Showing papers on "Chromosome published in 2016"
TL;DR: Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.
Abstract: The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.
441 citations
TL;DR: This study elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.
Abstract: Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.
371 citations
TL;DR: A crucial role is demonstrated for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC–seq) and RNA sequencing, and deletion of the boundary disrupts mega-domain formation.
Abstract: X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors.
355 citations
TL;DR: This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development.
Abstract: The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell’s chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.
324 citations
01 Jan 2016
TL;DR: A model that accounts for the generation of a wide variety of chromosomal aberrations-rearrangements, resulting from the various ways in which the overreplicated strands can undergo recombination is proposed.
Abstract: We propose that a fundamental problem in the faithful replication of complex chromosomes of higher eukaryotes is the proper control of both the number and timing of the multiple initiations of replication on single chromosomes. When replication patterns are disrupted by any of a variety of agents, overreplication of DNA can occur. We propose a model that accounts for the generation of a wide variety of chromo- somal aberrations-rearrangements, resulting from the various ways in which the overreplicated strands can undergo recom- bination. We also discuss certain implications of the generation of chromosomal alterations in higher eukaryotes as they may relate to cancer chemotherapy, cancer progression, aging, and rapid speciation-evolution.
185 citations
TL;DR: A probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells and incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states.
Abstract: Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.
175 citations
TL;DR: The data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity, which suggests ongoing chromosome instability that other platforms fail to detect.
Abstract: Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification.
167 citations
TL;DR: It is shown that chromosomal compartments, topological domains, contact insulation and long-range loops, all defined by ensemble Hi-C maps, are governed by distinct cell cycle dynamics, while DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2.
Abstract: Chromosomes in proliferating metazoan cells undergo dramatic structural metamorphoses every cell cycle, alternating between a highly condensed mitotic structure facilitating chromosome segregation, and a decondensed interphase structure accommodating transcription, gene silencing and DNA replication. These cyclical structural transformations have been evident under the microscope for over a century, but their molecular-level analysis is still lacking. Here we use single-cell Hi-C to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle.We show that chromosomal compartments, topological domains (TADs), contact insulation and long-range loops, all defined by ensemble Hi-C maps, are governed by distinct cell cycle dynamics. In particular, DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2. Analysing whole genome 3D structural models using haploid cell data, we discover a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data creates an essential new paradigm for the re-interpretation of chromosome conformation maps through the prism of the cell cycle.
162 citations
TL;DR: The structural model arising from this study provides a unique high-resolution view of the complex chromosomal architecture in senescent cells.
Abstract: Replicative cellular senescence is a fundamental biological process characterized by an irreversible arrest of proliferation. Senescent cells accumulate a variety of epigenetic changes, but the three-dimensional (3D) organization of their chromatin is not known. We applied a combination of whole-genome chromosome conformation capture (Hi-C), fluorescence in situ hybridization, and in silico modeling methods to characterize the 3D architecture of interphase chromosomes in proliferating, quiescent, and senescent cells. Although the overall organization of the chromatin into active (A) and repressive (B) compartments and topologically associated domains (TADs) is conserved between the three conditions, a subset of TADs switches between compartments. On a global level, the Hi-C interaction matrices of senescent cells are characterized by a relative loss of long-range and gain of short-range interactions within chromosomes. Direct measurements of distances between genetic loci, chromosome volumes, and chromatin accessibility suggest that the Hi-C interaction changes are caused by a significant reduction of the volumes occupied by individual chromosome arms. In contrast, centromeres oppose this overall compaction trend and increase in volume. The structural model arising from our study provides a unique high-resolution view of the complex chromosomal architecture in senescent cells.
119 citations
01 Dec 2016
109 citations
01 Jan 2016
TL;DR: The chromosome banding pattern has been analyzed in clones of mouse myeloid leukemic cells that differ in their ability to be induced to differentiate by the protein in-ducer MGI (macrophage and granulocyte inducer) as mentioned in this paper.
Abstract: The chromosome banding pattern has been analyzed in clones of mouse myeloid leukemic cells that differ in their ability to be induced to differentiate by the protein in- ducer MGI (macrophage and granulocyte inducer). None of the clones had a completely normal diploid banding pattern. The clones studied were either MGI+ (that can be induced to form Fc and C3 rosettes), a stage in the differentiation of myeloid cells, or MG1- (that cannot be induced to form these rosettes). All six cultured clones of MG1- cells from myeloid leukemias independently produced in six separate animals showed a loss of a piece of one chromosome 2 and this abnormal chromosome was maintained in leukemias derived from the cultured cells. This loss was not found in MGI+ clones or lymphoid leukemias. Five MGI+ mutants, derived from an MGI- clone with a loss of a piece of one chromosome 2, one normal chromosome 12, and two translocated chromosomes 12, maintained the abnor- mal chromosome 2 but lost either the one normal or one of these translocated chromosome 12. These results indicate that chro- mosomes 2 and 12 carry genes that control the differentiation of myeloid leukemic cells and that inducibility by MGI is con- trolled by the balance between these genes. We suggest that these chromosomes also carry genes that control the malignancy of these cells.
TL;DR: It is shown that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences.
Abstract: The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.
TL;DR: This review will consider the evidence for dosage compensation and the molecular mechanisms implicated, and several modes of dosage compensation have evolved.
TL;DR: It is found that unlike COs, NCOs are insensitive to the centromere effect and do not demonstrate interference, which has multiple implications for the understanding of how meiotic recombination is regulated to ensure proper chromosome segregation and maintain genome stability.
Abstract: A century of genetic analysis has revealed that multiple mechanisms control the distribution of meiotic crossover events. In Drosophila melanogaster, two significant positional controls are interference and the strongly polar centromere effect. Here, we assess the factors controlling the distribution of crossovers (COs) and noncrossover gene conversions (NCOs) along all five major chromosome arms in 196 single meiotic divisions to generate a more detailed understanding of these controls on a genome-wide scale. Analyzing the outcomes of single meiotic events allows us to distinguish among different classes of meiotic recombination. In so doing, we identified 291 NCOs spread uniformly among the five major chromosome arms and 541 COs (including 52 double crossovers and one triple crossover). We find that unlike COs, NCOs are insensitive to the centromere effect and do not demonstrate interference. Although the positions of COs appear to be determined predominately by the long-range influences of interference and the centromere effect, each chromosome may display a different pattern of sensitivity to interference, suggesting that interference may not be a uniform global property. In addition, unbiased sequencing of a large number of individuals allows us to describe the formation of de novo copy number variants, the majority of which appear to be mediated by unequal crossing over between transposable elements. This work has multiple implications for our understanding of how meiotic recombination is regulated to ensure proper chromosome segregation and maintain genome stability.
TL;DR: It can be concluded that, due to accumulation of repetitive DNA and transposons, the Y and W chromosomes can increase in size during the initial phase of their differentiation.
Abstract: The Y and W chromosomes of mammals and birds are known to be small because most of their genetic content degenerated and were lost due to absence of recombination with the X or Z, respectively. Thus, a picture has emerged of ever-shrinking Ys and Ws that may finally even fade into disappearance. We review here the large amount of literature on sex chromosomes in vertebrate species and find by taking a closer look, particularly at the sex chromosomes of fishes, amphibians and reptiles where several groups have evolutionary younger chromosomes than those of mammals and birds, that the perception of sex chromosomes being doomed to size reduction is incomplete. Here, sex-determining mechanisms show a high turnover and new sex chromosomes appear repeatedly. In many species, Ys and Ws are larger than their X and Z counterparts. This brings up intriguing perspectives regarding the evolutionary dynamics of sex chromosomes. It can be concluded that, due to accumulation of repetitive DNA and transposons, the Y and W chromosomes can increase in size during the initial phase of their differentiation.
TL;DR: The pseudoautosomal region (PAR) is a unique segment of sequence homology between differentiated sex chromosomes where recombination occurs during meiosis, of evolutionary, genetic and biomedical significance and a ‘research hotspot' in eutherian genomes.
Abstract: The pseudoautosomal region (PAR) is a unique segment of sequence homology between differentiated sex chromosomes where recombination occurs during meiosis. Molecular and functional properties of the PAR are distinctive from the autosomes and the remaining regions of the sex chromosomes. These include a higher rate of recombination than genome average, bias towards GC-substitutions and increased interindividual nucleotide divergence and mutations. As yet, the PAR has been physically demarcated in only 28 eutherian species representing 6 mammalian orders. Murid rodents have the smallest, gene-poorest and most diverged PARs. Other eutherian PARs are largely homologous but differ in size and gene content, being the smallest in equids and human/simian primates and much larger in other eutherians. Because pseudoautosomal genes escape X inactivation, their dosage changes with sex chromosome aneuploidies, whereas phenotypic effects of the latter depend on the size and gene content of the PAR. Thus, X monosomy is more viable in mice, humans and horses than in species with larger PARs. Presently, little is known about the functions of PAR genes in individual species, though human studies suggest their involvement in early embryonic development. The PAR is, thus, of evolutionary, genetic and biomedical significance and a 'research hotspot' in eutherian genomes.
TL;DR: How aneuploidy shapes gene expression and how it may contribute to the phenotypes of cancer cells is summarized.
Abstract: Unbalanced chromosome content, so-called aneuploidy, is a hallmark of cancer cells. Changes in the copy numbers of chromosomes or large chromosomal regions significantly alter the expression of several hundreds of genes that are gained or lost. At the same time, aneuploidy per se affects the transcription of many genes throughout the entire genome, as several pathways are activated or inhibited in response to changes in chromosome copy number. In recent years, a large amount of quantitative genome, transcriptome and proteome data has enabled comparison of the changes in gene expression observed in aneuploid cancer cells, as well as in model aneuploid cells with defined karyotypes. Here, we summarize how aneuploidy shapes gene expression and how it may contribute to the phenotypes of cancer cells.
TL;DR: Evidence is provided indicating that the genome of Wolbachia bacterial endosymbionts was horizontally transferred into a chromosome of the common pillbug Armadillidium vulgare, which resulted in this chromosome evolving as a new female (W) sex chromosome, which represents a remarkable mechanism underpinning the birth of sex chromosomes.
Abstract: Sex determination is a fundamental developmental pathway governing male and female differentiation, with profound implications for morphology, reproductive strategies, and behavior. In animals, sex differences between males and females are generally determined by genetic factors carried by sex chromosomes. Sex chromosomes are remarkably variable in origin and can differ even between closely related species, indicating that transitions occur frequently and independently in different groups of organisms. The evolutionary causes underlying sex chromosome turnover are poorly understood, however. Here we provide evidence indicating that Wolbachia bacterial endosymbionts triggered the evolution of new sex chromosomes in the common pillbug Armadillidium vulgare. We identified a 3-Mb insert of a feminizing Wolbachia genome that was recently transferred into the pillbug nuclear genome. The Wolbachia insert shows perfect linkage to the female sex, occurs in a male genetic background (i.e., lacking the ancestral W female sex chromosome), and is hemizygous. Our results support the conclusion that the Wolbachia insert is now acting as a female sex-determining region in pillbugs, and that the chromosome carrying the insert is a new W sex chromosome. Thus, bacteria-to-animal horizontal genome transfer represents a remarkable mechanism underpinning the birth of sex chromosomes. We conclude that sex ratio distorters, such as Wolbachia endosymbionts, can be powerful agents of evolutionary transitions in sex determination systems in animals.
TL;DR: This work reviews here how crossing-over occurs and how it is prevented in and around the centromeres and promises to explain how potentially deleterious crossovers are avoided in certain chromosomal regions while allowing beneficial crossover in others.
TL;DR: Surprisingly, transfer of the mobile pathogenicity chromosome always accompanied this exchange of core chromosomes and was accompanied by the loss of the homologous region from the recipient strain.
Abstract: Horizontal transfer of supernumerary or lineage-specific (LS) chromosomes has been described in a number of plant pathogenic filamentous fungi. So far it was not known whether transfer is restricted to chromosomes of certain size or properties, or whether 'core' chromosomes can also undergo horizontal transfer. We combined a directed and a non-biased approach to determine whether such restrictions exist. Selection genes were integrated into the genome of a strain of Fusarium oxysporum pathogenic on tomato, either targeted to specific chromosomes by homologous recombination or integrated randomly into the genome. By testing these strains for transfer of the marker to another strain we could confirm transfer of a previously described mobile pathogenicity chromosome. Surprisingly, we also identified strains in which (parts of) core chromosomes were transferred. Whole genome sequencing revealed that this was accompanied by the loss of the homologous region from the recipient strain. Remarkably, transfer of the mobile pathogenicity chromosome always accompanied this exchange of core chromosomes.
TL;DR: Drosophila has been a model for the study of this dosage compensation and has brought key strengths, including classical genetics, the exceptional cytology of polytene chromosomes, and more recently, comprehensive genomics, providing valuable insights into mechanisms for the establishment and maintenance of chromatin domains, and for the coordinate regulation of transcription.
Abstract: The sex chromosomes have special significance in the history of genetics. The chromosomal basis of inheritance was firmly established when Calvin Bridges demonstrated that exceptions to Mendel's laws of segregation were accompanied at the cytological level by exceptional sex chromosome segregation. The morphological differences between X and Y exploited in Bridges' experiments arose as a consequence of the evolution of the sex chromosomes. Originally a homologous chromosome pair, the degeneration of the Y chromosome has been accompanied by a requirement for increased expression of the single X chromosome in males. Drosophila has been a model for the study of this dosage compensation and has brought key strengths, including classical genetics, the exceptional cytology of polytene chromosomes, and more recently, comprehensive genomics. The impact of these studies goes beyond sex chromosome regulation, providing valuable insights into mechanisms for the establishment and maintenance of chromatin domains, and for the coordinate regulation of transcription.
TL;DR: Results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa.
Abstract: The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.
TL;DR: Advances in DNA sequencing methods and the development of high-resolution microscopy with which to analyse chromosome dynamics in live cells have been fundamental in building a more refined view of the existing links between DNA damage and micronuclei.
Abstract: Micronuclei (MN) have generally been considered a consequence of DNA damage and, as such, have been used as markers of exposure to genotoxic agents. However, advances in DNA sequencing methods and the development of high-resolution microscopy with which to analyse chromosome dynamics in live cells have been fundamental in building a more refined view of the existing links between DNA damage and micronuclei. Here, we review recent progress indicating that defects of micronuclei affect basic nuclear functions, such as DNA repair and replication, generating massive damage in the chromatin of the MN. In addition, the physical isolation of chromosomes within MN offers an attractive mechanistic explanation for chromothripsis, a massive local DNA fragmentation that produces complex rearrangements restricted to only one or a few chromosomes. When micronuclear chromatin is reincorporated in the daughter cell nuclei, the under-replicated, damaged or rearranged micronuclear chromatin might contribute to genome instability. The traditional conception of micronuclei has been overturned, as they have evolved from passive indicators of DNA damage to active players in the formation of DNA lesions, thus unravelling previously unforeseen roles of micronuclei in the origins of chromosome instability.
TL;DR: Associations between heterochromatic regions are a major component of the chromosome conformation in Neurospora, but two widely studied keyheterochromatin proteins are not necessary, implying that undefined protein factors play key roles in maintaining overall chromosome organization.
Abstract: Eukaryotic genomes are organized into chromatin domains with three-dimensional arrangements that presumably result from interactions between the chromatin constituents-proteins, DNA, and RNA-within the physical constraints of the nucleus. We used chromosome conformation capture (3C) followed by high-throughput sequencing (Hi-C) with wild-type and mutant strains of Neurospora crassa to gain insight into the role of heterochromatin in the organization and function of the genome. We tested the role of three proteins thought to be important for establishment of heterochromatin, namely, the histone H3 lysine 9 methyltransferase DIM-5, Heterochromatin Protein 1 (HP1), which specifically binds to the product of DIM-5 (trimethylated H3 lysine 9 [H3K9me3]), and DIM-3 (importin alpha), which is involved in DIM-5 localization. The average genome configuration of the wild-type strain revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, subtelomeres, and interspersed heterochromatin. Surprisingly, loss of either H3K9me3 or HP1 had only mild effects on heterochromatin compaction, whereas dim-3 caused more drastic changes, specifically decreasing interactions between constitutive heterochromatic domains. Thus, associations between heterochromatic regions are a major component of the chromosome conformation in Neurospora, but two widely studied key heterochromatin proteins are not necessary, implying that undefined protein factors play key roles in maintaining overall chromosome organization.
TL;DR: This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.
Abstract: The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.
TL;DR: The origin of mitochondria was the origin of vertical lineage inheritance, and sex was required to keep vertically evolving lineages viable by rescuing the incipient eukaryotic lineage from Muller’s ratchet.
Abstract: Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically evolving lineages viable by rescuing the incipient eukaryotic lineage from Muller's ratchet. The origin of mitochondria was, in this view, the decisive incident that precipitated symbiosis-specific cell biological problems, the solutions to which were the salient features that distinguish eukaryotes from prokaryotes: A nuclear membrane, energetically affordable ATP-dependent protein-protein interactions in the cytosol, and a cell cycle involving reduction division and reciprocal recombination (sex).
TL;DR: DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories, suggesting differences in the structures of these regions.
Abstract: Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.
TL;DR: The data provide the first documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome and highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.
Abstract: Recognition of the X chromosome by the dosage compensation complex in Drosophila relies on the sequence and shape of PionX sites. In male Drosophila, the male-specific lethal dosage compensation complex (MSL-DCC) discriminates the X chromosome from autosomes and doubles the transcription output of the X chromosome selectively. Some sequence elements are known to be involved in targeting the DCC to the X chromosome, but it has not been clear how X-chromosomal sequence elements are selected from the thousands of similar sequences in the genome. Here, Peter Becker and colleagues show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. The MSL2 subunit uses two distinct DNA interaction surfaces to distinguish a subset of MSL2 binding sites — termed PionX — which are defined not only by additional sequence features but also by a distinct DNA conformation (base roll). These sites originated early during X chromosome evolution. The results are an example of how transcription factors can distinguish a minority of functional DNA elements from a large pool of similar but non-functional sequences. The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known1. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power2,3. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further4. Here, using an in vitro genome-wide DNA binding assay, we show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces—the CXC and proline/basic-residue-rich domains—to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which we name PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. Our data provide the first, to our knowledge, documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.
TL;DR: To investigate the origin of these B chromosomes, a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing showed that B chromosomes have not yet reached the Paranapanema River basin and are mitotically unstable.
Abstract: B chromosomes constitute a heterogeneous mixture of genomic parasites that are sometimes derived intraspecifically from the standard genome of the host species, but result from interspecific hybridization in other cases. The mode of origin determines the DNA content, with the B chromosomes showing high similarity with the A genome in the first case, but presenting higher similarity with a different species in the second. The characid fish Moenkhausia sanctaefilomenae harbours highly invasive B chromosomes, which are present in all populations analyzed to date in the Parana and Tiete rivers. To investigate the origin of these B chromosomes, we analyzed two natural populations: one carrying B chromosomes and the other lacking them, using a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing (Illumina HiSeq2000). Our results showed that i) B chromosomes have not yet reached the Paranapanema River basin; ii) B chromosomes are mitotically unstable; iii) there are two types of B chromosomes, the most frequent of which is lightly C-banded (similar to euchromatin in A chromosomes) (B1), while the other is darkly C-banded (heterochromatin-like) (B2); iv) the two B types contain the same tandem repeat DNA sequences (18S ribosomal DNA, H3 histone genes, MS3 and MS7 satellite DNA), with a higher content of 18S rDNA in the heterochromatic variant; v) all of these repetitive DNAs are present together only in the paracentromeric region of autosome pair no. 6, suggesting that the B chromosomes are derived from this A chromosome; vi) the two B chromosome variants show MS3 sequences that are highly divergent from each other and from the 0B genome, although the B2-derived sequences exhibit higher similarity with the 0B genome (this suggests an independent origin of the two B variants, with the less frequent, B2 type presumably being younger); and vii) the dN/dS ratio for the H3.2 histone gene is almost 4-6 times higher for B chromosomes than for A chromosome sequences, suggesting that purifying selection is relaxed for the DNA sequences located on the B chromosomes, presumably because they are mostly inactive.
TL;DR: In the presence of maize B chromosome, the transcription of A genome genes was altered, with more impact by the increase of the B chromosome number, and the B-located transcriptionally active genes showed high similarity to their A-genome homologues, and retrotransposons on B chromosome also have partial homology to A genome sequences.
Abstract: The dispensable maize (Zea mays L.) B chromosome is highly heterochromatic and widely believed to be devoid of functional genes. Although low-copy B chromosome causes no obvious phenotype variation, its existence might influence A genome gene expression. Previous studies suggested that B chromosomes are evolved from standard chromosomes; therefore, they might contain genic regions showing homology with A chromosome sequences. Our data suggested that maize B chromosome influences the A-genome transcription with stronger effect associated with an increase in copy number of B chromosome. In total 130 differently expressed genes were detected in comparison between with and without B chromosome lines. These differentially expressed genes are mainly involved in cell metabolism and nucleotide binding. Using Starter + B, we amplified ten B chromosome loci with high sequence similarity to A-genome genes. Fluorescence in situ hybridization (FISH) confirmed that at least four ~5 kb-sized genes are located on the B chromosome. In addition, through de novo assembly of the reads not unmapped to maize B73 reference genome together with PCR validation, we found three B-located LTR; in particular, one of them, the 3.2 kb comp75688, is expressed in a B-dosage dependent manner. We found that in the presence of maize B chromosome, the transcription of A genome genes was altered, with more impact by the increase of the B chromosome number. The B-located transcriptionally active genes showed high similarity to their A-genome homologues, and retrotransposons on B chromosome also have partial homologous to A genome sequences. Our data shed more lights on the genome structure and evolution of the maize B chromosome.