Satoru Fujimoto

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.

Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces

We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions. AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region. Approximately 30 paralogues were identified in the Arabidopsis genome. Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif. Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding. AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin. Biochemically, AHL1 was also found in the nuclear matrix fraction. In the M phase, AHL1 was localized on the chromosomal surface. The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization. Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain. In addition, AHL1 is located on the surface of chromosomes during mitosis.

Genome and Chromosome Dimensions of Lotus japonicus

Lotus Japonicus, Miyakojima MG-20 and Gifu B-129. The genome sizes of Miyakojima and Gifu were determined as 472.1 and 442.8 Mbp, respectively. Both the accessions were diploid (2n=12) and six chromosomes were identified and characterized based on the condensation patterns and the locations of rDNA loci. The obvious polymorphism observed in the genome size and the chromosome morphology between the two accessions, revealed specific accumulation of heterochromatin in Miyakojima or elimination in Gifu. The chromosomes L. japonicus were numbered according to their length. A quantitative chromosome map was also developed by the imaging methods using the digital data of the condensation pattern. 45S rDNA loci were localized on chromosomes A and F, and 5S rDNA locus was localized on chromosome A by fluorescence in situ hybridization (FISH). Identification of the chromosome and genome sizes and development of the quantitative chromosome map represent significant contribution to the L. japonicus genome project as the basic information.

Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana

Highlight Transcription activator-like effectors fused to fluorescent proteins can visualize repetitive genomic sequences including centromere, telomere, and rDNA sequences for analysing chromatin dynamics in living plant cells.

Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X

Abstract Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

Crystal structure of Pyrococcus horikoshii PPC protein at 1.60 Å resolution

AHL1 (AT-hook Motif and Nucleus Localized protein 1) is a protein localizing at the nuclear matrix and originally identified in Arabidopsis thaliana by using a random GFP-cDNA fusion method. A. thaliana AHL1 (AtAHL1) consists of an AT-hook motif and PPC domain (Plants and Prokaryotes Conserved domain). It was revealed based on the deletion of AtAHL1 that the hydrophobic region of PPC domain is essential for its nuclear localization [Fig. 1(A)]. Phylogenetic analysis of PPC revealed that this domain is conserved among AHL1 homologues and also in bacteria and archaea; whereas neither yeasts nor animals has a protein with this domain [Fig. 1(B)]. To infer the function of PPC, our study aims at the clarification of three-dimensional (3-D) structure of this protein. We first tried to over-express A. thaliana PPC (AtPPC) (gi: 23506149) using Escherichia coli system. However, AtPPC was obtained only as an inclusion body in all conditions tested. Recent structural studies on the counterpart of eukaryotic proteins, which were found in thermophilic archaea or bacteria, provide structural insight for original eukaryotic proteins. We therefore turned our attention to PPC from Pyrococcus horikoshii, a hyperthermophilic archea. P. horikoshii is a hyperthermophilic archaea, proteins produced by this archaea are highly stable against heat and chemical denaturants, thus are suitable for structural and functional analysis over a wide temperature range and various solution conditions. P. horikoshii PPC protein (PhPPC) (gi: 3257212) possesses nearly full-length sequence and consists of 143 amino acids. In the present study, 3-D structure of PhPPC with high resolution was determined, providing the information on functional aspects of this protein.

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

An ion beam–induced Arabidopsis mutant with marked chromosomal rearrangement

Abstract Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2–derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.

Mapping of T-DNA and Ac / Ds by TAIL-PCR to Analyze Chromosomal Rearrangements

Insertion mutagenesis using known DNA sequences such as T-DNA and transposons is an important tool for studies on gene function in plant sciences. The transposons Activator (Ac)/Dissociation (Ds) have been systematically used to manipulate plant chromosomes. For both of these applications, the recovery of genomic DNA sequences flanking the insertions is required to estimate the sizes and/or scales of the reconstituted chromosomes. In this chapter, we describe the protocols for thermal asymmetric interlaced PCR (TAIL-PCR) for isolation of genomic sequences flanking DNA inserts in plant genomes.

Does BLM helicase unwind nucleosomal DNA

RecQ helicases maintain chromosome stability by resolving several highly specific DNA structures. BLM, the protein mutated in Blooms syndrome, is a member of the RecQ helicase family, and possesses both DNA-unwinding and strand-annealing activity. In this study, we have investigated the unwinding activity of BLM on nucleosomal DNA, the natural nuclear substrate for the enzyme. We generated a DNA template including a strong nucleosome-positioning sequence flanked by forked DNA, which is reportedly one of the preferred DNA substrates for BLM. BLM did not possess detectable unwinding activity toward the forked DNA substrate. However, the truncated BLM, lacking annealing activity, unwound it partially. In the presence of the single-stranded DNA-binding protein RPA, the unwinding activity of both the full-length and the truncated BLMs was promoted. Next, the histone octamer was reconstituted onto the forked DNA to generate a forked mononucleosome. Full-length BLM did not unwind the nucleosomal DNA, but truncated BLM unwound it partially. The unwinding activity for the mononucleosome was not promoted dramatically with RPA. These results indicate that full-length BLM may require additional factors to unwind nucleosomal DNA in vivo, and that RPA is, on its own, unable to perform this auxiliary function.