RNAi-independent de novo DNA methylation revealed in Arabidopsis mutants of chromatin remodeling gene DDM1

Methylation of histone H3 lysine 9 (H3K9me) and small RNAs are associated with constitutively silent chromatin in diverse eukaryotes including plants. In plants, silent transposons are also marked by cytosine methylation, especially at non‐CpG sites. Transposon‐specific non‐CpG methylation in plants is controlled by small RNAs and H3K9me. Although it is often assumed that small RNA directs H3K9me, interaction between small RNA and H3K9me has not been directly demonstrated in plants. We have previously shown that a mutation in the chromatin remodeling gene DDM1 (DECREASE IN DNA METHYLATION 1) induces a global decrease but a local increase of cytosine methylation and accumulation of small RNA at a locus called BONSAI. Here we show that de novo BONSAI methylation does not depend on RNAi but does depend on H3K9me. In mutants of H3K9 methyltransferase gene KRYPTONITE or the H3K9me‐dependent DNA methyltransferase gene CHROMOMETHYALSE3, the ddm1‐induced de novo cytosine methylation was abolished for all three contexts (CpG, CpHpG and CpHpH). Furthermore, RNAi mutants showed strong developmental defects when combined with the ddm1 mutation. Our results revealed unexpected interactions of epigenetic modifications that may be conserved among diverse eukaryotes.     

Relationship between cultivar difference in the sensitivity of net photosynthesis to ozone and reactive oxygen species scavenging system in Japanese winter wheat (Triticum aestivum)

To clarify the relationship between cultivar difference in the sensitivity of net photosynthesis to ozone (O(3) ) and the reactive oxygen species (ROS) scavenging system in wheat (Triticum aestivum), we investigated the effects of chronic exposure to ambient levels of O(3) on gas exchange rates, activity and concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), activity of ROS scavenging enzymes and concentration of antioxidants of the flag leaf in two Japanese winter wheat cultivars (Norin 61 and Shirogane-komugi). Although the net photosynthetic rate of the flag leaf in Norin 61 was not significantly reduced by exposure to O(3) , that in Shirogane-komugi was significantly reduced by the exposure to O(3) during the anthesis and early grain-filling stages. In the two cultivars, stomatal diffusive conductance to H(2) O of the flag leaf was not significantly affected by the exposure to O(3) . The exposure to O(3) induced significant reductions in the activity and concentration of Rubisco, activities of catalase (CAT) and monodehydroascorbate reductase (MDAR) and concentrations of reduced form of ascorbate and total glutathione of the flag leaf in Shirogane-komugi. It was concluded that the sensitivity of net photosynthesis of flag leaf to O(3) is higher in Shirogane-komugi than in Norin 61, and the difference in the sensitivity to O(3) between the two cultivars is mainly due to that in the effects of O(3) on the detoxification ability of ROS, mainly determined by the activity of ROS scavenging enzymes, such as CAT and MDAR.     

DNA methylation in plants: relationship to small RNAs and histone modifications, and functions in transposon inactivation

DNA methylation is a type of epigenetic marking that strongly influences chromatin structure and gene expression in plants and mammals. Over the past decade, DNA methylation has been intensively investigated in order to elucidate its control mechanisms. These studies have shown that small RNAs are involved in the induction of DNA methylation, that there is a relationship between DNA methylation and histone methylation, and that the base excision repair pathway has an important role in DNA demethylation. Some aspects of DNA methylation have also been shown to be shared with mammals, suggesting that the regulatory pathways are, in part at least, evolutionarily conserved. Considerable progress has been made in elucidating the mechanisms that control DNA methylation; however, many aspects of the mechanisms that read the information encoded by DNA methylation and mediate this into downstream regulation remain uncertain, although some candidate proteins have been identified. DNA methylation has a vital role in the inactivation of transposons, suggesting that DNA methylation is a key factor in the evolution and adaptation of plants.     

Functional Redundancy in HIV-1 Viral Particle Assembly

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.     

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 2

Modification of the C-2 position of a benzofuran derivative 6 (RO-09-4609), an N-myristoyltransferase (Nmt) inhibitor, has led us to discover antifungal agents that are active in a murine systemic candidiasis model. The drug design is based on the analysis of a crystal structure of a Candida Nmt complex with 2. The optimization has been guided by various biological evaluations including a quasi in vivo assay and pharmacokinetic analysis.     

Enhanced green fluorescent protein as a useful tag for rapid identification of homozygous transgenic mice

We developed a technique that simplifies the process of confirming homozygous transgenics at preimplantation stages, which are the earliest stages used in test breeding, using enhanced green fluorescent protein as a tag. All the blastocysts obtained by mating with the combination of Tg/Tg male (homozygous for transgene) x +/+ female exhibited fluorescence.     

Common variants in the COL4A4 gene confer susceptibility to lattice degeneration of the retina

Lattice degeneration of the retina is a vitreoretinal disorder characterized by a visible fundus lesion predisposing the patient to retinal tears and detachment. The etiology of this degeneration is still uncertain, but it is likely that both genetic and environmental factors play important roles in its development. To identify genetic susceptibility regions for lattice degeneration of the retina, we performed a genome-wide association study (GWAS) using a dense panel of 23,465 microsatellite markers covering the entire human genome. This GWAS in a Japanese cohort (294 patients with lattice degeneration and 294 controls) led to the identification of one microsatellite locus, D2S0276i, in the collagen type IV alpha 4 (COL4A4) gene on chromosome 2q36.3. To validate the significance of this observation, we evaluated the D2S0276i region in the GWAS cohort and in an independent Japanese cohort (280 patients and 314 controls) using D2S0276i and 47 single nucleotide polymorphisms covering the region. The strong associations were observed in D2S0276i and rs7558081 in the COL4A4 gene (Pc = 5.8 × 10(-6), OR = 0.63 and Pc = 1.0 × 10(-5), OR = 0.69 in a total of 574 patients and 608 controls, respectively). Our findings suggest that variants in the COL4A4 gene may contribute to the development of lattice degeneration of the retina.     

Origins and Properties of Dental,Thymic, and Bone Marrow Mesenchymal Cells and Their Stem Cells

Mesenchymal cells arise from the neural crest (NC) or mesoderm. However, it is difficult to distinguish NC-derived cells from mesoderm-derived cells. Using double-transgenic mouse systems encoding P0-Cre, Wnt1-Cre, Mesp1-Cre, and Rosa26EYFP, which enabled us to trace NC-derived or mesoderm-derived cells as YFP-expressing cells, we demonstrated for the first time that both NC-derived (P0- or Wnt1-labeled) and mesoderm-derived (Mesp1-labeled) cells contribute to the development of dental, thymic, and bone marrow (BM) mesenchyme from the fetal stage to the adult stage. Irrespective of the tissues involved, NC-derived and mesoderm-derived cells contributed mainly to perivascular cells and endothelial cells, respectively. Dental and thymic mesenchyme were composed of either NC-derived or mesoderm-derived cells, whereas half of the BM mesenchyme was composed of cells that were not derived from the NC or mesoderm. However, a colony-forming unit-fibroblast (CFU-F) assay indicated that CFU-Fs in the dental pulp, thymus, and BM were composed of NC-derived and mesoderm-derived cells. Secondary CFU-F assays were used to estimate the self-renewal potential, which showed that CFU-Fs in the teeth, thymus, and BM were entirely NC-derived cells, entirely mesoderm-derived cells, and mostly NC-derived cells, respectively. Colony formation was inhibited drastically by the addition of anti-platelet–derived growth factor receptor-β antibody, regardless of the tissue and its origin. Furthermore, dental mesenchyme expressed genes encoding critical hematopoietic factors, such as interleukin-7, stem cell factor, and cysteine-X-cysteine (CXC) chemokine ligand 12, which supports the differentiation of B lymphocytes and osteoclasts. Therefore, the mesenchymal stem cells found in these tissues had different origins, but similar properties in each organ.     

Preparation of mouse monoclonal antibody for RB1CC1 and its clinical application

RB1-inducible coiled-coil 1 (RB1CC1; also known as FIP200) plays important roles in several biological pathways such as cell proliferation and autophagy. Evaluation of RB1CC1 expression can provide useful clinical information on various cancers and neurodegenerative diseases. In order to realize the clinical applications, it is necessary to establish a stable supply of antibody and reproducible procedures for the laboratory examinations. In the present study, we have generated mouse monoclonal antibodies for RB1CC1, and four kinds of antibodies (N1-8, N1-216, N3-2, and N3-42) were found to be optimal for clinical applications such as ELISA and immunoblots and work as well as the pre-existing polyclonal antibodies. N1-8 monoclonal antibody provided the best recognition of RB1CC1 in the clinico-pathological examination of formalin-fixed paraffin-embedded tissues. These monoclonal antibodies will help to generate new opportunities in scientific examinations in biology and clinical medicine.