De novo induction of a DNA-histone H3K9 methylation loop on synthetic human repetitive DNA in cultured tobacco cells.

Genetic modifications in plants are crucial tools for fundamental and applied research. Transgene expression usually varies among independent lines or their progeny and is associated with the chromatin structure of the insertion site. Strategies based on understanding how to manipulate the epigenetic state of the inserted gene cassette would help to ensure transgene expression. Here, we report a strategy for chromatin manipulation by the artificial tethering of epigenetic effectors to a synthetic human centromeric repetitive DNA (alphoid DNA) platform in plant Bright-Yellow-2 (BY-2) culture cells. By tethering DNA-methyltransferase (Nicotiana tabacum DRM1), we effectively induced DNA methylation and histone methylation (H3K9me2) on the alphoid DNA platform. Tethering of the Arabidopsis SUVH9, which has been reported to lack histone methyltransferase activity, also induced a similar epigenetic state on the alphoid DNA in BY-2 cells, presumably by activating the RNA-dependent DNA methylation (RdDM) pathway. Our results emphasize that the interplay between DNA and histone methylation mechanisms is intrinsic to plant cells. We also found that once epigenetic modification states were induced by the tethering of either DRM1 or SUVH9, the modification was maintained even when the direct tethering of the effector was inhibited. Our system enables the analysis of more diverse epigenetic effectors and will help to elucidate the chromatin assembly mechanisms of plant cells.

CENP-B creates alternative epigenetic chromatin states permissive for CENP-A or heterochromatin assembly.

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce de novo CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites. Excessive heterochromatin invasion into centromere chromatin suppresses CENP-A assembly. It is unclear how CENP-B controls such different chromatin states. Here, we show that the CENP-B acidic domain recruits histone chaperones and many chromatin modifiers, including the H3K36 methylase ASH1L, as well as the heterochromatin components Suv39h1 and HP1 (HP1α, ß and γ, also known as CBX5, CBX1 and CBX3, respectively). ASH1L facilitates the formation of open chromatin competent for CENP-A assembly on alphoid DNA. These results indicate that CENP-B is a nexus for histone modifiers that alternatively promote or suppress CENP-A assembly by mutually exclusive mechanisms. Besides the DNA-binding domain, the CENP-B acidic domain also facilitates CENP-A assembly de novo on transfected alphoid DNA. CENP-B therefore balances CENP-A assembly and heterochromatin formation on satellite DNA.

Mass spectrometry-based metabolomics to identify taurine-modified metabolites in heart.

Taurine is an abundant beta-amino acid found in high concentration in mammalian tissues. Taurine possesses many beneficial functions in mammalian cells. There are also a variety of taurine-conjugated products formed between taurine and bile acids, fatty acids, chloramine, mitochondrial tRNA, etc., and some of these have been identified as functional compounds. In the present study, we identified taurine-conjugated metabolites using LC-MS-based metabolome analysis of heart extracts prepared from hearts of wild-type and taurine transporter-knockout (TauTKO) mice, the latter being severely taurine deficient. Comparison analysis of metabolites identified taurine-containing dipeptides, including glutamyltaurine, aspartyltaurine, isoleucyltaurine, and leucyltaurine, which are present in wild-type but not TauTKO hearts. Acyltaurines (taurine-conjugated fatty acids) and taurine-conjugated bile acids were also detected, with levels unchanged in the TauTKO heart in comparison to the wild-type heart. These results demonstrate that taurine exists not only in the standard free form within the heart, but also in multiple conjugated forms, whose functions in the heart remain to be discovered.

Introduction of a long synthetic repetitive DNA sequence into cultured tobacco cells.

Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.

Combination of CENP-B Box Positive and Negative Synthetic Alpha Satellite Repeats Improves De Novo Human Artificial Chromosome Formation.

Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin.

Genome features of common vetch (Vicia sativa) in natural habitats.

Wild plants are often tolerant to biotic and abiotic stresses in their natural environments, whereas domesticated plants such as crops frequently lack such resilience. This difference is thought to be due to the high levels of genome heterozygosity in wild plant populations and the low levels of heterozygosity in domesticated crop species. In this study, common vetch (Vicia sativa) was used as a model to examine this hypothesis. The common vetch genome (2n = 14) was estimated as 1.8 Gb in size. Genome sequencing produced a reference assembly that spanned 1.5 Gb, from which 31,146 genes were predicted. Using this sequence as a reference, 24,118 single nucleotide polymorphisms were discovered in 1243 plants from 12 natural common vetch populations in Japan. Common vetch genomes exhibited high heterozygosity at the population level, with lower levels of heterozygosity observed at specific genome regions. Such patterns of heterozygosity are thought to be essential for adaptation to different environments. The resources generated in this study will provide insights into de novo domestication of wild plants and agricultural enhancement.

Analysis of Complex DNA Rearrangements during Early Stages of HAC Formation.

Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

Metabolite annotations based on the integration of mass spectral information.

A large number of metabolites are found in each plant, most of which have not yet been identified. Development of a methodology is required to deal systematically with unknown metabolites, and to elucidate their biological roles in an integrated 'omics' framework. Here we report the development of a 'metabolite annotation' procedure. The metabolite annotation is a process by which structures and functions are inferred for metabolites. Tomato (Solanum lycopersicum cv. Micro-Tom) was used as a model for this study using LC-FTICR-MS. Collected mass spectral features, together with predicted molecular formulae and putative structures, were provided as metabolite annotations for 869 metabolites. Comparison with public databases suggests that 494 metabolites are novel. A grading system was introduced to describe the evidence supporting the annotations. Based on the comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that are frequently found in tomato fruit tissues, and predicted novel metabolic pathways for flavonoids and glycoalkaloids. These results demonstrate that metabolite annotation facilitates the systematic analysis of unknown metabolites and biological interpretation of their relationships, which provide a basis for integrating metabolite information into the system-level study of plant biology.

KAGIANA: an excel-based tool for retrieving summary information on Arabidopsis genes.

Various public databases provide Arabidopsis gene information via the internet. It is useful to abstract information obtained from such databases. We have developed the KAGIANA tool, which allows a user to retrieve summary information obtained from selective databases and to access pages for a gene of interest in those databases. The tool is based on Microsoft Excel and provides several macro programs for gene expression analyses. It can assist plant biologists in accessing omics information for plant biology. The KAGIANA tool is freely available at http://pmnedo.kazusa.or.jp/kagiana/.

PCR detection of type I polyketide synthase genes in myxobacteria.

The diversity of type I modular polyketide synthase (PKS) was explored by PCR amplification of DNA encoding ketosynthase and acyltransferase domains in myxobacteria. The sequencing of the amplicons revealed that many PKS genes were distantly related to the published sequences. Thus, myxobacteria may be excellent resources for novel and diverse polyketides.

Dikaryotic cell division of the fission yeast Schizosaccharomyces pombe.

Dikaryons, cells with two haploid nuclei contributed by the members of a mating pair, are part of the life cycle of many filamentous fungi, but the molecular mechanisms underlying the division of dikaryons are largely unknown. We found that the fission yeast Schizosaccharomyces pombe has a latent ability to divide as a dikaryon. Cells capable of restarting the mitotic cycle with two nuclei were prepared by transient inactivation of the septation initiation network. Close pairing of the two nuclei before mitosis was dependent on minus-end-directed kinesin Klp2p and was essential for propagation as a dikaryon. The two spindles extended in opposite directions, keeping their old spindle pole bodies at the prospective site of cell division until the mid-anaphase. The spindles then overlapped, exchanging the inner nuclei. Finally, twin mitosis was followed by a single cytokinesis, producing two daughter dikaryons carrying copies of the original pair of nuclei.

Inter-subspecies hybrid dikaryons of oyster mushroom independently isolated in Vietnam and Japan.

Two strains of the mushroom Pleurotus, isolated from nature in Vietnam and Japan, contained a similar combination of two distinct rDNA internal transcribed spacer (ITS) sequences. They were perhaps hybrid dikaryons between P. cystidiosus subsp. abalonus and a novel P. cystidiosus subspecies. These mushrooms produce dikaryotic arthroconidia. This unique asexual reproduction might allow stable maintenance of a particular pair of nuclei.

Lub1 participates in ubiquitin homeostasis and stress response via maintenance of cellular ubiquitin contents in fission yeast.

Ubiquitin-dependent proteolysis plays a pivotal role in stress responses. To investigate the mechanisms of these cellular processes, we have been studying Schizosaccharomyces pombe mutants that have altered sensitivities to various stress conditions. Here, we showed that Lub1, a homologue of Ufd3p/Zzz4p/Doa1p in budding yeast, is involved in the regulation of ubiquitin contents. Disruption of the lub1+ gene resulted in monoubiquitin as well as multiubiquitin depletion without change in mRNA level and in hypersensitivity to various stress conditions. Consistently, overexpression of genes encoding ubiquitin suppressed the defects associated with lub1 mutation, indicating that the phenotypes of the lub1 mutants under stress conditions were due to cellular ubiquitin shortage at the posttranscriptional level. In addition, the lub1-deleted cells showed aberrant functions in ubiquitin/proteasome-dependent proteolysis, with accelerated degradation of ubiquitin. Also Cdc48, a stress-induced chaperon-like essential ATPase, was found to interact with Lub1, and this association might contribute to the stabilization of Lub1. Our results indicated that Lub1 is responsible for ubiquitin homeostasis at the protein level through a negative regulation of ubiquitin degradation.

The 14-kDa dynein light chain-family protein Dlc1 is required for regular oscillatory nuclear movement and efficient recombination during meiotic prophase in fission yeast.

A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1 loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.

Microtubule-organizing center formation at telomeres induces meiotic telomere clustering.

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering requires the interaction of telomeres with the nuclear membrane proteins SUN (Sad1/UNC-84) and KASH (Klarsicht/ANC-1/Syne homology). The mechanism by which telomeres gather remains elusive. In this paper, we show that telomere clustering in fission yeast depends on microtubules and the microtubule motors, cytoplasmic dynein, and kinesins. Furthermore, the γ-tubulin complex (γ-TuC) is recruited to SUN- and KASH-localized telomeres to form a novel microtubule-organizing center that we termed the "telocentrosome." Telocentrosome formation depends on the γ-TuC regulator Mto1 and on the KASH protein Kms1, and depletion of either Mto1 or Kms1 caused severe telomere clustering defects. In addition, the dynein light chain (DLC) contributes to telocentrosome formation, and simultaneous depletion of DLC and dynein also caused severe clustering defects. Thus, the telocentrosome is essential for telomere clustering. We propose that telomere-localized SUN and KASH induce telocentrosome formation and that subsequent microtubule motor-dependent aggregation of telocentrosomes via the telocentrosome-nucleated microtubules causes telomere clustering.

The Sec14 family glycerophospholipid-transfer protein is required for structural integrity of the spindle pole body during meiosis in fission yeast.

The fission yeast spo20+ gene encodes a glycerophospholipid-transfer protein. spo20 mutants are unable to assemble the forespore membrane properly. Here we studied the structural integrity of the spindle pole body (SPB) in spo20-H6 mutants during meiosis. Meiotic cells expressing a GFP-tagged SPB marker protein, Spo15-GFP, showed an excess number of SPBs, some of which were not localized to the spindle poles and were termed 'pseudo-SPBs'. Formation of spindles for meiosis I was significantly delayed in spo20-H6 cells, although the morphology of spindles and segregation of the sister chromatids seemed normal. The SPB of spo20-H6 contained meiosis-specific outer plaques, though outermost layers were less evident. Time-lapse studies of spo20-H6 cells showed that the pseudo-SPBs originated from normal SPBs at the spindle poles during meiosis I. Among the SPB components tested, Spo15, Spo13, Sad1 and Cut12 were localized to the pseudo-SPBs, but Sid4 was not always present. Alp4, a component of the gamma-tubulin complex, was also present in about 40% of the pseudo-SPBs. The forespore membranes initiated from both the SPBs and the pseudo-SPBs. We conclude that Spo20 plays a role in maintaining the structural integrity of the meiotic SPB, besides supplying membrane vesicles for forespore membrane assembly.