Impacts of chromatin dynamics and compartmentalization on DNA repair.
TL;DR: In this paper, the importance of the spatial organization of chromatin and proteins for the maintenance of genome integrity is discussed, and the role of membraneless nuclear bodies and transcription in DNA repair is discussed.
About: This article is published in DNA Repair.The article was published on 2021-06-19. It has received 7 citations till now. The article focuses on the topics: Chromatin & Histone.
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TL;DR: It is found that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects, and chromatin features have little to no effect on theoxicity of a single CRISPR/Cas9-induced DSB.
Abstract: Abstract Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.
5 citations
TL;DR: In this article, a review of commonly used tools to induce DNA damage under the microscope and discuss spatio-temporal analysis tools that can bring added information regarding protein dynamics at sites of damage.
Abstract: DNA repair requires a coordinated effort from an array of factors that play different roles in the DNA damage response from recognizing and signaling the presence of a break, creating a repair competent environment, and physically repairing the lesion. Due to the rapid nature of many of these events, live-cell microscopy has become an invaluable method to study this process. In this review we outline commonly used tools to induce DNA damage under the microscope and discuss spatio-temporal analysis tools that can bring added information regarding protein dynamics at sites of damage. In particular, we show how to go beyond the classical analysis of protein recruitment curves to be able to assess the dynamic association of the repair factors with the DNA lesions as well as the target-search strategies used to efficiently find these lesions. Finally, we discuss how the use of mathematical models, combined with experimental evidence, can be used to better interpret the complex dynamics of repair proteins at DNA lesions.
3 citations
TL;DR: The findings suggest that, within statistical error margin, positioning of Drosophila TADs at the nuclear envelope does not, by itself, systematically affect the average gene expression in these T ADs, while the expected negative correlation is confirmed.
Abstract: Multiple studies have shown a correlation between gene expression and positioning of genes at nuclear envelope (NE) lined by nuclear lamina. Here, we ask whether there is a causal, systematic connection between the expression level of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probability of TADs to be found at the NE. To investigate the connection, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with the genome-wide gene expression data for both the control and lamins depleted (LD) nuclei. We analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be near the NE. Our findings suggest that, within statistical error margin, positioning of Drosophila TADs at the nuclear envelope does not, by itself, systematically affect the average gene expression in these TADs, while the expected negative correlation is confirmed. Verifiable hypotheses of the underlying mechanism for the presence of correlation without causality are discussed. We introduce a TAD length normalized metric for the average transcription activity of genes in a TAD: number of Reads mapped to all genes in a TAD Per Kilobase of TAD length per Million reads mapped to all TADs (RPKMT).
2 citations
TL;DR: In this paper , the authors summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double-strand breaks, including nucleotide modifications, and bulky lesions, among others.
1 citations
TL;DR: The investigation of these mechanisms makes a significant contribution to the understanding of those related diseases and provides new ideas for clinical treatments.
Abstract: Mitosis and meiosis are crucial life activities that transmit eukaryotic genetic information to progeny in a stable and orderly manner. The formation and appearance of chromosomes, which are derived from chromatin, are the preconditions and signs of mitosis. When entering mitosis, interphase loose chromatin is highly spiralized and folded to form compact chromosomes. In recent years, it has been found that in addition to the well-known DNA, histones, and topoisomerase, a large protein complex called condensin plays an important role in the process of chromosome formation. Numerous studies have shown that the abnormal function of condensin can lead to incomplete or excessive concentration of chromatin, as well as disorder of genome organization process, abnormal transmission of genetic information, and ultimately lead to various diseases of individual, especially in nervous system diseases. In this review, the biological function of condensin and the potential pathogenic mechanism of condensin in nervous system diseases are briefly summarized. Therefore, the investigation of these mechanisms makes a significant contribution to the understanding of those related diseases and provides new ideas for clinical treatments.
1 citations
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TL;DR: The authors' improving understanding of DNA-damage responses is providing new avenues for disease management, and these responses are biologically significant because they prevent diverse human diseases.
Abstract: The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.
4,871 citations
TL;DR: This work has shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates and has proposed a physical framework for this organizing principle.
Abstract: In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.
3,294 citations
TL;DR: The findings together suggest that several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation, and represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process.
Abstract: BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.
2,432 citations
TL;DR: It is postulated that super-enhancers are phase-separated multimolecular assemblies, also known as biomolecular condensates, which provide a means to compartmentalize and concentrate biochemical reactions within cells.
Abstract: Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here, we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets and MED1-IDR droplets can compartmentalize and concentrate transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in control of key cell identity genes.
1,506 citations
TL;DR: Recent insights are reviewed into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.
1,427 citations