Tomoko Toyama

Relationship between DNA Methylation States and Transcription of Individual Isoforms Encoded by the Protocadherin-α Gene Cluster

The protocadherin-α (Pcdh-α) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-α genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-α gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-α isoforms and found that 1) the transcription of specific Pcdh-α isoforms correlated significantly with the methylation state of the promoter and the 5′ (but not the 3′) region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-α isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-α isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5′-regions of the Pcdh-αC1 and αC2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-α1 to -α12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5′-region of the first exon may help regulate differential Pcdh-α transcription and that hypermethylation is sufficient to repress transcription.

Identification of Plasmodium malariae, a Human Malaria Parasite, in Imported Chimpanzees

It is widely believed that human malaria parasites infect only man as a natural host. However, earlier morphological observations suggest that great apes are likely to be natural reservoirs as well. To identify malaria parasites in great apes, we screened 60 chimpanzees imported into Japan. Using the sequences of small subunit rRNA and the mitochondrial genome, we identified infection of Plasmodium malariae, a human malaria parasite, in two chimpanzees that were imported about thirty years ago. The chimpanzees have been asymptomatic to the present. In Japan, indigenous malaria disappeared more than fifty years ago; and thus, it is most likely inferred that the chimpanzees were infected in Africa, and P. malariae isolates were brought into Japan from Africa with their hosts, suggesting persistence of parasites at low level for thirty years. Such a long term latent infection is a unique feature of P. malariae infection in humans. To our knowledge, this is the first to report P. malariae infection in chimpanzees and a human malaria parasite from nonhuman primates imported to a nonendemic country.

Concatenated mitochondrial DNA of the coccidian parasite Eimeria tenella.

Apicomplexan parasites of the genus Plasmodium, pathogens causing malaria, and the genera Babesia and Theileria, aetiological agents of piroplasmosis, are closely related. However, their mitochondrial (mt) genome structures are highly divergent: Plasmodium has a concatemer of 6-kb unit and Babesia/Theileria a monomer of 6.6- to 8.2-kb with terminal inverted repeats. Fragmentation of ribosomal RNA (rRNA) genes and gene arrangements are remarkably distinctive. To elucidate the evolutionary origin of this structural divergence, we determined the mt genome of Eimeria tenella, pathogens of coccidiosis in domestic fowls. Analysis revealed that E. tenella mt genome was concatemeric with similar protein-coding genes and rRNA gene fragments to Plasmodium. Copy number was 50-fold of the nuclear genome. Evolution of structural divergence in the apicomplexan mt genomes is discussed.

Expression levels of Protocadherin-α transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions

The mouse protocadherin (Pcdh) clusters, Pcdh-alpha, -beta, and -gamma, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-alpha genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-alpha isoforms among several strains, we observed lower expression levels of Pcdh-alpha9 in BLG2 and BFM/2, and of Pcdh-alpha8 in C57BL/6 (B6) than in the other strains. For Pcdh-alpha8, high DNA methylation (72.7%) in the promoter region was found only in B6, whereas 36.4-44.3% methylation was seen in the other strains. On the other hand, the Pcdh-alpha9 DNA-methylation levels were similar (23.6-36.3%) among the strains regardless of the difference in expression levels. Interestingly, however, the Pcdh-alpha9 variable exon in both BLG2 and BFM/2 included a premature termination codon (PTC) generated by a nucleotide deletion or insertion. Treatment with emetine, a potent inhibitor of nonsense-mediated mRNA decay (NMD), increased the expression level of Pcdh-alpha9 from the BLG2-Pcdh-alpha locus. These data indicate that the transcription levels of mature Pcdh-alpha mRNAs are decreased by the DNA-methylation state of the Pcdh-alpha promoter regions and by the NMD pathway during RNA maturation. And we correct some previous data on Sugino, H., Toyama, T., Taguchi, Y., Esumi, S., Miyazaki, M., Yagi, T., (2004) Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines, Gene 337 91-103.

Gibberellin Biosynthetic Inhibitors Make Human Malaria Parasite Plasmodium falciparum Cells Swell and Rupture to Death

Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.

Relationship between DNA methylation and gene expression in the protocadherin gene cluster

P3-c04 G protein-coupled receptor (GPCR) signaling induces activity-dependent gene expression through the modulation of N-methyl-d-aspartate receptor (NMDA-R) in neurons Masaaki Tsuda 1 , Mamoru Fukuchi 1, Shinjiro Watanabe 1, Yuki Kuwana 1, Ichiro Takasaki 2, Akiko Tabuchi 1 1 Department Biol. Chem., Grad. Sch. of Med. and Pharm. Sci., University of Toyama, Toyama 2 Div. of Mol. Gen. Res., Life. Sci. Res. Ctr., University of Toyama, Toyama

Axons of medial habenular nucleus topographically sorted in the core of fasciculus retroflexus

opment are unknown. Here, we examined roles of 5-HT1A and 5-HT2A receptors in the dendrite formation of rat cerebral cortex using a dissociation culture. Cerebral cortex was dissected from rat embryos (Wistar/ST) at embryonic day 16 and dissociated cortical neurons were cultured up to 5 days. During the culture, neurons were treated chronically or acutely with selective receptor agonists. After the culture, the neurons were double-immunostained by antibodies against microtubule-associated protein 2 (MAP2) and glutamate decarboxylase 65/67 (GAD65/67). We found that both chronic and acute treatment with 5-HT1A agonist (8-OH DPAT) decreased the dendiritic length of GAD65/67 negative non-GABAergic neurons.