Chromosome Organization Research Articles

MksB is a novel mycobacterial condensin that orchestrates spatiotemporal positioning of replication machinery

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

Fluidification of entangled polymers by loop extrusion

Loop extrusion is one of the main processes shaping chromosome organization across the cell cycle, yet its role in regulating deoxyribonucleic acid (DNA) entanglement and nucleoplasm viscoelasticity remains overlooked. We simulate entangled solutions of linear polymers under the action of generic loop extruding factors (LEFs) with a model that fully accounts for topological constraints and LEF-DNA uncrossability. We discover that extrusion drives the formation of bottlebrushlike structures which significantly lower the entanglement and effective viscosity of the system through an active fluidification mechanism. Interestingly, this fluidification displays an optimum at one LEF every 300–3000 base pairs. In marked contrast with entangled linear chains, the viscosity of extruded chains scales linearly with polymer length, yielding up to 1000-fold fluidification in our system. Our results illuminate how intrachain loop extrusion contributes to actively modulate genome entanglement and viscoelasticity . Published by the American Physical Society 2024

Species of the Sections Hedysarum and Multicaulia of the Genus Hedysarum (Fabaceae): Taxonomy, Distribution, Chromosomes, Genomes, and Phylogeny.

The genus Hedysarum L. (Fabaceae) includes about 200 species of annual and perennial herbs distributed in Asia, Europe, North Africa, and North America. Many species of this genus are valuable medicinal, melliferous, and forage resources. In this review, we consider the taxonomic history of the genus Hedysarum, the chromosomal organization of the species from the sections Hedysarum and Multicaulia, as well as phylogenetic relationships between these sections. According to morphological, genetic, and phylogenetic data, the genus Hedysarum is divided into three main sections: Hedysarum (= syn. Gamotion), Multicaulia, and Stracheya. In species of this genus, two basic chromosome numbers, x = 7 (section Hedysarum) and x = 8 (sections Multicaulia and Stracheya), were determined. The systematic positions of some species within the sections are still uncertain due to their morphological similarities. The patterns of distribution of molecular chromosomal markers (45S rDNA, 5S rDNA, and different satellite DNAs) in karyotypes of various Hedysarum species made it possible to determine their ploidy status and also specify genomic relationships within the sections Hedysarum and Multicaulia. Recent molecular phylogenetic studies clarified significantly the taxonomy and evolutionary development of the genus Hedysarum.

Chromosome organization from the Ice Age.

Chromosome organization from the Ice Age.

Unique territorial and compartmental organization of chromosomes in the holocentric silkmoth.

The hallmarks of chromosome organization in multicellular eukaryotes are chromosome territories (CT), chromatin compartments, and insulated domains, including topologically associated domains (TADs). Yet, most of these elements of chromosome organization are derived from analyses of a limited set of model organisms, while large eukaryotic groups, including insects, remain mostly unexplored. Here we combine Hi-C, biophysical modeling, and microscopy to characterize the 3D genome architecture of the silkmoth, Bombyx mori. In contrast to other eukaryotes, B. mori chromosomes form highly separated territories. Similar to other eukaryotes, B. mori chromosomes segregate into active A and inactive B compartments, yet unlike in vertebrate systems, contacts between euchromatic A regions appear to be a strong driver of compartmentalization. Remarkably, we also identify a third compartment, called secluded "S," with a unique contact pattern. Each S region shows prominent short-range self-contacts and is remarkably devoid of contacts with the rest of the chromosome, including other S regions. Compartment S hosts a unique combination of genetic and epigenetic features, localizes towards the periphery of CTs, and shows developmental plasticity. Biophysical modeling reveals that the formation of such secluded domains requires highly localized loop extrusion within them, along with a low level of extrusion in A and B. Our Hi-C data supports predicted genome-wide and localized extrusion. Such a broad, non-uniform distribution of extruders has not been seen in other organisms. Overall, our analyses support loop extrusion in insects and highlight the evolutionary plasticity of 3D genome organization, driven by a new combination of known processes.

HIV-1 Vif disrupts phosphatase feedback regulation at the kinetochore, leading to a pronounced pseudo-metaphase arrest.

The human immunodeficiency virus type 1 (HIV-1) Virion Infectivity Factor (Vif) targets and degrades cellular APOBEC3 proteins, key regulators of intrinsic and innate antiretroviral immune responses, thereby facilitating HIV-1 infection. While Vif's role in degrading APOBEC3G is well-studied, Vif is also known to cause cell cycle arrest but the detailed nature of Vif's effects on the cell cycle has yet to be delineated. In this study, we employed high-temporal single-cell live imaging and super-resolution microscopy to monitor individual cells during Vif-induced cell cycle arrest. Our findings reveal that Vif does not affect the G2/M boundary as previously thought. Instead, Vif triggers a unique and robust pseudo-metaphase arrest, which is markedly distinct from the mild prometaphase arrest induced by the HIV-1 accessory protein, Vpr, known for modulating the cell cycle. During Vif-mediated arrest, chromosomes align properly to form a metaphase plate but later disassemble, resulting in polar chromosomes. Notably, unlike Vpr, Vif significantly reduces the levels of both Phosphatase 1 (PP1) and 2 (PP2) at kinetochores, which are key regulators of chromosome-microtubule interactions. These results reveal a novel function of Vif in kinetochore regulation that governs the spatial organization of chromosomes during mitosis.

Temperature regulates negative supercoils to modulate meiotic crossovers and chromosome organization.

Crossover recombination is a hallmark of meiosis that holds the paternal and maternal chromosomes (homologs) together for their faithful segregation, while promoting genetic diversity of the progeny. The pattern of crossover is mainly controlled by the architecture of the meiotic chromosomes. Environmental factors, especially temperature, also play an important role in modulating crossovers. However, it is unclear how temperature affects crossovers. Here, we examined the distribution of budding yeast axis components (Red1, Hop1, and Rec8) and the crossover-associated Zip3 foci in detail at different temperatures, and found that both increased and decreased temperatures result in shorter meiotic chromosome axes and more crossovers. Further investigations showed that temperature changes coordinately enhanced the hyperabundant accumulation of Hop1 and Red1 on chromosomes and the number of Zip3 foci. Most importantly, temperature-induced changes in the distribution of axis proteins and Zip3 foci depend on changes in DNA negative supercoils. These results suggest that yeast meiosis senses temperature changes by increasing the level of negative supercoils to increase crossovers and modulate chromosome organization. These findings provide a new perspective on understanding the effect and mechanism of temperature on meiotic recombination and chromosome organization, with important implications for evolution and breeding.

Rapid meiotic prophase chromosome movements in Arabidopsis thaliana are linked to essential reorganization at the nuclear envelope

Meiotic rapid prophase chromosome movements (RPMs) require connections between the chromosomes and the cytoskeleton, involving SUN (Sad1/UNC-84)-domain-containing proteins at the inner nuclear envelope (NE). RPMs remain significantly understudied in plants, with respect to their importance in the regulation of meiosis. Here, we demonstrate that Arabidopsis thaliana meiotic centromeres undergo rapid (up to 500 nm/s) and uncoordinated movements during the zygotene and pachytene stages. These centromere movements are not affected by altered chromosome organization and recombination but are abolished in the double mutant sun1 sun2. We also document the changes in chromosome dynamics and nucleus organization during the transition from leptotene to zygotene, including telomere attachment to SUN-enriched NE domains, bouquet formation, and nucleolus displacement, all of which were defective in sun1 sun2. These results establish A. thaliana as a model species for studying the functional implications of meiotic RPMs and demonstrate the mechanistic conservation of telomere-led RPMs in plants.

Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades’ radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

Unique territorial and compartmental organization of chromosomes in the holocentric silkmoth.

The hallmarks of chromosome organization in multicellular eukaryotes are chromosome territories (CT), chromatin compartments, and insulated domains, including topologically associated domains (TADs). Yet, most of these elements of chromosome organization are derived from analyses of a limited set of model organisms, while large eukaryotic groups, including insects, remain mostly unexplored. Here we combine Hi-C, biophysical modeling, and microscopy to characterize the 3D genome architecture of the silkworm, Bombyx mori. In contrast to other eukaryotes, B. mori chromosomes form highly separated territories. Similar to other eukaryotes, B. mori chromosomes segregate into active A and inactive B compartments, yet unlike in vertebrate systems, contacts between euchromatic A regions appear to be a strong driver of compartmentalization. Remarkably, we also identify a third compartment, called secluded S, with a unique contact pattern. Each S region shows prominent short-range self-contacts and is remarkably devoid of contacts with the rest of the chromosome, including other S regions. Compartment S hosts a unique combination of genetic and epigenetic features, localizes towards the periphery of CTs, and shows developmental plasticity. Biophysical modeling reveals that the formation of such secluded domains requires highly localized loop extrusion within them, along with a low level of extrusion in A and B. Our Hi-C data supports predicted genome-wide and localized extrusion. Such a broad, non-uniform distribution of extruders has not been seen in other organisms. Overall, our analyses support loop extrusion in insects and highlight the evolutionary plasticity of 3D genome organization, driven by a new combination of known processes.

A global transcriptional activator involved in the iron homeostasis in cyanobacteria.

Cyanobacteria use a series of adaptation strategies and a complicated regulatory network to maintain intracellular iron (Fe) homeostasis. Here, a global activator named IutR has been identified through three-dimensional chromosome organization and transcriptome analysis in a model cyanobacterium Synechocystis sp. PCC 6803. Inactivation of all three homologous IutR-encoding genes resulted in an impaired tolerance of Synechocystis to Fe deficiency and loss of the responses of Fe uptake-related genes to Fe-deplete conditions. Protein-promoter interaction assays confirmed the direct binding of IutR with the promoters of genes related to Fe uptake, and chromatin immunoprecipitation sequencing analysis further revealed that in addition to Fe uptake, IutR could regulate many other physiological processes involved in intracellular Fe homeostasis. These results proved that IutR is an important transcriptional activator, which is essential for cyanobacteria to induce Fe-deficiency response genes. This study provides in-depth insights into the complicated Fe-deficient signaling network and the molecular mechanism of cyanobacteria adaptation to Fe-deficient environments.

Heterogeneous flexibility can contribute to chromatin segregation in the cell nucleus.

The highly and slightly condensed forms of chromatin, heterochromatin and euchromatin, respectively, segregate in the cell nucleus. Heterochromatin is more abundant in the nucleus periphery. Here we study the mechanism of heterochromatin segregation by modeling interphase chromosomes as diblock ring copolymers confined in a rigid spherical shell using molecular dynamics simulations. In our model, heterochromatin and euchromatin are distinguished by their bending stiffnesses only, while an interaction potential between the spherical shell and chromatin is used to model lamin-associated proteins. Our simulations indicate that in the absence of attractive interactions between the nuclear shell and the chromatin, most heterochromatin segregates towards the nuclear interior due to the depletion of less flexible heterochromatin segments from the nuclear periphery. This inverted chromatin distribution,which is opposite to the conventional case with heterochromatin dominating at the periphery, is in accord with experimental observations in rod cells. This "inversion" is also found to be independent of the heterochromatin concentration and chromosome number. The chromatin distribution at the periphery found in vivo can be recovered by further increasing the bending stiffness of heterochromatin segments or by turning on attractive interactions between the nuclear shell and heterochromatin. Our results indicate that the bending stiffness of chromatin could be a contributor to chromosome organization along with differential effects of HP1α-driven phase segregation and of loop extruders and interactions with the nuclear envelope and topological constraints.

A role for condensin-mediator interaction in mitotic chromosomal organization.

Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensinmediated chromatin domains during mitosis and causes chromosomal segregation defects. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Furthermore, this study predicts that mediator-driven transcription of mitotically activated genes contributes to forming domain boundaries via phase separation. This study provides a novel insight into how genome-wide gene expression during mitosis is transformed into the functional chromosomal architecture suitable for chromosomal segregation.

Carica papaya L. sex chromosome review and physical mapping of the serk 2, svp-like and mdar 4 sequences

Physical mapping evidences the chromosome organization and structure. Despite the data about plant cytogenomics, physical mapping has been conducted from single-copy and/or low-copy genes for few species. Carica papaya cytogenomics has been accomplished from BAC-FISH and repeatome sequences. We aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya. The sequences were amplified and the amplicons sequenced, showing similarity in relation to serk 2, svp-like and mdar 4 genes. Carica papaya diploidy was confirmed and the mitotic chromosomes characterized. The chromosome 1 exhibited the secondary constriction pericentromeric to the centromere of the long arm. So, we concluded that it is the sex chromosomes. serk 2 was mapped in the long arm interstitial portion of the sex chromosomes, and the interphase nuclei showed two fluorescence signals. Considering these results and the sequencing data from the C. papaya sex chromosomes, svp-like and mdar 4 genes were mapped in the interstitial region of the sex chromosome long arm. Both sequences showed only one fluorescence signal in the interphase nuclei. The procedure adopted here can be reproduced for other single-copy and/or low-copy genes, allowing the construction of cytogenetic maps. In addition, we revisited the cytogenomics data about C. papaya sex chromosomes, presenting a revised point of view about the structure and evolution to these chromosomes.

Tracing the Chromatin: From 3C to Live-Cell Imaging.

Chromatin organization plays a key role in gene regulation throughout the cell cycle. Understanding the dynamics governing the accessibility of chromatin is crucial for insight into mechanisms of gene regulation, DNA replication, and cell division. Extensive research has been done to track chromatin dynamics to explain how cells function and how diseases develop, in the hope of this knowledge leading to future therapeutics utilizing proteins or drugs that modify the accessibility or expression of disease-related genes. Traditional methods for studying the movement of chromatin throughout the cell relied on cross-linking spatially adjacent sections or hybridizing fluorescent probes to chromosomal loci and then constructing dynamic models from the static data collected at different time points. While these traditional methods are fruitful in understanding fundamental aspects of chromatin organization, they are limited by their invasive sample preparation protocols and diffraction-limited microscope resolution. These limitations have been challenged by modern methods based on high- or super-resolution microscopy and specific labeling techniques derived from gene targeting tools. These modern methods are more sensitive and less invasive than traditional methods, therefore allowing researchers to track chromosomal organization, compactness, and even the distance or rate of chromatin domain movement in detail and real time. This review highlights a selection of recently developed methods of chromatin tracking and their applications in fixed and live cells.

Transcription regulates the spatio-temporal dynamics of genes through micro-compartmentalization

Although our understanding of the involvement of heterochromatin architectural factors in shaping nuclear organization is improving, there is still ongoing debate regarding the role of active genes in this process. In this study, we utilize publicly-available Micro-C data from mouse embryonic stem cells to investigate the relationship between gene transcription and 3D gene folding. Our analysis uncovers a nonmonotonic - globally positive - correlation between intragenic contact density and Pol II occupancy, independent of cohesin-based loop extrusion. Through the development of a biophysical model integrating the role of transcription dynamics within a polymer model of chromosome organization, we demonstrate that Pol II-mediated attractive interactions with limited valency between transcribed regions yield quantitative predictions consistent with chromosome-conformation-capture and live-imaging experiments. Our work provides compelling evidence that transcriptional activity shapes the 4D genome through Pol II-mediated micro-compartmentalization.

Principles of bacterial genome organization, a conformational point of view.

Bacterial chromosomes are large molecules that need to be highly compacted to fit inside the cells. Chromosome compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, and segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (Hi-C) in combination with other "omics" approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings using Hi-C and discuss the main features of bacterial genome folding.

Spatial Chromosome Organization and Adaptation of Escherichia coli under Heat Stress.

The spatial organization of bacterial chromosomes is crucial for cellular functions. It remains unclear how bacterial chromosomes adapt to high-temperature stress. This study delves into the 3D genome architecture and transcriptomic responses of Escherichia coli under heat-stress conditions to unravel the intricate interplay between the chromosome structure and environmental cues. By examining the role of macrodomains, chromosome interaction domains (CIDs), and nucleoid-associated proteins (NAPs), this work unveils the dynamic changes in chromosome conformation and gene expression patterns induced by high-temperature stress. It was observed that, under heat stress, the short-range interaction frequency of the chromosomes decreased, while the long-range interaction frequency of the Ter macrodomain increased. Furthermore, two metrics, namely, Global Compactness (GC) and Local Compactness (LC), were devised to measure and compare the compactness of the chromosomes based on their 3D structure models. The findings in this work shed light on the molecular mechanisms underlying thermal adaptation and chromosomal organization in bacterial cells, offering valuable insights into the complex inter-relationships between environmental stimuli and genomic responses.