Genome Folding Research Articles

Brownian dynamics simulations of mesoscale chromatin fibers

The relationship between chromatin architecture and function defines a central problem in biology and medicine. Many computational chromatin models with atomic, coarse-grained, mesoscale, and polymer resolution have been used to shed light onto the mechanisms that dictate genome folding and regulation of gene expression. The associated simulation techniques range from Monte Carlo to molecular, Brownian, and Langevin dynamics. Here, we present an efficient Compute Unified Device Architecture (CUDA) implementation of Brownian dynamics (BD) to simulate chromatin fibers at the nucleosome resolution with our chromatin mesoscale model. With the CUDA implementation for computer architectures with graphic processing units (GPUs), we significantly accelerate compute-intensive hydrodynamic tensor calculations in the BD simulations by massive parallelization, boosting the performance a hundred-fold compared with central processing unit calculations. We validate our BD simulation approach by reproducing experimental trends on fiber diffusion and structure as a function of salt, linker histone binding, and histone-tail composition, as well as Monte Carlo equilibrium sampling results. Our approach proves to be physically accurate with performance that makes feasible the study of chromatin fibers in the range of kb or hundreds of nucleosomes (small gene). Such simulations are essential to advance the study of biological processes such as gene regulation and aberrant genome-structure related diseases.

Causality in transcription and genome folding: Insights from X inactivation.

The spatial organization of genomes is becoming increasingly understood. In mammals, where it is most investigated, this organization ties in with transcription, so an important research objective is to understand whether gene activity is a cause or a consequence of genome folding in space. In this regard, the phenomena of X-chromosome inactivation and reactivation open a unique window of investigation because of the singularities of the inactive X chromosome. Here we focus on the cause-consequence nexus between genome conformation and transcription and explain how recent results about the structural changes associated with inactivation and reactivation of the X chromosome shed light on this problem.

Computational and Experimental Approaches to Study the RNA Secondary Structures of RNA Viruses.

Most pandemics of recent decades can be traced to RNA viruses, including HIV, SARS, influenza, dengue, Zika, and SARS-CoV-2. These RNA viruses impose considerable social and economic burdens on our society, resulting in a high number of deaths and high treatment costs. As these RNA viruses utilize an RNA genome, which is important for different stages of the viral life cycle, including replication, translation, and packaging, studying how the genome folds is important to understand virus function. In this review, we summarize recent advances in computational and high-throughput RNA structure-mapping approaches and their use in understanding structures within RNA virus genomes. In particular, we focus on the genome structures of the dengue, Zika, and SARS-CoV-2 viruses due to recent significant outbreaks of these viruses around the world.

Topoisomerase 2β and DNA topology during B cell development.

Topoisomerase 2β (TOP2B) introduces transient double strand breaks in the DNA helix to remove supercoiling structures and unwind entangled DNA strains. Advances in genomic technologies have enabled the discovery of novel functions for TOP2B in processes such as releasing of the paused RNA polymerase II and maintaining the genome organization through DNA loop domains. Thus, TOP2B can regulate transcription directly by acting on transcription elongation and indirectly by controlling interactions between enhancer and promoter regions through genome folding. The identification of TOP2B mutations in humans unexpectedly revealed a unique role of TOP2B in B-cell progenitors. Here we discuss the functions of TOP2B and the mechanisms leading to the B-cell development defect in patients with TOP2B deficiency.

CTCF and Its Partners: Shaper of 3D Genome during Development.

The 3D genome organization and its dynamic modulate genome function, playing a pivotal role in cell differentiation and development. CTCF and cohesin, acting as the core architectural components involved in chromatin looping and genome folding, can also recruit other protein or RNA partners to fine-tune genome structure during development. Moreover, systematic screening for partners of CTCF has been performed through high-throughput approaches. In particular, several novel protein and RNA partners, such as BHLHE40, WIZ, MAZ, Aire, MyoD, YY1, ZNF143, and Jpx, have been identified, and these partners are mostly implicated in transcriptional regulation and chromatin remodeling, offering a unique opportunity for dissecting their roles in higher-order chromatin organization by collaborating with CTCF and cohesin. Here, we review the latest advancements with an emphasis on features of CTCF partners and also discuss the specific functions of CTCF-associated complexes in chromatin structure modulation, which may extend our understanding of the functions of higher-order chromatin architecture in developmental processes.

The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism

Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin’s SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the three-dimensional genome in human cells. ESCO1 restricts the length of chromatin loops, and of architectural stripes emanating from CTCF sites. HDAC8 conversely promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation controls the interaction of cohesin with PDS5A to restrict chromatin loop length. Our data support a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding.

Essential role of Cp190 in physical and regulatory boundary formation.

Boundaries in animal genomes delimit contact domains with enhanced internal contact frequencies and have debated functions in limiting regulatory cross-talk between domains and guiding enhancers to target promoters. Most mammalian boundaries form by stalling of chromosomal loop-extruding cohesin by CTCF, but most Drosophila boundaries form CTCF independently. However, how CTCF-independent boundaries form and function remains largely unexplored. Here, we assess genome folding and developmental gene expression in fly embryos lacking the ubiquitous boundary-associated factor Cp190. We find that sequence-specific DNA binding proteins such as CTCF and Su(Hw) directly interact with and recruit Cp190 to form most promoter-distal boundaries. Cp190 is essential for early development and prevents regulatory cross-talk between specific gene loci that pattern the embryo. Cp190 was, in contrast, dispensable for long-range enhancer-promoter communication at tested loci. Cp190 is thus currently the major player in fly boundary formation and function, revealing that diverse mechanisms evolved to partition genomes into independent regulatory domains.

Genome architecture and totipotency: An intertwined relation during early embryonic development.

Chromosomes are not randomly packed and positioned into the nucleus but folded in higher-order chromatin structures with defined functions. However, the genome of a fertilized embryo undergoes a dramatic epigenetic reprogramming characterized by extensive chromatin relaxation and the lack of a defined three-dimensional structure. This reprogramming is followed by a slow genome refolding that gradually strengthens the chromatin architecture during preimplantation development. Interestingly, genome refolding during early development coincides with a progressive loss of developmental potential suggesting a link between chromatin organization and cell plasticity. In agreement, loss of chromatin architecture upon depletion of the insulator transcription factor CTCF in embryonic stem cells led to the upregulation of the transcriptional program found in totipotent cells of the embryo, those with the highest developmental potential. This essay will discuss the impact of genome folding in controlling the expression of transcriptional programs involved in early development and their plastic-associated features.

Genome-Directed Cell Nucleus Assembly.

Simple SummarySpeckles and other nuclear bodies, the nucleolus and perinucleolar zone, transcription/replication factories and the lamina-associated compartment, serve as a structural basis for various genomic functions. In turn, genome activity and specific chromatin 3D organization directly impact the integrity of intranuclear assemblies, initiating/facilitating their formation and dictating their composition. Thus, the large-scale nucleus structure and genome activity mutually influence each other. The cell nucleus is frequently considered a compartment in which the genome is placed to protect it from external forces. Here, we discuss the evidence demonstrating that the cell nucleus should be considered, rather, as structure built around the folded genome. Decondensing chromosomes provide a scaffold for the assembly of the nuclear envelope after mitosis, whereas genome activity directs the assembly of various nuclear compartments, including nucleolus, speckles and transcription factories.The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA’s genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the “box” in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses.

EBF1 nuclear repositioning instructs chromatin refolding to promote therapy resistance in T leukemic cells

Chromatin misfolding has been implicated in cancer pathogenesis; yet, its role in therapy resistance remains unclear. Here, we systematically integrated sequencing and imaging data to examine the spatial and linear chromatin structures in targeted therapy-sensitive and -resistant human Tcell acute lymphoblastic leukemia (T-ALL). We found widespread alterations in successive layers of chromatin organization including spatial compartments, contact domain boundaries, and enhancer positioning upon the emergence of targeted therapy resistance. The reorganization of genome folding structures closely coincides with the restructuring of chromatin activity and redistribution of architectural proteins. Mechanistically, the derepression and repositioning of the B-lineage-determining transcription factor EBF1 from the heterochromatic nuclear envelope to the euchromatic interior instructs widespread genome refolding and promotes therapy resistance in leukemic Tcells. Together, our findings suggest that lineage-determining transcription factors can instruct changes in genome topology as a driving force for epigenetic adaptations in targeted therapy resistance.

DNA Repair in Space and Time: Safeguarding the Genome with the Cohesin Complex.

DNA double-strand breaks (DSBs) are a deleterious form of DNA damage, which must be robustly addressed to ensure genome stability. Defective repair can result in chromosome loss, point mutations, loss of heterozygosity or chromosomal rearrangements, which could lead to oncogenesis or cell death. We explore the requirements for the successful repair of DNA DSBs by non-homologous end joining and homology-directed repair (HDR) mechanisms in relation to genome folding and dynamics. On the occurrence of a DSB, local and global chromatin composition and dynamics, as well as 3D genome organization and break localization within the nuclear space, influence how repair proceeds. The cohesin complex is increasingly implicated as a key regulator of the genome, influencing chromatin composition and dynamics, and crucially genome organization through folding chromosomes by an active loop extrusion mechanism, and maintaining sister chromatid cohesion. Here, we consider how this complex is now emerging as a key player in the DNA damage response, influencing repair pathway choice and efficiency.

Diurnal RNAPII-tethered chromatin interactions are associated with rhythmic gene expression in rice

BackgroundThe daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear.ResultsHere, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs.ConclusionOur findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

Integrating Multimorbidity into a Whole-Body Understanding of Disease Using Spatial Genomics.

Multimorbidity is characterized by multidimensional complexity emerging from interactions between multiple diseases across levels of biological (including genetic) and environmental determinants and the complex array of interactions between and within cells, tissues and organ systems. Advances in spatial genomic research have led to an unprecedented expansion in our ability to link alterations in genome folding with changes that are associated with human disease. Studying disease-associated genetic variants in the context of the spatial genome has enabled the discovery of transcriptional regulatory programmes that potentially link dysregulated genes to disease development. However, the approaches that have been used have typically been applied to uncover pathological molecular mechanisms occurring in a specific disease-relevant tissue. These forms of reductionist, targeted investigations are not appropriate for the molecular dissection of multimorbidity that typically involves contributions from multiple tissues. In this perspective, we emphasize the importance of a whole-body understanding of multimorbidity and discuss how spatial genomics, when integrated with additional omic datasets, could provide novel insights into the molecular underpinnings of multimorbidity.

Multi-scale phase separation by explosive percolation with single-chromatin loop resolution

The 2 m-long human DNA is tightly intertwined into the cell nucleus of the size of 10 μm. The DNA packing is explained by folding of chromatin fiber. This folding leads to the formation of such hierarchical structures as: chromosomal territories, compartments; densely-packed genomic regions known as Topologically Associating Domains (TADs), or Chromatin Contact Domains (CCDs), and loops. We propose models of dynamical human genome folding into hierarchical components in human lymphoblastoid, stem cell, and fibroblast cell lines. Our models are based on explosive percolation theory. The chromosomes are modeled as graphs where CTCF chromatin loops are represented as edges. The folding trajectory is simulated by gradually introducing loops to the graph following various edge addition strategies that are based on topological network properties, chromatin loop frequencies, compartmentalization, or epigenomic features. Finally, we propose the genome folding model - a biophysical pseudo-time process guided by a single scalar order parameter. The parameter is calculated by Linear Discriminant Analysis of chromatin features. We also include dynamics of loop formation by using Loop Extrusion Model (LEM) while adding them to the system. The chromatin phase separation, where fiber folds in 3D space into topological domains and compartments, is observed when the critical number of contacts is reached. We also observe that at least 80% of the loops are needed for chromatin fiber to condense in 3D space, and this is constant through various cell lines. Overall, our in-silico model integrates the high-throughput 3D genome interaction experimental data with the novel theoretical concept of phase separation, which allows us to model event-based time dynamics of chromatin loop formation and folding trajectories.

Tough Tissue Hi-C.

The ability to decipher the three-dimensional chromosome folding in many eukaryotes is a major asset in molecular biology. It is not only required to study the biological relevance of chromosome folding in cellular processes but also for the de novo assembly of genomes of nonmodel species. With lowering DNA sequencing costs, the latter has recently become interesting to many scientists, ranging from molecular biologists that aim to establish new model organisms, to evolutionary biologists and ecologists, interested in genome evolution and diversity. Hi-C is regarded as the method of choice to characterize three-dimensional genome folding and, thus, also has been integrated as a standard method in assembly pipelines. However, Hi-C is a demanding molecular biology technique, and its application can be considerably challenged by the tissue used. Hi-C relies on efficient and pure nuclei isolation, which is, especially in many plant species, inhibited by the tough nature of plant tissues and cell walls. The Hi-C protocol presented here has been optimized for such tissues and has been shown to generate Hi-C samples of sufficient quality in various plant and animal tissues.

Mapping Mammalian 3D Genomes by Micro-C.

3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.

Single-Molecule/Cell Analyses Reveal Principles of Genome-Folding Mechanisms in the Three Domains of Life.

Comparative structural/molecular biology by single-molecule analyses combined with single-cell dissection, mass spectroscopy, and biochemical reconstitution have been powerful tools for elucidating the mechanisms underlying genome DNA folding. All genomes in the three domains of life undergo stepwise folding from DNA to 30–40 nm fibers. Major protein players are histone (Eukarya and Archaea), Alba (Archaea), and HU (Bacteria) for fundamental structural units of the genome. In Euryarchaeota, a major archaeal phylum, either histone or HTa (the bacterial HU homolog) were found to wrap DNA. This finding divides archaea into two groups: those that use DNA-wrapping as the fundamental step in genome folding and those that do not. Archaeal transcription factor-like protein TrmBL2 has been suggested to be involved in genome folding and repression of horizontally acquired genes, similar to bacterial H-NS protein. Evolutionarily divergent SMC proteins contribute to the establishment of higher-order structures. Recent results are presented, including the use of Hi-C technology to reveal that archaeal SMC proteins are involved in higher-order genome folding, and the use of single-molecule tracking to reveal the detailed functions of bacterial and eukaryotic SMC proteins. Here, we highlight the similarities and differences in the DNA-folding mechanisms in the three domains of life.

Chromatin Network Analyses: Towards Structure-Function Relationships in Epigenomics.

Recent technological advances have allowed us to map chromatin conformation and uncover the genome’s spatial organization of the genome inside the nucleus. These experiments have revealed the complexities of genome folding, characterized by the presence of loops and domains at different scales, which can change across development and in different cell types. There is strong evidence for a relationship between the topological properties of chromatin contacts and cellular phenotype. Chromatin can be represented as a network, in which genomic fragments are the nodes and connections represent experimentally observed spatial proximity of two genomically distant regions in a specific cell type or biological condition. With this approach we can consider a variety of chromatin features in association with the 3D structure, investigating how nuclear chromatin organization can be related to gene regulation, replication, malignancy, phenotypic variability and plasticity. We briefly review the results obtained on genome architecture through network theoretic approaches. As previously observed in protein-protein interaction networks and many types of non-biological networks, external conditions could shape network topology through a yet unidentified structure-function relationship. Similar to scientists studying the brain, we are confronted with a duality between a spatially embedded network of physical contacts, a related network of correlation in the dynamics of network nodes and, finally, an abstract definition of function of this network, related to phenotype. We summarise major developments in the study of networks in other fields, which we think can suggest a path towards better understanding how 3D genome configuration can impact biological function and adaptation to the environment.

BRD4 orchestrates genome folding to promote neural crest differentiation.

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.

Uncovering the Principles of Genome Folding by 3D Chromatin Modeling.

Our understanding of how genomic DNA is tightly packed inside the nucleus, yet is still accessible for vital cellular processes, has grown dramatically over recent years with advances in microscopy and genomics technologies. Computational methods have played a pivotal role in the structural interpretation of experimental data, which helped unravel some organizational principles of genome folding. Here, we give an overview of current computational efforts in mechanistic and data-driven 3D chromatin structure modeling. We discuss strengths and limitations of different methods and evaluate the added value and benefits of computational approaches to infer the 3D structural and dynamic properties of the genome and its underlying mechanisms at different scales and resolution, ranging from the dynamic formation of chromatin loops and topological associated domains to nuclear compartmentalization of chromatin and nuclear bodies.