RESUMEN
Recent analyses revealed the essential function of chromatin structure in maintaining and regulating genomic information. Advancements in microscopy, nuclear structure observation techniques, and the development of methods utilizing next-generation sequencers (NGSs) have significantly progressed these discoveries. Methods utilizing NGS enable genome-wide analysis, which is challenging with microscopy, and have elucidated concepts of important chromatin structures such as a loop structure, a domain structure called topologically associating domains (TADs), and compartments. In this chapter, I introduce chromatin interaction techniques using NGS and outline the principles and features of each method.
Asunto(s)
Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Genómica/métodos , Estudio de Asociación del Genoma Completo/métodos , AnimalesRESUMEN
Hi-C is a popular ligation-based technique to detect 3D physical chromosome structure within the nucleus using cross-linking and next-generation sequencing. As an unbiased genome-wide assay based on chromosome conformation capture, it provides rich insights into chromosome structure, dynamic chromosome folding and interactions, and the regulatory state of a cell. Bioinformatics analyses of Hi-C data require dedicated protocols as most genome alignment tools assume that both paired-end reads will map to the same chromosome, resulting in large two-dimensional matrices as processed data. Here, we outline the necessary steps to generate high-quality aligned Hi-C data by separately mapping each read while correcting for biases from restriction enzyme digests. We introduce our own custom open-source pipeline, which enables users to select an aligner of their choosing with high accuracy and performance. This enables users to generate high-resolution datasets with fast turnaround and fewer unmapped reads. Finally, we discuss recent innovations in experimental techniques, bioinformatics techniques, and their applications in clinical testing for diagnostics.
Asunto(s)
Mapeo Cromosómico , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Programas Informáticos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biología Computacional/métodos , Humanos , Mapeo Cromosómico/métodos , Cromosomas/genética , Genómica/métodos , Cromatina/genética , Cromatina/químicaRESUMEN
Polymer modeling has been playing an increasingly important role in complementing 3D genome experiments, both to aid their interpretation and to reveal the underlying molecular mechanisms. This chapter illustrates an application of Hi-C metainference, a Bayesian approach to explore the 3D organization of a target genomic region by integrating experimental contact frequencies into a prior model of chromatin. The method reconstructs the conformational ensemble of the target locus by combining molecular dynamics simulation and Monte Carlo sampling from the posterior probability distribution given the data. Using prior chromatin models at both 1 kb and nucleosome resolution, we apply this approach to a 30 kb locus of mouse embryonic stem cells consisting of two well-defined domains linking several gene promoters together. Retaining the advantages of both physics-based and data-driven strategies, Hi-C metainference can provide an experimentally consistent representation of the system while at the same time retaining molecular details necessary to derive physical insights.
Asunto(s)
Teorema de Bayes , Cromatina , Simulación de Dinámica Molecular , Animales , Ratones , Cromatina/genética , Cromatina/química , Cromatina/metabolismo , Genoma , Genómica/métodos , Método de Montecarlo , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
Hi-C methods reveal 3D genome features but lack correspondence to dynamic chromatin behavior. PHi-C2, Python software, addresses this gap by transforming Hi-C data into polymer models. After the optimization algorithm, it enables us to calculate 3D conformations and conduct dynamic simulations, providing insights into chromatin dynamics, including the mean-squared displacement and rheological properties. This chapter introduces PHi-C2 usage, offering a tutorial for comprehensive 4D genome analysis.
Asunto(s)
Algoritmos , Cromatina , Programas Informáticos , Cromatina/genética , Cromatina/química , Cromatina/metabolismo , Humanos , Genómica/métodos , Genoma , Biología Computacional/métodosRESUMEN
Significance: Azimuth-resolved optical scattering signals obtained from cell nuclei are sensitive to changes in their internal refractive index profile. These two-dimensional signals can therefore offer significant insights into chromatin organization. Aim: We aim to determine whether two-dimensional scattering signals can be used in an inverse scheme to extract the spatial correlation length â c and extent δ n of subnuclear refractive index fluctuations to provide quantitative information on chromatin distribution. Approach: Since an analytical formulation that links azimuth-resolved signals to â c and δ n is not feasible, we set out to assess the potential of machine learning to predict these parameters via a data-driven approach. We carry out a convolutional neural network (CNN)-based regression analysis on 198 numerically computed signals for nuclear models constructed with â c varying in steps of 0.1 µ m between 0.4 and 1.0 µ m , and δ n varying in steps of 0.005 between 0.005 and 0.035. We quantify the performance of our analysis using a five-fold cross-validation technique. Results: The results show agreement between the true and predicted values for both â c and δ n , with mean absolute percent errors of 8.5% and 13.5%, respectively. These errors are smaller than the minimum percent increment between successive values for respective parameters characterizing the constructed models and thus signify an extremely good prediction performance over the range of interest. Conclusions: Our results reveal that CNN-based regression can be a powerful approach for exploiting the information content of two-dimensional optical scattering signals and hence monitoring chromatin organization in a quantitative manner.
Asunto(s)
Núcleo Celular , Cromatina , Redes Neurales de la Computación , Cromatina/química , Núcleo Celular/química , Análisis de Regresión , Dispersión de Radiación , Refractometría/métodos , Aprendizaje Automático , Humanos , AlgoritmosRESUMEN
Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.
Asunto(s)
Drosophila , Animales , Drosophila/genética , Drosophila melanogaster/genética , Cromatina/química , Cromatina/metabolismo , Cohesinas , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Estructuras Cromosómicas , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/químicaRESUMEN
Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.
Asunto(s)
ADN , Histonas , Nucleosomas , Histonas/química , Histonas/metabolismo , Histonas/genética , ADN/química , ADN/metabolismo , Nucleosomas/metabolismo , Nucleosomas/química , Cromatina/química , Cromatina/metabolismo , Cromatina/genética , Animales , HumanosRESUMEN
The six-subunit shelterin complex binds to mammalian telomeres and protects them from triggering multiple DNA damage response pathways. The loss of this protective function by shelterin can have detrimental effects on cells. In this review, we first discuss structural studies of shelterin, detailing the contributions of each subunit and inter-subunit interactions in protecting chromosome ends. We then examine the influence of telomeric chromatin dynamics on the function of shelterin at telomeres. These studies provide valuable insights and underscore the challenges that future research must tackle to attain high-resolution structures of shelterin.
Asunto(s)
Cromatina , Complejo Shelterina , Proteínas de Unión a Telómeros , Telómero , Telómero/metabolismo , Cromatina/metabolismo , Cromatina/química , Humanos , Complejo Shelterina/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/química , Animales , Daño del ADN , Unión ProteicaRESUMEN
Since the advent of Hi-C in 2009, a plethora of high-throughput sequencing methods have emerged to profile the three-dimensional (3D) organization of eukaryotic genomes, igniting the era of 3D genomics. In recent years, the genomic resolution achievable by these approaches has dramatically increased and several single-cell versions of Hi-C have been developed. Moreover, a new repertoire of tools not based on proximity ligation of digested chromatin has emerged, enabling the investigation of the higher-order organization of chromatin in the nucleus. In this review, we summarize the expanding portfolio of 3D genomic technologies, highlighting recent developments and applications from the past three years. Lastly, we present an outlook of where this technology-driven field might be headed.
Asunto(s)
Genómica , Humanos , Animales , Cromatina/metabolismo , Cromatina/química , Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento , GenomaRESUMEN
Cell biology emerges from spatiotemporally coordinated molecular processes. Recent advances in live-cell microscopy, fueled by a surge in optical, molecular, and computational technologies, have enabled dynamic observations from single molecules to whole organisms. Despite technological leaps, there is still an untapped opportunity to fully leverage their capabilities toward biological insight. We highlight how single-molecule imaging has transformed our understanding of biological processes, with a focus on chromatin organization and transcription in the nucleus. We describe how this was enabled by the close integration of new imaging techniques with analysis tools and discuss the challenges to make a comparable impact at larger scales from organelles to organisms. By highlighting recent successful examples, we describe an outlook of ever-increasing data and the need for seamless integration between dataset visualization and quantification to realize the full potential warranted by advances in new imaging technologies.
Asunto(s)
Microscopía , Humanos , Animales , Microscopía/métodos , Cromatina/metabolismo , Cromatina/química , Núcleo Celular/metabolismo , Imagen Individual de MoléculaRESUMEN
Biologists have the capability to edit a genome at the nanometer scale and then observe whether or not the edit affects the structure of a developing organ or organism at the centimeter scale. Our understanding of the underlying mechanisms driving this emergent phenomenon from a multiscale perspective remains incomplete. This review focuses predominantly on recent experimental developments in uncovering the mechanical interplay between the chromatin and cell scale since mechanics plays a major role in determining nuclear, cellular, and tissue structure. Here, we discuss the generation and transmission of forces through the cytoskeleton, affecting chromatin diffusivity and organization. Decoding such pieces of these multiscale connections lays the groundwork for solving the genotype-to-phenotype puzzle in biology.
Asunto(s)
Cromatina , Citoesqueleto , Cromatina/metabolismo , Cromatina/química , Humanos , Animales , Citoesqueleto/metabolismo , Ensamble y Desensamble de CromatinaRESUMEN
Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained.
Asunto(s)
Cromatina , G-Cuádruplex , Regiones Promotoras Genéticas , Cromatina/metabolismo , Cromatina/química , Cromatina/genética , Humanos , Histonas/metabolismo , Histonas/química , Histonas/genética , Elementos de Facilitación GenéticosRESUMEN
The population-averaged contact maps generated by the chromosome conformation capture technique provide important information about the average frequency of contact between pairs of chromatin loci as a function of the genetic distance between them. However, these datasets do not tell us anything about the joint statistics of simultaneous contacts between genomic loci in individual cells. This kind of statistical information can be extracted using the single-cell Hi-C method, which is capable of detecting a large fraction of simultaneous contacts within a single cell, as well as through modern methods of fluorescent labeling and super-resolution imaging. Motivated by the prospect of the imminent availability of relevant experimental data, in this work, we theoretically model the joint statistics of pairs of contacts located along a line perpendicular to the main diagonal of the single-cell contact map. The analysis is performed within the framework of an ideal polymer model with quenched disorder of random loops, which, as previous studies have shown, allows us to take into account the influence of the loop extrusion process on the conformational properties of interphase chromatin.
Asunto(s)
Cromatina , Interfase , Interfase/genética , Cromatina/química , Cromatina/genética , Cromosomas/química , Cromosomas/genética , Conformación de Ácido NucleicoRESUMEN
Single-cell ATAC-seq sequencing data (scATAC-seq) has been widely used to investigate chromatin accessibility on the single-cell level. One important application of scATAC-seq data analysis is differential chromatin accessibility (DA) analysis. However, the data characteristics of scATAC-seq such as excessive zeros and large variability of chromatin accessibility across cells impose a unique challenge for DA analysis. Existing statistical methods focus on detecting the mean difference of the chromatin accessible regions while overlooking the distribution difference. Motivated by real data exploration that distribution difference exists among cell types, we introduce a novel composite statistical test named "scaDA", which is based on zero-inflated negative binomial model (ZINB), for performing differential distribution analysis of chromatin accessibility by jointly testing the abundance, prevalence and dispersion simultaneously. Benefiting from both dispersion shrinkage and iterative refinement of mean and prevalence parameter estimates, scaDA demonstrates its superiority to both ZINB-based likelihood ratio tests and published methods by achieving the highest power and best FDR control in a comprehensive simulation study. In addition to demonstrating the highest power in three real sc-multiome data analyses, scaDA successfully identifies differentially accessible regions in microglia from sc-multiome data for an Alzheimer's disease (AD) study that are most enriched in GO terms related to neurogenesis and the clinical phenotype of AD, and AD-associated GWAS SNPs.
Asunto(s)
Cromatina , Análisis de la Célula Individual , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/estadística & datos numéricos , Humanos , Biología Computacional/métodos , Enfermedad de Alzheimer/genética , Modelos Estadísticos , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Simulación por Computador , Animales , Análisis de Secuencia de ADN/métodos , AlgoritmosRESUMEN
DNA loop extrusion plays a key role in the regulation of gene expression and the structural arrangement of chromatin. Most existing mechanistic models of loop extrusion depend on some type of ratchet mechanism, which should permit the elongation of loops while preventing their collapse, by enabling DNA to move in only one direction. STAG2 is already known to exert a role as DNA anchor, but the available structural data suggest a possible role in unidirectional DNA motion. In this work, a computational simulation framework was constructed to evaluate whether STAG2 could enforce such unidirectional displacement of a DNA double helix. The results reveal that STAG2 V-shape allows DNA sliding in one direction, but blocks opposite DNA movement via a linear ratchet mechanism. Furthermore, these results suggest that RAD21 binding to STAG2 controls its flexibility by narrowing the opening of its V-shape, which otherwise remains widely open in absence of RAD21. Therefore, in the proposed model, in addition to its already described role as a DNA anchor, the STAG2-RAD21 complex would be part of a ratchet mechanism capable of exerting directional selectivity on DNA sliding during loop extrusion. The identification of the molecular basis of the ratchet mechanism of loop extrusion is a critical step in unraveling new insights into a broad spectrum of chromatin activities and their implications for the mechanisms of chromatin-related diseases.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , ADN , ADN/química , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Unión Proteica , Conformación de Ácido Nucleico , Proteínas Nucleares/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Modelos Moleculares , Humanos , Simulación de Dinámica Molecular , Cromatina/química , Cromatina/genética , Cromatina/metabolismoRESUMEN
Pioneering transcription factors (TFs) can drive cell fate changes by binding their DNA motifs in a repressive chromatin environment. Recent structures illustrate emerging rules for nucleosome engagement: TFs distort the nucleosomal DNA to gain access or employ alternative DNA-binding modes with smaller footprints, they preferentially access solvent-exposed motifs near the entry/exit sites, and frequently interact with histones. The extent of TF-histone interactions, in turn, depends on the motif location on the nucleosome, the type of DNA-binding fold, and adjacent domains present. TF-histone interactions can phase TF motifs relative to nucleosomes, and we discuss how these complex and surprisingly diverse interactions between nucleosomes and TFs contribute to function.
Asunto(s)
Cromatina , ADN , Nucleosomas , Factores de Transcripción , ADN/metabolismo , ADN/química , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Cromatina/metabolismo , Cromatina/química , Nucleosomas/metabolismo , Nucleosomas/química , Humanos , Unión Proteica , Animales , Histonas/metabolismo , Histonas/químicaRESUMEN
Mammalian chromosomes form a hierarchical structure within the cell nucleus, from chromatin loops, megabase (Mb)-sized topologically associating domains (TADs) to larger-scale A/B compartments. The molecular basis of the structures of loops and TADs has been actively studied. However, the A and B compartments, which correspond to early-replicating euchromatin and late-replicating heterochromatin, respectively, are still relatively unexplored. In this review, we focus on the A/B compartments, discuss their close relationship to DNA replication timing (RT), and introduce recent findings on the features of subcompartments revealed by detailed classification of the A/B compartments. In doing so, we speculate on the structure, potential function, and developmental dynamics of A/B compartments and subcompartments in mammalian cells.
Asunto(s)
Núcleo Celular , Heterocromatina , Humanos , Animales , Núcleo Celular/metabolismo , Núcleo Celular/química , Heterocromatina/metabolismo , Heterocromatina/química , Replicación del ADN , Eucromatina/metabolismo , Eucromatina/química , Cromatina/metabolismo , Cromatina/químicaRESUMEN
The substrate for the repair of DNA damage in living cells is not DNA but chromatin. Chromatin bears a range of modifications, which in turn bind ligands that compact or open chromatin structure, and determine its spatial organization within the nucleus. In some cases, RNA in the form of RNA:DNA hybrids or R-loops modulates DNA accessibility. Each of these parameters can favor particular pathways of repair. Chromatin or nucleosome remodelers are key regulators of chromatin structure, and a number of remodeling complexes are implicated in DNA repair. We cover novel insights into the impact of chromatin structure, nuclear organization, R-loop formation, nuclear actin, and nucleosome remodelers in DNA double-strand break repair, focusing on factors that alter repair functional upon ablation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Animales , Cromatina/metabolismo , Cromatina/química , Nucleosomas/metabolismo , Nucleosomas/químicaRESUMEN
High-throughput chromosome conformation capture (Hi-C) technology captures spatial interactions of DNA sequences into matrices, and software tools are developed to identify topologically associating domains (TADs) from the Hi-C matrices. With structural information theory, SuperTAD adopted a dynamic programming approach to find the TAD hierarchy with minimal structural entropy. However, the algorithm suffers from high time complexity. To accelerate this algorithm, we design and implement an approximation algorithm with a theoretical performance guarantee. We implemented a package, SuperTAD-Fast. Using Hi-C matrices and simulated data, we demonstrated that SuperTAD-Fast achieved great runtime improvement compared with SuperTAD. SuperTAD-Fast shows high consistency and significant enrichment of structural proteins from Hi-C data of human cell lines in comparison with the existing six hierarchical TADs detecting methods.
Asunto(s)
Cromatina , Técnicas Genéticas , Programas Informáticos , Cromatina/química , Cromatina/genética , Simulación por Computador , Algoritmos , Entropía , GenomaRESUMEN
The 3D folding of a mammalian gene can be studied by a polymer model, where the chromatin fiber is represented by a semiflexible polymer which interacts with multivalent proteins, representing complexes of DNA-binding transcription factors and RNA polymerases. This physical model leads to the natural emergence of clusters of proteins and binding sites, accompanied by the folding of chromatin into a set of topologies, each associated with a different network of loops. Here, we combine numerics and analytics to first classify these networks and then find their relative importance or statistical weight, when the properties of the underlying polymer are those relevant to chromatin. Unlike polymer networks previously studied, our chromatin networks have finite average distances between successive binding sites, and this leads to giant differences between the weights of topologies with the same number of edges and nodes but different wiring. These weights strongly favor rosettelike structures with a local cloud of loops with respect to more complicated nonlocal topologies. Our results suggest that genes should overwhelmingly fold into a small fraction of all possible 3D topologies, which can be robustly characterized by the framework we propose here.