (Hung et al., 2021). Clusters establish in-trans associated contact domains (I-TADs) enriched, beyond copy number, in regulatory contacts among specific ecDNA regions, encompassing its PVT1-MYC fusions but not its other canonical MYC copy. That explains why the fusions originate most of ecDNA MYC transcripts (Hung et al., 2021), and shows that ecDNA clustering per se is important but not sufficient to amplify oncogene expression beyond copy-number, reconciling opposite views on the role of clusters (Hung et al., 2021; Zhu et al., 2021; Purshouse et al. 2022). Regulatory contacts become strongly enriched as soon as half a dozen ecDNAs aggregate, then saturate because of steric hindrance, highlighting that even cells with few ecDNAs can experience pathogenic MYC upregulations. To help drug design and therapeutic applications, with the model we dissect the effects of JQ1, a BET inhibitor. We find that JQ1 reverses ecDNA phase separation hence abolishing I-TADs and extra regulatory contacts, explaining how in COLO320-DM cells it reduces MYC transcription (Hung et al., 2021).
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Technology for measuring 3D genome topology is increasingly important for studying 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 that maps chromatin contacts genome-wide. We perform a detailed comparison of multiplex-GAM and Hi-C using mouse embryonic stem cells. 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 are currently underestimated in ligation-based approaches, and highlights the need for orthogonal advances in genome-wide contact mapping technologies.
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Cromatina , Genoma , Animales , Ratones , Cromatina/genética , Mapeo Cromosómico/métodos , Cromosomas , Genómica/métodosRESUMEN
Human chromosomes have a complex 3D spatial organization in the cell nucleus, which comprises a hierarchy of physical interactions across genomic scales. Such an architecture serves important functional roles, as genes and their regulators have to physically interact to control gene regulation. However, the molecular mechanisms underlying the formation of those contacts remain poorly understood. Here, we describe a polymer-physics-based approach to investigate the machinery shaping genome folding and function. In silico model predictions on DNA single-molecule 3D structures are validated against independent super-resolution single-cell microscopy data, supporting a scenario whereby chromosome architecture is controlled by thermodynamics mechanisms of phase separation. Finally, as an application of our methods, the validated single-polymer conformations of the theory are used to benchmark powerful technologies to probe genome structure, such as Hi-C, SPRITE, and GAM.
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Cromatina , Polímeros , Humanos , Polímeros/química , Cromosomas/genética , Núcleo Celular/química , ADN/genética , ADN/análisis , Cromosomas Humanos , FísicaRESUMEN
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.
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Cromatina , Placenta , Animales , Factor de Unión a CCCTC/metabolismo , Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos , Evolución Molecular , Femenino , Genoma , Mamíferos/metabolismo , Placenta/metabolismo , Embarazo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
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Membrana Nuclear , Células Madre Pluripotentes , Cromatina/genética , Cromatina/metabolismo , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Heterocromatina/metabolismo , Histonas/genética , Membrana Nuclear/metabolismo , Células Madre Pluripotentes/metabolismo , Complejo Represivo Polycomb 2/genéticaRESUMEN
Loop-extrusion and phase-separation have been proposed as mechanisms that shape chromosome spatial organization. It is unclear, however, how they perform relative to each other in explaining chromatin architecture data and whether they compete or co-exist at the single-molecule level. Here, we compare models of polymer physics based on loop-extrusion and phase-separation, as well as models where both mechanisms act simultaneously in a single molecule, against multiplexed FISH data available in human loci in IMR90 and HCT116 cells. We find that the different models recapitulate bulk Hi-C and average multiplexed microscopy data. Single-molecule chromatin conformations are also well captured, especially by phase-separation based models that better reflect the experimentally reported segregation in globules of the considered genomic loci and their cell-to-cell structural variability. Such a variability is consistent with two main concurrent causes: single-cell epigenetic heterogeneity and an intrinsic thermodynamic conformational degeneracy of folding. Overall, the model combining loop-extrusion and polymer phase-separation provides a very good description of the data, particularly higher-order contacts, showing that the two mechanisms can co-exist in shaping chromatin architecture in single cells.
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Cromatina , Polímeros , Cromosomas , Genoma , Humanos , Conformación Molecular , Polímeros/químicaRESUMEN
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.
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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.
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Cromatina , Polímeros , Animales , Cromosomas , Genoma , Mamíferos/genética , FísicaRESUMEN
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.
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Genes Homeobox , Discapacidad Intelectual , Animales , Niño , Proteínas de Homeodominio/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Ratones , Mutación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
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8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Factor de Unión a CCCTC/metabolismo , Elementos de Facilitación Genéticos , Epigénesis Genética , Cromatina/genética , ADN , Inestabilidad Genómica , Humanos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
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.
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Cromatina , Fenotipo , Animales , Cromatina/genética , Cromosomas , Humanos , Ratones , Física , PolímerosRESUMEN
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. Progress 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.
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Cromatina/química , Biopolímeros/química , Regulación de la Expresión Génica , Modelos Biológicos , MutaciónRESUMEN
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.
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Cromosomas , Polímeros , Núcleo Celular , CromatinaRESUMEN
The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.
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Encéfalo/citología , Células/clasificación , Ensamble y Desensamble de Cromatina , Cromatina/química , Cromatina/genética , Genes , Conformación Molecular , Animales , Sitios de Unión , Células/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Familia de Multigenes/genética , Neuronas/clasificación , Neuronas/metabolismo , Desnaturalización de Ácido Nucleico , Factores de Transcripción/metabolismoRESUMEN
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.