Organization Of Eukaryotic Genome Research Articles

Implications of the three-dimensional chromatin organization for genome evolution in a fungal plant pathogen

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly insulated in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus.

Towards unraveling the origins of eukaryotic nuclear genome organization.

With 3D genome mapping maturing over the past decade, studies exposed the differences between eukaryotic and prokaryotic genome organization. This raises the question of how the complex eukaryotic genome organization originated. Here, I explore potential pathways to answering this question, guided by our changing understanding of the origins of eukaryotes.

Three-dimensional chromatin architecture in plants – General features and novelties

Research on the three-dimensional (3D) structure of the genome and its distribution within the nuclear space has made a big leap in the last two decades. Work in the animal field has led to significant advances in our general understanding on eukaryotic genome organization. This did not only bring along insights into how the 3D genome interacts with the epigenetic landscape and the transcriptional machinery but also how 3D genome architecture is relevant for fundamental developmental processes, such as cell differentiation. In parallel, the 3D organization of plant genomes have been extensively studied, which resulted in both congruent and novel findings, contributing to a more complete view on how eukaryotic genomes are organized in multiple dimensions. Plant genomes are remarkably diverse in size, composition, and ploidy. Furthermore, as intrinsically sessile organisms without the possibility to relocate to more favorable environments, plants have evolved an elaborate epigenetic repertoire to rapidly respond to environmental challenges. The diversity in genome organization and the complex epigenetic programs make plants ideal study subjects to acquire a better understanding on universal features and inherent constraints of genome organization. Furthermore, considering a wide range of species allows us to study the evolutionary crosstalk between the various levels of genome architecture. In this article, we aim at summarizing important findings on 3D genome architecture obtained in various plant species. These findings cover many aspects of 3D genome organization on a wide range of levels, from gene loops to topologically associated domains and to global 3D chromosome configurations. We present an overview on plant 3D genome organizational features that resemble those in animals and highlight facets that have only been observed in plants to date.

Compartmentalization with nuclear landmarks yields random, yet precise, genome organization

The 3D organization of eukaryotic genomes plays an important role in genome function. While significant progress has been made in deciphering the folding mechanisms of individual chromosomes, the principles of the dynamic large-scale spatial arrangement of all chromosomes inside the nucleus are poorly understood. We use polymer simulations to model the diploid human genome compartmentalization relative to nuclear bodies such as nuclear lamina, nucleoli, and speckles. We show that a self-organization process based on a cophase separation between chromosomes and nuclear bodies can capture various features of genome organization, including the formation of chromosome territories, phase separation of A/B compartments, and the liquid property of nuclear bodies. The simulated 3D structures quantitatively reproduce both sequencing-based genomic mapping and imaging assays that probe chromatin interaction with nuclear bodies. Importantly, our model captures the heterogeneous distribution of chromosome positioning across cells while simultaneously producing well-defined distances between active chromatin and nuclear speckles. Such heterogeneity and preciseness of genome organization can coexist due to the nonspecificity of phase separation and the slow chromosome dynamics. Together, our work reveals that the cophase separation provides a robust mechanism for us to produce functionally important 3D contacts without requiring thermodynamic equilibration that can be difficult to achieve.

Tuning between Nuclear Organization and Functionality in Health and Disease.

The organization of eukaryotic genome in the nucleus, a double-membraned organelle separated from the cytoplasm, is highly complex and dynamic. The functional architecture of the nucleus is confined by the layers of internal and cytoplasmic elements, including chromatin organization, nuclear envelope associated proteome and transport, nuclear-cytoskeletal contacts, and the mechano-regulatory signaling cascades. The size and morphology of the nucleus could impose a significant impact on nuclear mechanics, chromatin organization, gene expression, cell functionality and disease development. The maintenance of nuclear organization during genetic or physical perturbation is crucial for the viability and lifespan of the cell. Abnormal nuclear envelope morphologies, such as invagination and blebbing, have functional implications in several human disorders, including cancer, accelerated aging, thyroid disorders, and different types of neuro-muscular diseases. Despite the evident interplay between nuclear structure and nuclear function, our knowledge about the underlying molecular mechanisms for regulation of nuclear morphology and cell functionality during health and illness is rather poor. This review highlights the essential nuclear, cellular, and extracellular components that govern the organization of nuclei and functional consequences associated with nuclear morphometric aberrations. Finally, we discuss the recent developments with diagnostic and therapeutic implications targeting nuclear morphology in health and disease.

Domain Model of Eukaryotic Genome Organization: From DNA Loops Fixed on the Nuclear Matrix to TADs

The article reviews the development of ideas on the domain organization of eukaryotic genome, with special attention on the studies of DNA loops anchored to the nuclear matrix and their role in the emergence of the modern model of eukaryotic genome spatial organization. Critical analysis of results demonstrating that topologically associated chromatin domains are structural-functional blocks of the genome supports the notion that these blocks are fundamentally different from domains whose existence was proposed by the domain hypothesis of eukaryotic genome organization formulated in the 1980s. Based on the discussed evidence, it is concluded that the model postulating that eukaryotic genome is built from uniformly organized structural-functional blocks has proven to be untenable.

A phylogenetic and proteomic reconstruction of eukaryotic chromatin evolution.

Histones and associated chromatin proteins have essential functions in eukaryotic genome organization and regulation. Despite this fundamental role in eukaryotic cell biology, we lack a phylogenetically-comprehensive understanding of chromatin evolution. Here, we combine comparative proteomics and genomics analysis of chromatin in eukaryotes and archaea. Proteomics uncovers the existence of histone post-translational modifications in Archaea. However, archaeal histone modifications are scarce, in contrast with the highly conserved and abundant marks we identify across eukaryotes. Phylogenetic analysis reveals that chromatin-associated catalytic functions (e.g., methyltransferases) have pre-eukaryotic origins, whereas histone mark readers and chaperones are eukaryotic innovations. We show that further chromatin evolution is characterized by expansion of readers, including capture by transposable elements and viruses. Overall, our study infers detailed evolutionary history of eukaryotic chromatin: from its archaeal roots, through the emergence of nucleosome-based regulation in the eukaryotic ancestor, to the diversification of chromatin regulators and their hijacking by genomic parasites.

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila.

Three-dimensional eukaryotic genome organization provides the structural basis for gene regulation. In Drosophila melanogaster, genome folding is characterized by somatic homolog pairing, where homologous chromosomes are intimately paired from end to end; however, how homologs identify one another and pair has remained mysterious. Recently, this process has been proposed to be driven by specifically interacting 'buttons' encoded along chromosomes. Here, we turned this hypothesis into a quantitative biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We tested our model using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. We show solid agreement between model predictions and experiments in the pairing dynamics of individual homologous loci. Our results strongly support a button-based mechanism of somatic homolog pairing in Drosophila and provide a theoretical framework for revealing the molecular identity and regulation of buttons.

The Myc‐Associated Zinc Finger Protein (MAZ) Controls Cohesin Positioning and Genome Organization Independently OR In Concert With CTCF

The Myc-associated zinc finger protein (MAZ) is a transcription factor that has been shown to regulate transcriptional initiation and termination. However, the underlying mechanisms of MAZ function in the context of chromatin is not fully understood. By applying biochemistry and genetics approaches, we show that MAZ binds to many genomic sites adjacent to the CCCTC-binding factor (CTCF), a protein that is best known for its insulation function in three-dimensional organization of eukaryotic genomes. We demonstrate that, similar to CTCF, MAZ physically interacts with the cohesin subunit Rad21. In addition, we find that MAZ shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and subsequently affects RNA alternative splicing. Our ChIP-seq analysis of the genome wide distribution of cohesins and CTCF reveals that depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ, showing that the binding of both CTCF and cohesin is more stable at the CTCF/MAZ double sites than at the sites occupied by CTCF alone. In an insulation assay like that used for CTCF, we find that binding of MAZ to sites between an enhancer and promoter results in downregulation of reporter gene expression, supporting a role for MAZ as a novel insulator protein. Hi-C analysis indicates that depletion of MAZ in cells not only disrupts local interactions within Topologically associating domains (TADs) but also reduces contacts that establish the boundaries of individual TADs. Taken together, our results suggest that MAZ shares properties with CTCF that allow it to act independently or synergistically in organizing higher order genome architectures, thereby controlling gene expression.

Genome-wide identification and expression pattern analysis of the ribonuclease T2 family in Eucommia ulmoides

The 2′,3′-cycling ribonuclease (RNase) genes are catalysts of RNA cleavage and include the RNase T2 gene family. RNase T2 genes perform important roles in plants and have been conserved in the genome of eukaryotic organisms. In this study we identified 21 EURNS genes in Eucommia ulmoides Oliver (E. ulmoides) and analyzed their structure, chromosomal location, phylogenetic tree, gene duplication, stress-related cis-elements, and expression patterns in different tissues. The length of 21 predicted EURNS proteins ranged from 143 to 374 amino acids (aa), their molecular weight (MW) ranged from 16.21 to 42.38 kDa, and their isoelectric point (PI) value ranged from 5.08 to 9.09. Two classifications (class I and class III) were obtained from the conserved domains analysis and phylogenetic tree. EURNS proteins contained a total of 15 motifs. Motif 1, motif 2, motif 3, and motif 7 were distributed in multiple sequences and were similar to the conserved domain of RNase T2. EURNS genes with similar structure and the predicted EURNS proteins with conserved motif compositions are in the same group in the phylogenetic tree. The results of RT-PCR and transcription data showed that EURNS genes have tissue-specific expression and exhibited obvious trends in different developmental stages. Gene duplication analysis results indicated that segment duplication may be the dominant duplication mode in this gene family. This study provides a theoretical basis for research on the RNase T2 gene family and lays a foundation for the further study of EURNS genes.

Polinton-like viruses are abundant in aquatic ecosystems

BackgroundPolintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited.ResultsHere, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae.ConclusionsOur study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups.4nQk6QRKxQCQHashEWMwNeVideo abstract

Hyperbolic rules of the cooperative organization of eukaryotic and prokaryotic genomes

The author's method of oligomer sums for analysis of oligomer compositions of eukaryotic and prokaryotic genomes is described. The use of this method revealed the existence of general rules for the cooperative oligomeric organization of a wide list of genomes. These rules are called hyperbolic because they are associated with hyperbolic sequences including the harmonic progression 1, 1/2, 1/3, .., 1/n. These rules are demonstrated by examples of quantitative analysis of many genomes from the human genome to the genomes of archaea and bacteria. The hyperbolic (harmonic) rules, speaking about the existence of algebraic invariants in full genomic sequences, are considered as candidates for the role of universal rules for the cooperative organization of genomes. The results concerns additionally the problem of the origin of life. The described phenomenological results were obtained as consequences of the previously published author's quantum-information model of long DNA sequences. The oligomer sums method was also applied to the analysis of long genes and viruses including the COVID-19 virus; this revealed, in characteristics of many of them, the phenomenon of such rhythmically repeating deviations from model hyperbolic sequences, which are associated with DNA triplets. In addition, an application of the oligomer sums method is shown to the analysis of amino acid sequences in long proteins like the protein Titin. The topics of the algebraic harmony in living bodies and of the quantum-information approach in biology are discussed.

Transcriptomic Profiling of Various Developmental Stages of Aphis Aurantii to Provide a Genetic Resource for Gene Expression and SSR Analysis.

The tea aphid, Aphis aurantii Boyer de Fonscolombe (Hemiptera: Aphididae), is found in regions where the tea plant grows, and has become one of the most important pests in tea gardens in the tropics and subtropics (Han et al., 2012; Deng et al., 2019). This species is also known as the black citrus aphid and destroys citrus orchards (Wang and Tsai, 2001). Ap. aurantii is a polyphagous aphid with over 190 genera hosts, including many other economically important plants in addition to tea and citrus, such as coffee, cacao, loquat, litchi, mango, and camellia (Carver, 1978; Deng et al., 2019). This aphid directly damages trees by sucking the phloem sap out of the shoot tip or from new fresh leaves and injecting its saliva, which causes phytotoxicity and stunting in the plant (Guidolin and Consoli, 2018). Moreover, this aphid secretes honeydew when sap-feeding, and sooty molds frequently grow on the honeydew, which hinders photosynthetic activity (Sevim et al., 2012). Aphids are wide-spread pests that feed on a wide range of fruits and vegetables, most of them are vectors of plant viruses (Huang and Qiao, 2014; Hulle et al., 2020). Except the tea aphid, there have been previous functional studies on genes associated with the development (Ding et al., 2017; Ye et al., 2019), reproduction (Shang et al., 2018; Ullah et al., 2019b), wing development (Shang et al., 2020b), response to the stress (Gao et al., 2018; Jing et al., 2018), and pest control (Mohammed et al., 2018; Ullah et al., 2019a) of other aphids pests (e.g., Ap. citricidus, Ap. gossypii, Acyrthosiphon pisum). Moreover, functional studies of the new insecticide targets in aphids were also conducted in these aphids (Ye et al., 2019; Shang et al., 2020a). However, most previously published papers have focused on the ecology (Alizadeh Kafeshani et al., 2018; Guidolin and Consoli, 2018; Li et al., 2019), control (Aslam et al., 2015; Gholamzadeh-Chitgar and Pourmoradi, 2017), and mitochondrial genome (Wang et al., 2019) in tea aphid Ap. aurantii. There have been few functional studies that focused on the development and reproduction of Ap. aurantii, because there are limited reference sequences. Quantitative and qualitative transcriptome analyses can reveal the integrated biochemical and physiological processes at a molecular level that are associated with specific aspects of the organism, such as identification of the critical genes during the different developmental stages in insects (Morandin et al., 2018; Liu et al., 2020). For instance, RNA-Seq was used to elucidate the underlying molecular mechanisms of metamorphic development of Henosepilachna vigintioctopunctata (Zhang et al., 2018). Using available insect genomes, comparative transcriptome analysis was conducted to analyze gene expression during all developmental stages of Zeugodacus cucurbitae (Wei et al., 2020), Bactrocera dorsalis (Liu et al., 2020). In addition, gene expression has been studied by RNA-Seq in multiple tissues to identify tissue-specific genes involved in female fertility in B. dorsalis [e.g., vitellogenin and vitelline membrane protein in female (Wei et al., 2018, 2019)]. In aphids, RNA-Seq was used to analyze the gene expression between dispersing and non-dispersing morphs (Shang et al., 2016, 2020b). Thus, RNA-Seq technology allows us to determine the gene expression to underlie certain biological functions of critical genes and identify potential targets of new environmentally friendly insecticides for pest control. Simple sequence repeats (SSRs), also known as microsatellites, are short, tandemly arranged, repeating motifs (1–6 bp), which are widely distributed throughout the genomes of eukaryotic organisms (Temnykh et al., 2001). SSRs are co-dominant, hypervariable, neutral, and reproducible molecular markers; therefore, they have become the most widely used molecular markers in population genetic and conservation studies to evaluate the level of genetic variation in a species (King, 2012). Transcriptomic sequencing is also a highly efficient approach to identify SSRs in insects with no accessible genome. This method was extensively used to identify and analyze the SSRs in Liposcelis entomophila (Wei et al., 2013) and H. vigintioctopunctata (Zhang et al., 2018). SSRs were also identified in specific tissues in B. dorsalis (Wei et al., 2015). In this study, RNA-Seq was conducted on samples from six stages of Ap. aurantii with four biological replicates. A comprehensive transcriptome was sequenced, the transcripts were de novo assembled, and the gene's functional annotation was performed. Gene expression during development and SSRs were analyzed. These results will be valuable for the future functional studies of genes involved in Ap. aurantii development, reproduction, and wing differentiation.

Weak interactions in higher-order chromatin organization.

The detailed principles of the hierarchical folding of eukaryotic chromosomes have been revealed during the last two decades. Along with structures composing three-dimensional (3D) genome organization (chromatin compartments, topologically associating domains, chromatin loops, etc.), the molecular mechanisms that are involved in their establishment and maintenance have been characterized. Generally, protein–protein and protein–DNA interactions underlie the spatial genome organization in eukaryotes. However, it is becoming increasingly evident that weak interactions, which exist in biological systems, also contribute to the 3D genome. Here, we provide a snapshot of our current understanding of the role of the weak interactions in the establishment and maintenance of the 3D genome organization. We discuss how weak biological forces, such as entropic forces operating in crowded solutions, electrostatic interactions of the biomolecules, liquid-liquid phase separation, DNA supercoiling, and RNA environment participate in chromosome segregation into structural and functional units and drive intranuclear functional compartmentalization.

Higher Order Organization of the mtDNA: Beyond Mitochondrial Transcription Factor A.

The higher order organization of eukaryotic and prokaryotic genomes is pivotal in the regulation of gene expression. Specifically, chromatin accessibility in eukaryotes and nucleoid accessibility in bacteria are regulated by a cohort of proteins to alter gene expression in response to diverse physiological conditions. By contrast, prior studies have suggested that the mitochondrial genome (mtDNA) is coated solely by mitochondrial transcription factor A (TFAM), whose increased cellular concentration was proposed to be the major determinant of mtDNA packaging in the mitochondrial nucleoid. Nevertheless, recent analysis of DNase-seq and ATAC-seq experiments from multiple human and mouse samples suggest gradual increase in mtDNA occupancy during the course of embryonic development to generate a conserved footprinting pattern which correlate with sites that have low TFAM occupancy in vivo (ChIP-seq) and tend to adopt G-quadruplex structures. These findings, along with recent identification of mtDNA binding by known modulators of chromatin accessibility such as MOF, suggest that mtDNA higher order organization is generated by cross talk with the nuclear regulatory system, may have a role in mtDNA regulation, and is more complex than once thought.

Evolution Without Genes: A Review

Evolution is a proven scientific fact. However, its mechanism is unknown. Modern Synthesis, the generally accepted theory of evolution is centered on species formation and population dynamics. According to Modern Synthesis, genes are considered the material basis of evolution. This circumstance is considered as the main obstacle in elucidating the mechanisms of evolution. It is proposed that any scientific hypothesis trying to explain the evolution of eukaryotes should meet the following requirement: all important evolutionary changes (nucleosomes, mitotic chromosomes, chromosome bands, cell nucleus, eukaryotic cells, sex, species, multicellular and homeothermic organisms, including modern humans) should occur based on one component of the genome. As is known, the genome of eukaryotic organisms consists of two components: euchromatin (coding DNA) and heterochromatin (noncoding DNA). A hypothesis is proposed that the evolution of eukaryotes occurred on the basis of a non-coding part of the genome. Therefore, with the advent of eukaryotic organisms, the material basis of evolution has evolved and noncoding DNAs have begun to play a leading role.

Organization of Chromatin by Intrinsic and Regulated Phase Separation

Eukaryotic chromatin is highly condensed but dynamically accessible to regulation and organized into subdomains. We demonstrate that reconstituted chromatin undergoes histone tail-driven liquid-liquid phase separation (LLPS) in physiologic salt and when microinjected into cell nuclei, producing dense and dynamic droplets. Linker histone H1 and internucleosome linker lengths shared across eukaryotes promote phase separation of chromatin, tune droplet properties, and coordinate to form condensates of consistent density in manners that parallel chromatin behavior in cells. Histone acetylation by p300 antagonizes chromatin phase separation, dissolving droplets invitro and decreasing droplet formation in nuclei. In the presence of multi-bromodomain proteins, such as BRD4, highly acetylated chromatin forms a new phase-separated state with droplets of distinct physical properties, which can be immiscible with unmodified chromatin droplets, mimicking nuclear chromatin subdomains. Our data suggest a framework, based on intrinsic phase separation of the chromatin polymer, for understanding the organization and regulation of eukaryotic genomes.

C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries.

Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods.

Investigation of Latent Periodicity Phenomenon in the Genomes of Eukaryotic Organisms

Investigation of Latent Periodicity Phenomenon in the Genomes of Eukaryotic Organisms

DOT1L and H3K79 Methylation in Transcription and Genomic Stability.

The organization of eukaryotic genomes into chromatin provides challenges for the cell to accomplish basic cellular functions, such as transcription, DNA replication and repair of DNA damage. Accordingly, a range of proteins modify and/or read chromatin states to regulate access to chromosomal DNA. Yeast Dot1 and the mammalian homologue DOT1L are methyltransferases that can add up to three methyl groups to histone H3 lysine 79 (H3K79). H3K79 methylation is implicated in several processes, including transcription elongation by RNA polymerase II, the DNA damage response and cell cycle checkpoint activation. DOT1L is also an important drug target for treatment of mixed lineage leukemia (MLL)-rearranged leukemia where aberrant transcriptional activation is promoted by DOT1L mislocalisation. This review summarizes what is currently known about the role of Dot1/DOT1L and H3K79 methylation in transcription and genomic stability.