Chromatin Folding Research Articles

Synthesis of ADP-Ribosylated Histones Reveals Site-Specific Impacts on Chromatin Structure and Function.

ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.

Hi-C 3.0: Improved Protocol for Genome-Wide Chromosome Conformation Capture.

The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi‐C is a chromosome conformation capture (3C)‐based technology to detect pair‐wise chromatin interactions genome‐wide, and has become a benchmark tool to study genome organization. In Hi‐C, chromatin conformation is first captured by chemical cross‐linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high‐throughput sequencing, which enables the mapping of pair‐wise chromatin interactions genome‐wide. Over the past decade, “next‐generation” sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in‐depth guide to performing an up‐to‐date Hi‐C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross‐linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi‐C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Fixation of nuclear conformationBasic Protocol 2: Chromosome conformation captureBasic Protocol 3: Hi‐C sequencing library preparation

Molecular Epigenetics: Chemical Biology Tools Come of Age.

The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.

Liquid-Liquid Phase Separation in Chromatin.

In eukaryotic cells, protein and RNA factors involved in genome activities like transcription, RNA processing, DNA replication, and repair accumulate in self-organizing membraneless chromatin subcompartments. These structures contribute to efficiently conduct chromatin-mediated reactions and to establish specific cellular programs. However, the underlying mechanisms for their formation are only partly understood. Recent studies invoke liquid-liquid phase separation (LLPS) of proteins and RNAs in the establishment of chromatin activity patterns. At the same time, the folding of chromatin in the nucleus can drive genome partitioning into spatially distinct domains. Here, the interplay between chromatin organization, chromatin binding, and LLPS is discussed by comparing and contrasting three prototypical chromatin subcompartments: the nucleolus, clusters of active RNA polymerase II, and pericentric heterochromatin domains. It is discussed how the different ways of chromatin compartmentalization are linked to transcription regulation, the targeting of soluble factors to certain parts of the genome, and to disease-causing genetic aberrations.

Non-coding RNAs in chromatin folding and nuclear organization.

One of the most intriguing questions facing modern biology concerns how the genome directs the construction of cells, tissues, and whole organisms. It is tempting to suggest that the part of the genome that does not encode proteins contains architectural plans. We are still far from understanding how these plans work at the level of building tissues and the body as a whole. However, the results of recent studies demonstrate that at the cellular level, special non-coding RNAs serve as scaffolds for the construction of various intracellular structures. The term "architectural RNAs" was proposed to designate this subset of non-coding RNAs. In this review, we discuss the role of architectural RNAs in the construction of the cell nucleus and maintenance of the three-dimensional organization of the genome.

HMGB1 coordinates SASP‐related chromatin folding and RNA homeostasis on the path to senescence

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome‐wide in two primary cell lines. We integrated ChIP‐seq and Hi‐C with graph theory to uncover clustering of HMGB1‐marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross‐Linking and Immuno‐Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA‐binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP‐relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell‐autonomous and paracrine senescence.

Robust and efficient single-cell Hi-C clustering with approximate k-nearest neighbor graphs.

MotivationHi-C technology provides insights into the 3D organization of the chromatin, and the single-cell Hi-C method enables researchers to gain knowledge about the chromatin state in individual cell levels. Single-cell Hi-C interaction matrices are high dimensional and very sparse. To cluster thousands of single-cell Hi-C interaction matrices, they are flattened and compiled into one matrix. Depending on the resolution, this matrix can have a few million or even billions of features; therefore, computations can be memory intensive. We present a single-cell Hi-C clustering approach using an approximate nearest neighbors method based on locality-sensitive hashing to reduce the dimensions and the computational resources.ResultsThe presented method can process a 10 kb single-cell Hi-C dataset with 2600 cells and needs 40 GB of memory, while competitive approaches are not computable even with 1 TB of memory. It can be shown that the differentiation of the cells by their chromatin folding properties and, therefore, the quality of the clustering of single-cell Hi-C data is advantageous compared to competitive algorithms.Availability and implementationThe presented clustering algorithm is part of the scHiCExplorer, is available on Github https://github.com/joachimwolff/scHiCExplorer, and as a conda package via the bioconda channel. The approximate nearest neighbors implementation is available via https://github.com/joachimwolff/sparse-neighbors-search and as a conda package via the bioconda channel.Supplementary information Supplementary data are available at Bioinformatics online.

Nucleosome plasticity is a critical element of chromatin liquid\u2013liquid phase separation and multivalent nucleosome interactions

Liquid–liquid phase separation (LLPS) is an important mechanism that helps explain the membraneless compartmentalization of the nucleus. Because chromatin compaction and LLPS are collective phenomena, linking their modulation to the physicochemical features of nucleosomes is challenging. Here, we develop an advanced multiscale chromatin model—integrating atomistic representations, a chemically-specific coarse-grained model, and a minimal model—to resolve individual nucleosomes within sub-Mb chromatin domains and phase-separated systems. To overcome the difficulty of sampling chromatin at high resolution, we devise a transferable enhanced-sampling Debye-length replica-exchange molecular dynamics approach. We find that nucleosome thermal fluctuations become significant at physiological salt concentrations and destabilize the 30-nm fiber. Our simulations show that nucleosome breathing favors stochastic folding of chromatin and promotes LLPS by simultaneously boosting the transient nature and heterogeneity of nucleosome–nucleosome contacts, and the effective nucleosome valency. Our work puts forward the intrinsic plasticity of nucleosomes as a key element in the liquid-like behavior of nucleosomes within chromatin, and the regulation of chromatin LLPS.

Promoter G-quadruplex folding precedes transcription and is controlled by chromatin

BackgroundFour-stranded G-quadruplexes (G4s) are DNA secondary structures in the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of promoter G4s, transcription and chromatin state.ResultsTranscriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed by G4 ChIP-seq, does not depend on transcriptional activity. We then show that chromatin compaction can lead to loss of promoter G4s and is accompanied by a corresponding loss of RNA polymerase II (Pol II), thus establishing a link between G4 formation and chromatin accessibility. Furthermore, pre-treatment of cells with a G4-stabilising ligand mitigates the loss of Pol II at promoters induced by chromatin compaction.ConclusionsOverall, our findings show that G4 folding is coupled to the establishment of accessible chromatin and does not require active transcription.

Chromatin tracing and multiplexed imaging of nucleome architectures (MINA) and RNAs in single mammalian cells and tissue.

The genome is hierarchically organized into several 3D architectures, including chromatin loops, domains, compartments and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization varies in different cell types in mammalian tissue and how organization affects gene expression remains a major challenge. Previous approaches have been limited by a lack of capacity to directly trace chromatin folding in 3D and to simultaneously measure genomic organization in relation to other nuclear components and gene expression in the same single cells. We have developed an image-based 3D genomics technique termed 'chromatin tracing', which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. More recently, we also developed multiplexed imaging of nucleome architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli and copy numbers of numerous RNA species in the same single cells in mammalian tissue. Here, we provide detailed protocols for chromatin tracing in cell lines and MINA in mammalian tissue, which take 3-4 d for experimental work and 2-3 d for data analysis. We expect these developments to be broadly applicable and to affect many lines of research on 3D genomics by depicting multiscale genomic architectures associated with gene expression, in different types of cells and tissue undergoing different biological processes.

Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV. SETDB1 coordinates histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

Interactions Between Nucleosomes: From Atomistic Simulation to Polymer Model.

The organization of genomes in space and time dimension plays an important role in gene expression and regulation. Chromatin folding occurs in a dynamic, structured way that is subject to biophysical rules and biological processes. Nucleosomes are the basic unit of chromatin in living cells, and here we report on the effective interactions between two nucleosomes in physiological conditions using explicit-solvent all-atom simulations. Free energy landscapes derived from umbrella sampling simulations agree well with recent experimental and simulation results. Our simulations reveal the atomistic details of the interactions between nucleosomes in solution and can be used for constructing the coarse-grained model for chromatin in a bottom-up manner.

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.

Liquid–liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

RNA Biogenesis Instructs Functional Inter-Chromosomal Genome Architecture.

Three-dimensional (3D) genome organization has emerged as an important layer of gene regulation in development and disease. The functional properties of chromatin folding within individual chromosomes (i.e., intra-chromosomal or in cis) have been studied extensively. On the other hand, interactions across different chromosomes (i.e., inter-chromosomal or in trans) have received less attention, being often regarded as background noise or technical artifacts. This viewpoint has been challenged by emerging evidence of functional relationships between specific trans chromatin interactions and epigenetic control, transcription, and splicing. Therefore, it is an intriguing possibility that the key processes involved in the biogenesis of RNAs may both shape and be in turn influenced by inter-chromosomal genome architecture. Here I present the rationale behind this hypothesis, and discuss a potential experimental framework aimed at its formal testing. I present a specific example in the cardiac myocyte, a well-studied post-mitotic cell whose development and response to stress are associated with marked rearrangements of chromatin topology both in cis and in trans. I argue that RNA polymerase II clusters (i.e., transcription factories) and foci of the cardiac-specific splicing regulator RBM20 (i.e., splicing factories) exemplify the existence of trans-interacting chromatin domains (TIDs) with important roles in cellular homeostasis. Overall, I propose that inter-molecular 3D proximity between co-regulated nucleic acids may be a pervasive functional mechanism in biology.

Global Landscape of 3D Architectural Anchors from Genome-Wide Analysis of Specific Chromatin Interactions

Chromatin folding and interactions play essential roles in fundamental processes such as regulation of gene expression and cellular specialization. While Hi-C measurements often detect numerous interactions (∼108), our previous results showed that only 2-9% of Hi-C interactions, which we call specific interactions, are required to generate ensembles of 3D single-cell chromatin conformations that collectively exhibit the observed Hi-C contact patterns. In this study, we carried out a genome-wide comprehensive analysis of these specific interactions.

A DNA Sequence Based Polymer Model for Chromatin Folding.

The recent development of sequencing technology and imaging methods has provided an unprecedented understanding of the inter-phase chromatin folding in mammalian nuclei. It was found that chromatin folds into topological-associated domains (TADs) of hundreds of kilo base pairs (kbps), and is further divided into spatially segregated compartments (A and B). The compartment B tends to be located near to the periphery or the nuclear center and interacts with other domains of compartments B, while compartment A tends to be located between compartment B and interacts inside the domains. These spatial domains are found to highly correlate with the mosaic CpG island (CGI) density. High CGI density corresponds to compartments A and small TADs, and vice versa. The variation of contact probability as a function of sequential distance roughly follows a power-law decay. Different chromosomes tend to segregate to occupy different chromosome territories. A model that can integrate these properties at multiple length scales and match many aspects is highly desired. Here, we report a DNA-sequence based coarse-grained block copolymer model that considers different interactions between blocks of different CGI density, interactions of TAD formation, as well as interactions between chromatin and the nuclear envelope. This model captures the various single-chromosome properties and partially reproduces the formation of chromosome territories.

Nuclear IL-33 restrains the early conversion of fibroblasts to an extracellular matrix-secreting phenotype

Interleukin (IL)-33 is a cytokine that appears to mediate fibrosis by signaling via its receptor ST2 (IL-33R/IL1RL1). It is also, however, a protein that after synthesis is sorted to the cell nucleus, where it appears to affect chromatin folding. Here we describe a novel role for nuclear IL-33 in regulating the fibroblast phenotype in murine kidney fibrosis driven by unilateral ureteral obstruction. Transcriptional profiling of IL-33-deficient kidneys 24 h after ligation revealed enhanced expression of fibrogenic genes and enrichment of gene sets involved in extracellular matrix formation and remodeling. These changes relied on intracellular effects of IL-33, because they were not reproduced by treatment with a neutralizing antibody to IL-33 that prevents IL-33R/ST2L receptor signaling nor were they observed in IL-33R/ST2-deficient kidneys. To further explore the intracellular function of IL-33, we established transcription profiles of human fibroblasts, observing that knockdown of IL-33 skewed the transcription profile from an inflammatory towards a myofibroblast phenotype, reflected in higher levels of COL3A1, COL5A1 and transgelin protein, as well as lower expression levels of IL6, CXCL8, CLL7 and CCL8. In conclusion, our findings suggest that nuclear IL-33 in fibroblasts dampens the initial profibrotic response until persistent stimuli, as enforced by UUO, can override this protective mechanism.

High-resolution single-cell 3D-models of chromatin ensembles during Drosophila embryogenesis

Single-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5–6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.

Order and stochasticity in the folding of individual Drosophila genomes

Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.

New insights on epigenetic mechanisms supporting axonal development: histone marks and miRNAs.

Mechanisms supporting axon growth and the establishment of neuronal polarity have remained largely disconnected from their genetic and epigenetic fundamentals. Recently, post-transcriptional modifications of histones involved in chromatin folding and transcription, and microRNAs controlling translation have emerged as regulators of axonal specification, growth, and guidance. In this article, we review novel evidence supporting the concept that epigenetic mechanisms work at both transcriptional and post-transcriptional levels to shape axons. We also discuss the role of splicing on axonal growth, as one of the most (if not the most) powerful post-transcriptional mechanism to diversify genetic information. Overall, we think exploring the gap between epigenetics and axonal growth raises new questions and perspectives to the development of axons in physiological and pathological contexts.