304 Genes Genet. Syst. (2014) 89

General sessions (1A-01 – 3E-13)

1A NAGAKI, Kiyotaka1, TANAKA, Keisuke2, 1A ADACHI, Shun1,2, MURAKAWA, Yasuhiro1,3, -01 MATSUSHIMA, Ryo1, KOBAYASHI, Hisato2, -03 HIRAGA, Sota1 (1Dept. Radiation Genet., Grad. Sch. MURATA, Minoru1 (1Inst. Plant Sci. and Resources, Med., Kyoto Univ., 2Medical Research Project, Inst. Okayama Univ., 2NODAI Genome Res. Center, Tokyo Health BioSci., The Univ. Tokushima Grad. Sch., Univ. Agr.) 3Max Delbruck Center for Molecular Medicine Berlin- Buch) Development of a novel method for identifying centromeric DNA sequences Involvement of SecA, SecY and AcpP in chromosome partition of Escherichia coli ChIP using anti-CENH3 antibodies is a powerful tool for isolation of centromeric DNAs. However, ChIP requires high quality We have found evidence that SecA (secretory), SecY and AcpP antibodies, and it is sometimes difficult to raise high quality (acyl carrier protein) are involved in chromosomal partition of E. antibodies, which is an obstacle for the isolation. Here, we report coli, acting together with MukBEF, DNA gyrase and Topo IV. a novel method for the isolation, based on the fact that the sizes When each protein was inactivated, cells exhibited the partition between canonical and centromeric nucleosomes are different. In minus phenotype. Inactivation of SecA caused reduction of the maize and rice, shorter DNA fragments on the CENH3 plasmid superhelicity and increase of plasmid concatemers. This nucleosomes than those on the canonical H3 nucleosomes have effect closely resembled the effect observed under inactivation of been reported. To know its universality, we isolated chromatins both MukB and DNA gyrase. SecA, SecY and AcpP localized as from rice, tobacco and welsh onion, and digested them into mono- helical structures with condensed parts. Time-lapse experiments nucleosome sizes. The digested DNA was separated using agarose of GFP-fused proteins showed that the condensed parts of protein gels. Monomeric size and shorter DNA fragments were purified, moved for a short time within the cell. The dynamic localization and used as templates of qPCR. In the qPCR, centromeric DNAs of these proteins was inhibited by inactivation of each protein. were significantly concentrated in the shorter fractions. In The addition of 1 mM sodium azide, which inhibited the ATPase addition, we found the shorter fragments enrichment with the activity of SecA, perturbed immediately the dynamic localization centromeric DNA sequences by sequence-length comparison after of SecY-GYP. The addition of 1 mM sodium azide resulted in Illumina sequencing. These results imply shorter DNA fragments abnormal subcellular localization of MukB, SeqA (sequestration) on centromeric nucleosomes are a universal feature, which must and oriC DNA. The novel chromosome partitioning mechanism be useful for identifying centromeric DNAs without anti-CENH3 that is performed by SecA, SecY and AcpP presumably acts as a antibodies. ‘molecular tether’ between chromosome and inner cell membrane.

1A MURATA, Minoru1, KANATANI, Asaka1, 1A OHNO, Yuko1, ISHII, Kojiro1,2 (1Grad. Sch. Front. -02 KASHIHARA, Kazunari1, HIRONAKA, Akiko1, -04 BioSci., Osaka Univ., 2Institute for Academic NAGAKI, Kiyotaka1 (1Group of Nuclear Genomics, Initiatives, Osaka Univ.) Inst. Plant Sci. and Resources, Okayama Univ.) Immediate response to chromosome aneuploidy Generation of a stable 1-Mb-sized artificial ring chromosome “Euploidy” represents a complete set of chromosomes which bear in Arabidopsis an entire gene set, or the genome, and therefore becomes a Plant artificial chromosomes (PACs) have been anticipated as prerequisite for every cellular program. However, equal segrega- new vectors for the development of new crops. Recently, we have tion and perfect inheritance of the chromosome set is an developed a novel method for generating PACs in the model plant exhaustive labor and accompanied with a considerable risk of Arabidopsis thaliana using the Cre/LoxP and Ac/Ds systems. The failure at every cell division. It remains enigmatic why generated PAC, “AtARC1 (A. thaliana artificial ring chromosome eukaryotes should persist in carrying the genome in separate 1)”, contains only a short centromere domain consisting of 180-bp molecules for euploidy establishment. We have prepared and repeats approximately 250 kb in length, and its flanking 2.6-Mb analyzed the chromosomally-engineered fission yeast in which region of chromosome 2 long arm. AtARC1 is stable during centromere region of a given chromosome can specifically be mitosis in the absence of selection and transmissible to the next removed and thereby acentric chromosomes can be conditionally generation through meiosis. No phenotypic abnormalities ap- induced. We found that such conditional acentrics are mostly peared in plants when an AtARC1 was added to the wild-type. missegregated into the daughter cells, rather than being lost However, because AtARC1 contains approximately 150 protein- immediately. This chromosome-specific missegregation allows a coding genes that derived from the long arm of chromosome 2, the temporal expansion and accumulation of aneuploid cells in the dosage effects are not negligible. In this study, therefore, smaller growing culture. Importantly, these conditional aneuploids are artificial ring chromosomes were attempted to be generated as made freshly. We revealed a specific response associated with the reported previously for AtARC1. As a result, a 1-Mb-sized ring immediate aneuploid cells. We will discuss the causes and chromosome was successfully generated. This AtARC2 was also consequences of such an immediate cellular response to chromo- stable during mitosis and transmissible to the next generation, so some aneuploidy. it is more suitable as a chromosome vector than AtARC1. Genes Genet. Syst. (2014) 89 305

1A TSUNEWAKI, Koichiro1,MORI,Naoki2, 1A HIRANO, Hiro-Yuki1, SATO, Dai-Suke1 (1Dept. Biol -05 YOTSUMOTO, Tatsuya2, TAKUMI, Shigeo2 (1Kyoto -07 Sci., Grad. Sch. Sci., Univ. Tokyo) Univ. (Emeritus), 2Lab. Plant Genet., Grad. Sch. Agr. Sci., Kobe Univ.s, Kobe Univ.) Genetic interaction among genes responsible for spikelet morphology in rice Genetic effect of the Aegilops caudata plasmon on manifes- Plants in the grass family have a unique inflorescence unit, called tation of the Ae. cylindrica genome a spikelet. In general, the spikelet consists of several florets and a We are carrying a genetic research to examine genetic autonomy pair of glumes. The floret contains the floral organs, including a of the plasmon from the co-existing genome by reconstructing an pistil, stamens and lodicules, enclosed by the lemma and palea. Aegilops caudata plant from its own genome (CC) and the Unlike general grass spikelets, rice spikelets has only one floret plasmon that passed half of a century in common wheat (genome and the glumes are highly degenerated. Therefore, the lemma AABBDD). For its reconstruction, it became necessary to transfer and plea are most prominent spikelet organs in rice. This rice the caudata plasmon from the alloplasmic wheat with this spikelet structure enables us to find many mutants that have plasmon to Ae. cylindrica (CCDD) for bridging the plasmon abnormal morphology of spikelet organs, such as the lemma, transfer from the 6x wheat to 2x Ae. caudata. For this purpose, palea and sterile lemmas. The sterile lemma is a specific organ we bred an alloplasmic line of Ae. cylindrica,(caudata)-CCDD, by that is found in rice, but not in other grasses. We analyzed three transferring the caudata plasmon in the 50th backcross gener- mutants such as g1, eg1 and th1 that have defects in spikelet ation of the alloplasmic wheat, (caudata)-AABBDD, to Ae. morphology. The sterile lemma was homeotically transformed cylindrica. We observed 13 characteristics for 5 generations into the lemma in the g1mutant, and extra lemma was produced during its breeding, comparing those characteristics in each in the eg1 mutant. In the th1mutant, the widths of the lemma generation with those of its euplasmic line. They differed only in and plea were reduced. We generated double mutant of g1 th1 one point, namely, male semi-sterility expression in the (cauda- and eg1 th1. The th1 mutation affected the extra lemma of eg1as ta)-CCDD. Comparison of 44 SSR loci in two organellar genomes seen in the original lemma, whereas it did not affect the between the alloplasmic and euplasmic Ae. cylindrica lines transformed lemma of g1. This difference may be related to the revealed 16 chloroplast and 6 mitochondrial SSR loci differing origin of the two ectopic lemmas. between them.

1A HONMA, Yujiro1, TAGUCHI, Kazunori2, HIYAMA, 1A NASUDA, Shuhei1, WATANABE, Shota1, IGARASHI, -06 Hajime1, YUI-KURINO, Rika1, HAMADA, Hiroyuki1, -08 Taro1, YOSHIOKA, Motohiro1, TSUJIMOTO, KUBO, Tomohiko1 (1Grad. Sch. Agr., Hokkaido U., Hisashi2, ENDO, Takashi R.1 (1Lab. Plant Genet., 2NARO, Hokkaido Agricultural Research Center) Grad. Sch. Agr., Kyoto Univ., 2Arid Land Research Center, Tottori Univ.) Characterization of candidate genes for restorer-of-fertility 2 of cytoplamsic male sterility in sugar beet (Beta vulgaris L.) Radiation hybrid mapping of the genes related to the gametocidal action in wheat Cytoplasmic male sterility (CMS) is maternally inherited male sterility. Plant CMS is associetd with mitochondrial ORF specific Several species of the genus Aegilops, wild relatives of wheat, to CMS plant. However, CMS expression suppressed by nuclear carry gametocidal (Gc) genes. The Gc gene Gc3-C1 derived from gene termed restorer-of-fertility (Rf). Hence, molecular mecha- chromosome 3C of Ae. triuncialis (2n = 4x = 28, CCUU) induces nism involved in the interaction between CMS mitochondria and chromosomal breakage in a wheat cultivar ’Chinese Spring’ (CS) Rf is an intersting subject. but not in ’Norin 26’ (N26), because N26 carries the suppressor Owen-type CMS is widely used for the hybrid breeding in sugar gene Igc1 to Gc3-C1 on chromosome 3B. We have genetically beet (Beta vulgaris L.). The CMS-associated mitochondrial ORF mapped Igc1 to pericentromeric bins and found co-segregating of Owen-type CMS is preSatp6, an N-terminal extension of atp6. DNA markers. In this study, we employed radiation hybrid (RH) On the other hand, although an Oma1-like metallo peptidase mapping and insertion site-based polymorphism (ISBP) markers gene has been identified as sugar beet Rf (Rf1), presence of located on chromosome 3B for accurate determination of the another Rf was suggested for a long time. We have been locus. We crossed disomic substitution 3C”(3B”) of CS with the interested in this uncharacterized Rf. irradiated N26 pollen for production of a RH population. We A selection of rf1rf1 was found to have an ability to restore male found no ISBP markers co-segregated with the Igc1 gene. The fertility to CMS plants, from which we have genetically identified results imply two hypotheses that (1) chromosome 3B of CS is anovelRf,namedRf2. Genetic mapping and nucleotide necessary for Gc action by Gc3-C1, and (2) the chromosomal sequencing of Rf2 region were performed, and bioinformatics region not represented by the markers carries the Igc1 gene. and gene expression analysis revealed 17 genes. Molecular Analysis of fertilities of the plants with and without chromosome organization of Rf2 region was compared between Rf2 and rf2 3B of CS did not support the former hypothesis. plants, and we identified a candidate gene that had a number of alterations. 306 Genes Genet. Syst. (2014) 89

1A KOBAYASHI, Fuminori1, KANAMORI, Hiroyuki1, 1B YOSHIDA, Takanori1, FURIHATA, Hazuka1, -09 TANAKA, Tsuyoshi1, KATAGIRI, Satoshi1, -01 TARUTANI, Yoshiaki2, KAKUTANI, Tetsuji2, KARASAWA, Wataru1, WATANABE, Shota2, KAWABE, Akira1 (1Lab. Population Genet., Dept. KURITA, Kanako1, KATAYOSE, Yuichi1, OGIHARA, Bioresource and Envi. Sci., Faclt. Life Sci., Kyoto Yasunari3, ENDO, Takashi R2, DOLEZEL, Jaroslav4, Sangyo Univ., 2Dept. Integrated Genetics, Natl. Inst. HAYAKAWA, Katsuyuki5, NASUDA, Shuhei2, WU, Genet.) Jianzhong1, HANDA, Hirokazu1 (1Agrogenomics Res. Center, Natl. Inst. AgroBiol. Sci., 2Grad. Sch. Agr., DNA methylations of nuclear chloroplast/mitochondrial Kyoto Univ., 3Kihara Inst. Biol. Res., Yokohama City DNAs and its inheritance pattern in plant nuclear genomes 4 Univ., Inst. Experimental Botany, Olomouc, Czech Stability of nuclear genome in plant species is faced with many 5 Republic, Nisshin Flour Milling Inc.) disturbances caused by foreign DNAs. Recent studies revealed that epigenetic mechanisms are involved in maintaining genome Construction of BAC physical map and analysis of genomic stability and repression of foreign DNAs. Here we reported the structure of wheat chromosome 6B pattern of DNA methylation on nuclear mitochondrial DNA-like The International Wheat Genome Sequencing Consortium sequences (NUMTs) in plant nuclear genomes and their inher- (IWGSC) is conducting the physical mapping and the genomic itance patterns. These sequences may act as foreign DNAs that sequencing of common wheat cultivar ‘Chinese Spring’. The disturb genome stability, and thus, become a target of genome Japanese research group as a member of IWGSC is in charge of defense system. We observed high DNA methylation in some the chromosome 6B. Using BAC libraries constructed from young NUMTs regardless of their flanking regions. The methyl- chromosome 6B-specific DNA, the BAC contigs were constructed that has an estimated chromosomal coverage of more than 91% of ation levels were decreased through time after integration. We the entire length of chromosome 6B. The contigs corresponding to also analyzed methylome data of epigenetic mutant lines in A. 87% of the entire chromosome were assigned in order along a thaliana and found decay of non-CG methylation in ddm1 and radiation hybrid map of chromosome 6B. The establishment and suvh4 mutants. These results showed similar pattern to nuclear analysis of the physical map led to the discovery of several plastid DNA-like sequences (NUPTs), suggesting same epigenetic important features concerning the genomic compositions and machinery acting on both NUPTs and NUMTs. The result of structure of chromosome 6B, including the fine chromosomal crossing experiments showed, in part, de novo DNA methylation localization and organization of Nor-B2, the chromosomal of NUMT associated with their organelle genotype. distribution patterns of syntenic genes and the evolutional history of chromosomal rearrangements among grass species. The use of marker information from syntenic genes led to the identification of chromosomal segments that were highly con- served or frequently rearranged, which is very useful for our understanding of the complexity of genome evolution in wheat.

1A NAITO, Ken1, KIKUCHU, Shinji2, WANG, Lixia3, 1B BO, Yina1, YOSHIDA, Takanori1, KAWABE, Akira1 -10 ISEMURA, Takehisa1, ISOBE, Sachiko4, KAGA, -02 (1Div. Life Sci.s, Kyoto Sangyo Univ.) Akito1, TOMOOKA, Norihiko1 (1Natl. Inst. AgroBiol. Sci., 2Grad. Sch. of Horticulture, Chiba Univ., The Evolutionary Analysis of the Type I MADS-box gene 3Chinese Academy of Agricultural Sciences, 4Kazusa family in Brassica rapa and relative species DNA Res. Inst.) Usually the expressions of maternally and paternally inherited alleles are the same, but the expression of some genes is derived Biased geographical distribution of reciprocal translocation primarily from one parental allele, depending on whether it was in the wild azuki bean inherited from the male or female parent. This phenomenon Using an F2 population derived from cultivated and wild azuki called genomic imprinting is observed in flowering plants and bean, we detected a reciprocal translocation of chromosomes. mammals. In plants, genomic imprinting primarily occurs in the Since a seed size QTL was identified near the presumed endosperm of developing seeds. In this study, we analyzed the translocation site, we hypothesized that the translocation caused DNA sequence variation of 14 type I MADS-box genes which is bigger seed and might have been selected for human use. To test the candidate imprinted genes in Brassica rapa and relative this hypothesis, we performed cytogenetic approach in addition to species. These candidate imprinted genes are related to the linkage analysis using 20 wild and cultivated accessions. imprinted genes in A. thaliana. The purpose of this study is to As a result, we found the translocation had not occurred in the confirm the molecular evolution pattern of imprinted genes cultivated accession, but in the wild accessions. However, observed in the type I MADS-box genes of Arabidopsis species. interestingly, we found the geographical distribution of the We identified the candidate imprinted genes in Brassica rapa and translocated chromosomes were extremely biased. The wild azuki relative species are duplicated and evolved faster than the ones bean accessions collected from West or Central Japan contained which not imprinted. The results in this study suggested that non-translocated accession, while those from East Japan con- there are correlation among genomic imprinting, gene duplica- tained the translocated chromosomes. The results exhibited no tion, and rapid evolution which have the same tendency with the exception. results of Arabidopsis species. Although azuki bean is self-pollinating, genetic exchange between wild and cultivated gene pools had often been observed. As such, the geographically biased distribution of the trans- location might be due to natural selection, rather than geographic isolation. Genes Genet. Syst. (2014) 89 307

1B HOSAKA, Aoi1, INAGAKI, soichi2,4, SAZE, hidetoshi3, 1B SHIMODA, Nobuyoshi2, HIROSE, Kentaro1, IZAWA, -03 KAKUTANI, tetsuji1,4 (1Dept. Genet., The Grad. Univ. -05 Toshiaki3, YOKOI, Hayato4, HASHIMOTO, Naohiro2, Adv. Stud., 2Dept. Plant Biol., College of Biol. Sci.s, KIKUCHI, Yutaka1 (1Dept. Biol. Sci., Grad. Sch. Sci., 3Plant Epigenetics Unit, Okinawa Inst. Sci. Tech., Hiroshima Univ., 2Dept. Regenerative Med., Natl. 4Dept. Integrated Genetics, Natl. Inst. Genet.) Inst. Longevity Sci., National Center for Geriatrics and Gerontology, 3Max Planck Inst. Biochemistry, Bidirectional interactions between the histone H3K9 deme- 4Grad. Sch. Agr. Sci., Tohoku Univ.) thylase IBM1 (INCREASE IN BONSAI METHYLATION 1) and transcription Is demethylation by deaminase and glycosylase occurred in zebrafish embryos? The DNA methylation is one of epigenetic modification and this modification is thought to be involved in regulation of tran- scription. Although it is well analyzed that methyltransferases function in DNA methylation, mechanisms of DNA demethyla- tion is still unresolved in animals. Recently, numerous studies showed that Ten-eleven-translocation (Tet) protein and DNA repair system are proposed to be active DNA demethylation mechanism in animals. On the other hand, a previous study has revealed that utilized zebrafish embryos provided a potent mechanism for active demethylation in which two proteins, deaminase and glycosylase, are involved in the phenomenon. We will present the function of these proteins in active demethylation by using zebrafish embryos.

1B ITO, Tasuku1,2, TARUTANI, Yoshiaki1, TOYODA, 1B CHIGI, Yuta1,2, SASAKI, Hiroyuki1, SADO, Takashi2,3 -04 Atsushi3, FUJIYAMA, Asao3, SAZE, Hidetoshi4, -06 (1Div. Epigenomics and Dev., Dept. Molec. Genet., KAKUTANI, Tetsuji1,2 (1Dept. Integrated Genetics, Med. Inst. Bioregulation, Kyushu Univ., 2Grad. Sch. of Natl. Inst. Genet., 2Dept. Biol. Sci.s, Grad. Sch. Sci., Med. Sci., Kyushu Univ., 3Dept. Advanced Bioscience, The Univ. Tokyo, 3Center for Genetic Resource Grad. Sch. Agr., Kinki Univ.) Information, Natl. Inst. Genet., 4Plant Epigenetics Unit, The Okinawa Inst. Sci. Tech.) Toward understanding of the mechanism involved in nuclear retention of Xist RNA Dual effect of ddm1 (decrease in DNA methylation1) X chromosome inactivation in female mammals is a mechanism mutation during the process of repeated self-pollinations to compensate for dosage difference of X-linked genes between In Arabidopsis genome, DNA methylation in transposable the sexes. This is mediated by X inactive-specific transcript (Xist) elements is maintained by DDM1 (DECREASE IN DNA RNA, which is expressed from the future inactive X and coats the METHYLATION 1), which encodes SWI2/SNF2 chromatin entire chromosome in cis. Xist RNA is a 17-kb long noncoding remodeling factor. ddm1 mutant shows loss of DNA methylation RNA (lncRNA), which is subjected to mRNA-like processing. in transposable elements (TEs) in both CpG and non-CpG Since Xist RNA functions in the nucleus with being accumulated context. Interestingly, self-pollinated ddm1 mutants show vari- on the inactive X chromosome, it has been considered that it is ous developmental defects caused by transposition of TEs or not exported to the cytoplasm. However, the molecular mecha- changes in expression of genes. In addition, while the genome nism of how Xist RNA escapes the pathway of nuclear export is originates from globally hypo-methylated ddm1 background, largely unknown. local increase of DNA methylation occurs in self-pollinated Recently, we found the 2.4 kb of Xist sequence from the ddm1 mutant. The machinery for the local increase of DNA transcription start site might be involved in nuclear retention methylation remains unclear. Genome-wide bisulfite sequencing of the RNA. To further study the effect of the 2.4 kb fragment, we of multiple lines of self-pollinated ddm1 revealed that there is have developed an assaying system, where a fusion RNA transgenerational effect during the repeated self-pollinations of consisting of a test fragment and the IRES-puromycin resistance ddm1 mutant. gene is expressed in NIH3T3 cells to examine the viability of cells in the presence of puromycin. The results demonstrate that the 2.4 kb Xist fragment indeed has an effect to prevent an export of the reporter RNA to the cytoplasm. 308 Genes Genet. Syst. (2014) 89

1B NAKAJIMA, Tatsuro1,2, HOKI, Yuko1, SASAKI, 1B SHIURA, Hirosuke1, ABE, Kuniya1 (1RIKEN BioRes. -07 Hiroyuki1, SADO, Takashi1,3 (1Div. Epigenomics and -09 Cent.) Dev., Med. Inst. Bioregulation, Kyushu Univ., 2Grad. Sch. of Med. Sci., Kyushu Univ., 3Dept. Advanced In vivo analysis of Xist/Tsix expression dynamics during peri- Bioscience, Grad. Sch. Agr., Kinki Univ.) implantation development in mice In female mice, the imprinted X chromosome inactivation (XCI), The defects in X-inactivation by partially dysfunctional Xist which is established in pre-implantation stage, is erased in allele embryonic lineage, and then random XCI is established during X-inactivation is a mechanism for dosage compensation of X- peri-implantation development. However, details of these proc- linked genes in female mammals. An X-linked noncoding RNA, esses remain elusive. To trace the changes in XCI, we examined Xist, plays an essential role in this process. We previously showed expression of Xist and Tsix, which are thought to be an indicator that the XistIVS allele was peculiar in that it apparently initiated of inactive and active X, respectively, in each cell at different but failed to establish and maintain X-inactivated state during developmental stages by using RNA-FISH method. We found that mouse development. The XistIVS allele contains an additional Xist repression (~E4.5) precedes Tsix reactivation (E4.75~5.0) on unrelated 0.9-kb intron in the 5’ region, which is spliced out but Xi, indicating that completion of X reactivation lags behind the still leaves a 16-base insertion in the spliced RNA. Here, we Xist erasure and Tsix expression is not prerequisite for Xist further characterized this partially dysfunctional allele. RNA- repression. Then, random XCI proceeds from E5.0 with increas- FISH showed that X-linked genes, which had been repressed at ing expression level of Xist. Intriguingly, ~15% cells showed Xist the blastocyst stage, were misexpressed on the X coated with bialelic expression in the process. mutant Xist RNA in the trophoblast, suggesting XistIVS RNA was Xist/Tsix dynamics described here have not been observed in in defective in establishing the eventual X-inactivated state. In vitro XCI analyses using differentiation system of ES cells. It is differentiating ES cells, on the other hand, the XistIVS allele did thus important to comprehensively analyze XCI mechanism not become upregulated, suggesting that transcriptional regu- taking advantages of both in vivo and in vitro system. lation of the XistIVS allele was impaired in ES cells. These findings may suggest that the regulatory mechanisms of Xist upregulation are different between the embryonic and extraem- bryonic tissues.

1B SAKAKIBARA, Yuki1, SASAKI, Hiroyuki1, BREWITT, 1B OHHATA, Tatsuya1,2, MATSUMOTO, Mika1, LEEB, -08 Marnie2, SADO, Takashi1,3 (1Div. Epigenomics and -10 Martin2, SHIBATA, Shinwa4, KITAGAWA, Kyoko1, Dev., Med. Inst. Bioreg., Kyushu Univ., 2The Walter NIIDA, Hiroyuki1, KITAGAWA, Masatoshi1, WUTZ, and Eliza Hall Inst. Med. Res., 3Dept. Advanced Anton2,3 (1Hamamatsu University Sch. Med., 2WT and Bioscience, Faclt. Agr., Kinki Univ.) MRC Stem Cell Institute, University of Cambridge, 3Inst. Molec. Health Sci., ETH, Zurich, X chromosome inactivation in mouse embryos deficient for 4Pharmaceuticals and medical devices agency) SmcHD1 Molecular dissection of Xist repression mechanisms in mouse In female mammals, one of the two X chromosomes is inactivated ES cells during early embryogenesis to compensate for dosage difference of X-linked genes between the sexes. It has been suggested that X One of the two X-chromosomes is inactivated in female mammals. chromosome inactivation is established and maintained by This is so called X-chromosome inactivation and Xist triggers epigenetic modifications of DNA and histones, which were chromosome wide gene silencing. Tsix is an antisense tran- mediated by various proteins recruited to the X chromosome scription of Xist and acts as a negative regulator of Xist undergoing inactivation. It has been shown that functional expression. Here, we investigate the molecular mechanism of deficiency of one of such proteins, SmcHD1, causes a failure in Xist repression in undifferentiated embryonic stem cells (ESCs). the maintenance of X chromosome inactivation and subsequent Disruption of the function of both Tsix and Polycomb repressive female-specific lethality at the mid-gestation stage. Here, we complex 2 (PRC2) achieved by an Eed mutation caused hyper- explored epigenetic status of the inactive X as well as its activation of Xist. The hyperactivation of Xist is independent of inactivated state in mouse embryo and embryonic fibroblast increased Rnf12 expression and is not accompanied by loss of deficient for SmcHD1. We found that histone modifications in the Oct4 from its binding site at Xist intron 1. Notably, in this case mutant were unexpectedly indistinguishable from those in wild- PRC2 acts independent of PRC1 as evidenced by analysis of type. However, replication timing of the inactive X, which is chromatin modifications. In contrast, H3K36me3 was recruited usually confined to late S phase, was shifted earlier in the at the Xist promoter in a Tsix-dependent manner in parallel with mutant. These findings suggest the importance of replication DNA methylation. In conclusion, several epigenetic marks control mediated by SmcHD1 in the maintenance of the X- contribute to Xist repression at the onset of X inactivation and inactive state. suggesting a separation in Tsix dependent and independent mechanisms. Genes Genet. Syst. (2014) 89 309

1B OHISHI, Hiroaki1, UNOKI, Motoko1, FUKUDA, Kei1, 1C MASUDA, Yuji1,2, KANAO, Rie1, OHMORI, Haruo3, -11 SADO, Takashi1,2, SASAKI, Hiroyuki1 (1Div. -01 HANAOKA, Fumio3, MASUTANI, Chikahide1 (1Dept. Epigenomics and Dev., Med. Inst. Bioregulation, Genome Dynamics, Res. Inst. of Env. Med., Nagoya Kyushu Univ., 2Laboratory for epigenetics and Univ., 2Dept. Toxicogenomics, Nagoya Univ. Grad. development, Kinki Univ.) Sch. Med., 3Dept. Life Sci., Faclt. Sci., Gakushuin Univ.) Zfp57 is Essential for Transcriptional Regulation of Mono- allelic Gene Expression in Ground-State Embryonic Stem Analysis of PCNA interacting motifs of DNA polymerase Cells DNA is constantly damaged by a wide variety of exogenous and Genomic imprinting is an epigenetic phenomenon in which endogenous agents. Although most of such DNA lesions inhibit parentally inherited DNA methylation marks at imprinting DNA synthesis, cells are equipped with molecular mechanisms to control regions (ICRs) mediate parent-of-origin-specific mono- continue DNA replication in the presence of DNA lesions. One of allelic expression. It is known that Zfp57, which is a causative such DNA damage tolerance mechanisms is translesion DNA gene of transient neonatal diabetes characterized by DNA synthesis (TLS). Y-family DNA polymerases carrying out TLS hypomethylation at ICRs, maintains DNA methylation at ICRs contain both PCNA- and ubiquitin-binding motifs, so that TLS is by recognition of its methylated consensus sequences at the considered to be promoted by direct interactions between Y- regions through its RH motif in the second zinc finger domain. family DNA polymerases and mono-ubiquitinated PCNA present Recently, it is reported that there are a number of monoallelically in DNA-damaged cells. We’ve been investigating the interaction expressed genes in addition to imprinted genes in specific cell between PCNA and human pol , one of the Y-family TLS types including embryonic stem cells (ESCs). However, tran- polymerases, by biochemical and cell-biological approaches and scriptional regulators for the genes were poorly understood. We we’ll discuss such results. thought that Zfp57 was one of the possible regulators and generated three Zfp57 mutant F1 (JF1/B6) ES cell-lines by CRISPR/Cas9 system, two of which lack the entire zinc finger domains and the other only lacks the RH motif. We identified 102 monoallelically expressed genes in wild-type ESCs by allele specific mRNA-seq analysis, and showed that Zfp57 regulates expression of more than half of these genes by the RH motif dependent or independent manners.

1B FUKUDA, Kei1, ICHIYANAGI, Kenji1, NAGANO, 1C FURUKOHRI, Asako1, IKEDA, Mio1, NISHIKAWA, -12 Masashi2, YANAGAWA, Yojiro2, TAKAESU, Noboru3, -02 Yoshito1, AKIYAMA, Masahiro Tatsumi1, IMAI, Hiroo4, SASAKI, Hiroyuki1 (1Div. of KATAYAMA, Tsutomu2, FUCHS, Robert P.3, MAKI, Epigenomics and Development, Med. Inst. Bioreg., Hisaji1 (1Grad. Sch. Biol. Sci., Nara Inst. Sci. Tech., Kyushu Univ., 2Lab. Theriogenology, Grad. School 2Grad. Sch. Pharm. Sci., Kyushu Univ., 3Cancer Vet. Med., Hokkaido Univ., 3Maruyama Zoo, 4Dept. Research Center of Marseille, CNRS, Marseille, Cell. Mol. Biol., Prim. Res. Inst., Kyoto. Univ.) France) Epigenomic evolution of primate germ cells The role of Escherichia coli DNA polymerase Pol IV at the stalled replication fork Changes in gene expression play an important role in the evolutionary divergence of phenotype, and can result from All organisms have several DNA polymerases for translesion epigenetic as well as genetic changes. However, it remains synthesis (TLS), a crucial cellular process for overcoming DNA unknown how epigenetic changes arise during evolution. We damage during DNA replication. Escherichia coli Pol IV was have analyzed the DNA methylomes of human, chimpanzee and discovered as a well-conserved, prototype of eukaryotic TLS macaque tissues to understand the relationships between the polymerase, but its cellular function is still under discussion epigenetic and genetic changes in primates. Small regions because of its incapacity to overcome most of common exogenous showing species-specific methylation differences are largely DNA damage such as UV-induced or chemical carcinogen non-overlapping between the sperm and somatic cells, suggesting adducts. that most methylation differences in somatic cells arise during Previously, we found that Pol IV has an ability to take over the development. In the sperm methylome, many large genomic primer terminus from replicative polymerase Pol III in a domains are hypomethylated only in the human lineage. Most of concentration-dependent manner, resulting the active polymer- the human-specific hypomethylated domains (HMDs) in the ase switch between two polymerases. Here, we reconstituted the sperm lie in lamina-associated domains and show enrichment polymerase switch reaction at the replication fork in vitro by in the sex chromosomes, suggesting that sub-nuclear localization using Escherichia coli replication machinery and Pol IV. We or chromatin structure may be important for the formation of the found that the switch between two polymerases is observed at the sperm HMDs. fork depending on the Pol IV concentration. We also showed that the stalled replication fork at a lesion is recovered by the polymerase switch and TLS by Pol IV. Based on our findings, the biological significance of Pol IV in the cell will be discussed. 310 Genes Genet. Syst. (2014) 89

1C NISHIMURA, Miki1, AIHARA, Reiko1, HIRASAWA, 1C UEFUNE, Haruka1, SAKAI, Akiko1, NUNOSE, -03 Yukei2, HIRATSU, Keiichirou3, NUNOSHIBA, -05 Shohei1, MAKI, Hisaji1 (1Grad. Sch. Biol. Sci., Nara Tatsuo1,2 (1Grad. Sch. Arts and Sci., International Inst. Sci. Tech.) Christian Univ., 2College of Liberal Arts, International Christian Univ., 3Dept. Appl. Chem., Natl. Defense Cellular level of oxygen radicals is increased in Escherichia Acad.) colicells grown under hypoxia condition To maintain the genetic stability, organisms are equipped with Expression mechanism of the photolyase gene in Thermus various mechanisms of DNA repair. Using an E. coli mutM thermophilus mutYdouble-mutant strain, which is defective in repair of a major Ultraviolet rays cause formation of pyrimidine dimers in DNA. oxidative DNA damage, 8-oxoG and its mismatch base pair, we These dimers are repaired by photolyase encoded by phr via have been analyzing oxidative mutations in cells grown under photoreactivation. Both activity of purified Thermus thermophi- different oxygen conditions, such as hypoxic or aerobically grown lusphotolyase and in vivosurvival recovery have been confirmed. or treated with a sudden shift of oxygen level. Interestingly, the However, the expression mechanism is unknown. Our previous growth conditions affected significantly the oxidative mutation studies have confirmed photoreactivating ability in T. thermo- frequency. Environmental factors probably influence the cellular philuswild type strain, but not in phr deficient strain. In this level of oxygen radicals by changing the metabolisms in cells. study, we found that illumination of cells during cultivation Here, to determine oxygen radicals levels in cells grown under before UV exposure stimulated photoreactivating ability, sug- different conditions, we analyzed expression levels of a hydrogen gesting a correlation between Phr production and light. However, peroxide-responsive protein fused with GFP by flow cytometry. no light-dependent promoter activity was observed using our Surprisingly, the hydrogen peroxide level in hypoxic grown cells reporter vector pRA-57. We found that in T. thermophilusHB27, was much higher than that in aerobically grown cells. Therefore, the phris adjacent to crtB. The crtBencodes for carotenoids and is E. colicells may consume more oxygen under hypoxia condition regulated by the repressor LitR. Derepression of LitR by light than anaerobic respiration. Another possible explanation is that induces transcription of TTP55, which activates crtB tran- cells grown under hypoxia condition may affect autoxidation and/ scription. Overexpression of TTP55 showed increase in both or terminal oxidases of electron transfer chain. carotenoids and photoreactivating ability, strongly suggesting that phr is expressed as a member of an operon with crtB.

1C INOKUCHI, Hachiro1, ITO, Riyoko2, SEKIGUCHI, 1C YOSHIKAWA, Yukihiro1, MATSUI, Ako1, ZHANG- -04 Mutsuo3 (1Research Center for Control of Aging, -06 AKIYAMA, Qiu-Mei1 (1Grad. Sch. Sci., Kyoto Univ.) Fukuoka Dental College, 2Dept. Functional Bioscience, Fukuoka Dental College, 3Adv. Sci. Res. Cent., Over-expression of hOgg1 results in increased sensitivity of Fukuoka Dental College) HeLaS3 cells to g-rays and hydrogen peroxide Reactive oxygen species (ROS) are generated by exogenous Search for the genes required for accurate genetic expression sources such as ionizing radiation and various chemical oxidants under oxidative stress as well as generated in living cells by normal cellular metabolism. In Escherichia coli, 8-oxo-7,8-dihydroguanine-related phenotypic 8-oxo-7,8-dihydroguanine (8-oxoG) is a major oxidative damaged suppression of lacZ amber is enhanced by mutations in the genes lesion, which could cause cellular mutations and genome related to the prevention of abnormal protein synthesis under instability. In eukaryotic cells, Ogg1 protein recognizes and oxidative stress. A genome-wide search for genes clarified that repairs 8-oxoG in DNA. In the present study, we constructed a some mutations cause enhanced levels of phenotypic suppression, stable HeLaS3 cell line with over-expressed hOgg1 protein based on the mutT deficient genetic background under aerobic (HeLaS3/hOgg1). We determined the survival of HeLaS3 and conditions. The involvement of the genes for Gmk, RpoB and HeLaS3/hOgg1 cells to UV, heavy ion particles, g-rays and RpoC, DnaB, DnaN and MsbA proteins, which were worked in hydrogen peroxide. The results showed that HeLaS3/hOgg1 cells mRNA synthesis, DNA replication and in preserving the were more sensitive to g-rays and H2O2, while there was no membrane structure, was found. In this time, by isolating and difference to UV and heavy ion particles. We further determined characterizing the more mutants related in phenotypic suppres- the levels of 8-oxoG foci and of chromosomal double-strand breaks sion, we reported the involvement of the genes for PrfA and PrfB by detecting g-H2AX foci formation in DNA. We discuss here proteins, which participate in protein synthesis. A series of the about the repair mechanisms against ROS in Ogg1-overexpress- studies revealed a dexterous mechanism in gene expression by ing cells. which bacterial cells protect themselves against oxidative damages. Genes Genet. Syst. (2014) 89 311

1C HAYASHI, Yuichiro1, MIYAJI, Masahiro1, KATO, 1C TAKANO, Noriko1, OHNO, Mizuki1, NAKABEPPU, -07 Yuichi1, AKIYAMA, Qiu-mei Zhang1 (1Lab. Stress -09 Yusaku2,3, NAKATSU, Yoshimichi1, TSUZUKI, Reponse Biology, Grad. Sch. Sci., Kyoto Univ.) Teruhisa1 (1Dept. Med. Biophysics and Radiation Biol., Faclt. Med. Sci., Kyushu Univ., 2Div. Analysis of human DIMT1L and TFB1M homologous to Neurofunctional Genomics, Med. Inst. Bioregulation, KsgA, a DNA glycosylase in E.coli Kyushu Univ., 3Res. Cent. for Nucleotide Pool, Kyushu Oxidized DNA causes mutation and stopping DNA replication, Univ.) leading to apoptosis or cancer. To prevent them, organism have Analysis of oxidative stress-induced tumorigenesis and Base Excision Repair(BER) which repairs damaged bases. DNA mutagenesis in Mutyh-deficient mice glycosylase removes target in the first step in BER. KsgA was originally identified as 16S methyltransferase in E.coli. In the To assess the role of MUTYH in oxidative DNA damage-induced previous study, we found that KsgA has DNA glycosylase activity mutagenesis and tumorigenesis, we analyzed tumor incidence that removes cytosine paired with oxidized thymine, Thymine- and mutation frequency in the small intestine of wild-type mice glycol and 5-formyluracil and that ksgA mutation increase and MUTYH-deficient mice. Oral administration of potassium spontaneous mutation frequency. KsgA has human homologs, bromate (KBrO3), at doses of 0.1, 0.15% in drinking water for 16 DIMT1L and TFB1M which also have methyltransferase activity. weeks, effectively induced epithelial tumors in the small intestine To reveal that these homologs have DNA glycosylase activity, we of MUTYH-deficient mice, while no tumor was found in the mice performed nicking assay and refampicin assay. Consequently, We treated with the 0.05% solution. We found a dose-response found that DIMT1L and TFB1M have DNA glycosylase activity relationship between the average number of tumor and the dose which removes cytosine paired with thymineglycol or 5-formylur- of KBrO3 in MUTYH-deficient mice. To analysis oxidative stress- acil like KsgA. Furthermore, it was revealed that KsgA and its induced mutagenesis, mice were given KBrO3 containing water human homologs remove 5-hydroxycytosine. The effect of for 4 weeks. Using rpsL-transgene as a reporter, we analyzed rifampicin assay showed that TFB1M suppresses spontaneous mutation frequency and mutation spectra both in MUTYH- mutation frequency. These result suggests that DIMT1L and deficient mice and wild-type mice. The mutation frequencies at TFB1M work as DNA glycosylase in BER. the dose of 0.05, 0.1 and 0.15% were 1.1-, 1.5- and 1.9-fold higher, respectively, in MUTYH-deficient mice than those in wild-type mice. Notably, the most frequently detected mutation in MUTYH-deficient mice was the G:C to T:A transversion that is known to be caused by 8-oxoguanine in DNA.

1C FUNAKOSHI, Masafumi1, IGARASHI, Kento2, 1C MANAL, Zorigtbaatar1, YAMAMOTO, Ayumi1 (1Dept. -08 KATO, Seiji1, ZHANG-AKIYAMA, Qiu-Mei1 (1Stress -10 Chem. and Biol. Eng., Hachinohe Natl. College of Response Biology, Div. Biol. Sci., Grad. Sch. Sci., Kyoto Tech.) Univ., 2Grad. Sch. Front. Sci.s, The Univ. Tokyo, Lab. Genome Stability) Induction of DNA damage, chromosomal aberration, and oxidative stress in human lymphoblastoid cells exposed to How the accumulation of AP sites affect the early develop- phenyl hydroquinone, an Ames test-negative carcinogen ment? Phenyl hydroquinone (PHQ), a hepatic metabolite of the Ames In this study, we focused on Apurinic/apyrimidinic (AP) sites, test-negative carcinogen o-phenyl phenol, causes bladder cancer which induce genomic instability if left unrepaired. AP sites are in rats and mice. Several conflicting reports have stated that produced continuously in living cells even under normal PHQ does or does not cause DNA damage, oxidative stress, and conditions, and disruption of AP site repair is known to induce chromosomal aberration in yeast and mammalian cells. The severe phenotype. For example, AP endonucleases are key mechanism of carcinogenesis induced by PHQ thus remains enzymes in repairing AP sites, and defect of mAPEX1, which is unclear. Here, we demonstrated its effect on human lympho- a major AP endonuclease in mouse, leads to embryonic lethality. blastoid TK6 cells, which express wild-type p53, a key protein of This result indicate that AP site repair play an important role in DNA damage checkpoint and DNA repair. Treatment of TK6 with the early development. In this study, we use Ciona intestinalis, 0 to 30 ?M PHQ induced dose-dependent increases of DNA which is a model organism in developmental biology, to inves- damage and chromosomal aberration. Furthermore, the presence tigate the impact of AP sites accumulation during the early of intracellular reactive oxygen species was stimulated by PHQ developmental stage. To accomplish this purpose, we used treatment. Finally, DNA damage and chromosomal aberration dominant negative (DN) protein of CiAPEX1,whichisC. caused by PHQ were inhibited by N-acetyl-L-cysteine, an intestinalis homolog of APEX1. DN-CiAPEX1 inhibits WT repair antioxidant chemical. These results indicate that PHQ induces activity competitively, and unrepaired AP sites accumulate. As a DNA damage and chromosome damage through the generation of result, we reveled that inhibition of CiAPEX1 caused develop- reactive oxygen species. Our results may be helpful to under- ment retardation in C.intestinalis. On the other hand, we stand the biological effects of Ames test-negative carcinogens, searched other AP site repair enzyme, and identified CiAPEX2 and indicate the importance of re-evaluation of the safety of PHQ. and ribosomal protein CiP0. Ribosomal protein P0 having AP site repair activity has been reported only in Drosophila so far. This result suggests that C.intestinalis has more complicated AP site repair system than mammalian. 312 Genes Genet. Syst. (2014) 89

1C KISHIMOTO, Aiko1, AKAGI, Junichi2, MATSUI, 1D TAKAHASHI, Keisuke1, KATOH, Kazutaka2,3, -11 Takeshi1, SAKAI, Wataru1,2, SUGASAWA, Kaoru1,2 -01 IWABE, Naoyuki1 (1Dept. Biophys., Grad. Sch. Sci., (1Dept. Biol., Grad. Sch. Sci., Kobe Univ., 2Biosignal Kyoto Univ., 2IFReC, Osaka Univ., 3CBRC, AIST) Res. Cent., Org. Adv. Sci. and Tech., Kobe Univ.) Molecular phylogenetic analyses of deuterostomes: evolu- Studies on the de-ubiquitylation mechanism of the xeroder- tionary position of cephalochordates ma pigmentosum group C protein Recent molecular phylogenetic analyses have suggested that the The xeroderma pigmentosum group C (XPC) protein plays a key closest living relatives of vertebrates are urochordates, not role in recognizing DNA damage for global genome nucleotide cephalochordates. There were, however, considerable uncertain- excision repair (GG-NER). Previously, we showed that XPC ties about the phylogeneitc position of cephalochordates in the undergoes reversible ubiquitination mediated by the CRL4DDB2 previous analyses: Putnam (2008) suggested that cephalochor- E3 ligase upon UV irradiation of cells. However, the biological dates were the sister group of vertebrate-urochordate clade with significance of this ubiquitination and the mechanism underlying 78.4% bootstrap support under maximum likelihood (ML) ubiquitin turnover on XPC after UV damage have remained method, while Delsuc (2006) showed the close relationship of unclear. Therefore, we attempted to identify the de-ubiquitinat- cephalochordates and echinoderms with 89% ML bootstrap ing enzyme (DUB) of XPC. As a result of the siRNA library screen support. In this study, taking account of the effects of LBA, we covering 118 DUBs, we succeeded in finding a candidate for the estimated the phylogenetic trees of deuterostomes including DUB. In this meeting, we report the physical interaction of XPC species of Echinodermata and Hemichordata by ML method, with the DUB, and the altered UV-damage responses of XPC by using highly-reliable 102 orthologous genes of transcription, suppression of the DUB expression. Our findings suggest possible translation, and replication systems and glycolytic enzymes. Our roles and molecular mechanisms of XPC de-ubiquitination in GG- results were as follows: 1) Both vertebrates-urochordates and NER. hemichordate-echinoderm clades were strongly supported. 2) Chordate monophyly did not receive significant statistical support, because other tree topologies, such as monophyly of cephalochordates and hemichordate-echinoderm clade, were not rejected in AU, KH, and SH tests.

1C OGURA, Keiji1, HARADA, Chihiro2, FUJIKAWA, 1D TAKEZAKI, Naoko1 (1Life Sci. Res. Cent., Kagawa -12 Katuyoshi1, TANAKA, Ignacia1,ICHINOHE, -02 Univ.) Kazuaki1, KOMURA, Junichiro1, TANAKA, Satoshi1 (1Dept. Radiobiology, Institute for Environmental Sci., Construction of phylogeny with multi-gene data 2 Japan Animal Care) Recently due to availability of genome wide data, phylogenetic trees are often constructed by using data consisting of many Transgenerational effects in Mice Exposed to Continuous genes. This study investigates the effect of phylogenetic con- Low-Dose-Rate Gamma-Rays, Genome Mutation Study struction methods, substitution models and missing data on the C57BL/6J male mice were exposed to low-dose-rate (LDR) gamma coelacanth phylogeny using two recently published data sets of rays (20 mGy/22 h/day and 0.05mGy/22 h/day) for 400 days from Amemiya et al. (2003) and Liang et al. (2003). In addition the rate 8 weeks of age and studied trans-generational effects on DNA difference and interval of divergence of the species are examined copy number aberrations (CNAs) using oligo-microarray compa- for these data. rative genome hybridization (CGH) (Agilent Tech.). We analyzed, so far, a total of 391genomes from mice (111 progenies from 20 pairs of parents in 20 mGy/22 h/day irradiated group, 46 progenies from 6 pairs of parents in 0.05 mGy/22 h/day irradiated group, and 140 progenies from 21 pairs of parents in non- irradiated group). As a result, we detected CNAs in 24 mice from 111 mice in 20 mGy/22 h/day irradiated group, five mice from 46 mice in 0.05 mGy/22 h/day irradiated group and 16 mice from 140 mice in non-irradiated control group. In addition, we found mice with hypermutation only in 20 mGy/22 h/day irradiated group and 0.05 mGy/22 h/day irradiated group. This study was performed under contract with the Aomori Prefectural Govern- ment, Japan. Genes Genet. Syst. (2014) 89 313

1D IWAMOTO, Eisuke1, TAMURA, Koichiro1 (1Dept. 1D IEIRI, Yuki1, TESHIMA, Kosuke2, TACHIDA, -03 Biol. Sci.s, Tokyo Metropolitan Univ.) -05 Hidenori2 (1The Lab. Evolutionary Genetics, Grad. Sch. of System Life Sci.s, Kyushu Univ., 2Faclt. Sci., Biased phylogenetic tree estimation by evolutionary pattern Kyushu Univ.) changes and its correction by data-filtering A Method to Choose An Appropriate Demographic Model In molecular phylogenetic analyses, a phylogenetic tree is Using Hierarchical Clustering and Discriminant Analysis inferred from a sequence alignment of macromolecules such as nucleotides and amino acids on the basis of mathematical model Constructing the population model of organisms is the first step of molecular evolution. Today, most of the tree-making methods to discover the evolutionary process, but the choice of a assume a constancy of substitution pattern of macromolecules population model is still a major concern in population genetics. throughout evolutionary processes. The violation of this assump- In this study, therefore, we propose a new method to suggest an tion may make the accuracy of phylogenetic tree reconstruction appropriate population model out of several models without worse. In this study, we have developed a method to improve the conducting a vast simulation. accuracy of phylogenetic tree reconstruction by identifying and At first, simultaneous distribution of summary statistics was removing sequence blocks, e.g., genes and/or codon positions, generated from simulated data sets under several models. Next, which potentially cause systematic biases in phylogenetic tree by conducting hierarchical clustering and discriminant analysis, reconstruction. Comparing the original distance matrix to the combinations of summary statistics were classified into catego- patristic distance matrix, we identified the sequence blocks that ries of each model. A suitable model of data was suggested based caused discrepancies between the original and patristic matrices. on the cluster they occur and the result of the discriminant Our computer simulations demonstrated that the removal of such analysis. blocks could substantially improve the accuracy of phylogenetic As a simple example, we consider the case where an appropriate tree reconstruction, when such blocks were present in the model is to be suggested out of five models: panmixia, two- dataset. However, when such block was absent, the method was islands, three-islands, admixture, and split models. Test samples not effective. were generated using simulation under the five models and investigated if this method indicates an appropriate model. It was assumed samples were obtained from a single population. It was shown that our method could suggest an appropriate model in more than 70% of the test cases.

1D YONEZAWA, Kouki1, IGARASHI, Manabu2, OHARA, 1D IKEZAKI, Yuka1, SUYAMA, Yoshihisa2 , -04 Yasuo3, ITO, Kimihito2 (1Dept. Computer Bioscience, -06 MIDDLETON, Beth A.3, TSUMURA, Yoshihiko4, Nagahama Inst. Bio-Sci. Tech., 2Research Center for TESHIMA, Kousuke5, TACHIDA, Hidenori5, Zoonosis Control, Hokkaido Univ., 3Mitsubishi Space KUSUMI, Junko6 (1Lab. Evolutionary Genet., Dept. Software Co. Ltd.) Systems Life Sci.s, Grad. Sch. Systems Life Sci.s, Kyushu Univ., 2Field Sci. Center, Grad. Sch. Agr. Sci., Closest-Neighbor Resampling for Huge Biological Sequence Tohoku Univ., 3USGS National Wetlands Res. Center, Datasets 4Forestry Forest Products Res. Inst., 5Dept. Biol., 6 A large number of nucleotide sequences of various pathogens are Faclt. Sci.s, Kyushu Univ., Dept. Environmental available in public databases. The cost of the computational Changes, Faclt. Social and Cultural Studies, Kyushu analysis of these growing datasets has increased. Moreover, Univ.) because of differences in surveillance activities, the number of Inference of the history and population structure of Taxo- sequences present in databases varies from one country to dium distichum, a coniferous tree in North America, based on another and from year to year. Therefore, it is important to amplicon sequence analysis study resampling methods to reduce the sampling bias. Hence we previously developed a resampling method – called the closest- Taxodium distichum is a coniferous tree widely distributed in neighbor trimming method – in which a given number of southeastern North America. Two of its varieties, bald-cypress sequences from a large number of sequence dataset are and pond-cypress are distinguished by morphological and habitat resampled. Also we have developed an application of CNT, differences. In addition, geographical differentiation was previ- ously suggested by an SSR analysis. To analyze both the available at the following address: http://citrus.nagahama-i-bio. geographical and variety differentiations, we determined the ac.jp/resampling/ sequences of 48 genes in 96 individuals of T. distichum sampled from the Mississippi River, Texas and Florida regions. Bayesian clustering analyses revealed differentiation between the Missis- sippi-Texas and Florida regions. The average F_ST values between the regions and between varieties were 0.058 and 0.091, respectively, but 5 loci showed high F_ST values (> 0.2) between varieties. Two of the 5 loci were identified as candidate genes for directional selection by a test based on the hierarchical island model. Finally, we inferred the histories of geographical and variety divergence based on the isolation with migration model using Jaatha. Estimated splitting times of geographical and variety divergence were very old (about 4 MYA), however, migration seemed to have reduced the level of differentiation. 314 Genes Genet. Syst. (2014) 89

1D KAWAI, Yosuke1, SATO, Yukuto1, YAMAGUCHI, 1D MAKINO, Takashi1 (1Grad. Sch. of Life Sci., Tohoku -07 Yumi1, NARIAI, Naoki1, SUGIMOTO, Sachiyo1, -09 Univ.) MIMORI, Takahiro1,KOJIMA,Kaname1, NAGASAKI, Masao1 (1Tohoku Medical Megabank Accumulation of deleterious mutations on genomes of Organization, Tohoku Univ.) domestic species Human beings have domesticated animals, plants and insects, Nonparametric inference of population demography from and selected phenotypes artificially for offering benefits to SNP data humans. Beneficial alleles on a genome would have been fixed Demographic history of population largely affects the genetic in a domestic population during domestication process, although variation in genomic scale. Although the diffusion approximation the number of them is not many. Genomes of domesticated of Wright-Fisher model can incorporate the natural selection into species are less polymorphic caused by bottlenecks, and natural population genetic analysis, a precise estimation of demographic selection on the genome would be relaxed by the artificial history is needed to separate the influence of recent change in selection. We tested a hypothesis in which deleterious mutations population size and that of natural selection. Such inferences are accumulated in domestic species compared to their wild have been limited to those using the demographic model with relatives using extensive single nucleotide variation datasets of relatively simplified demographic model. Recent progress in high- domestic animals, plants and insect and their wild relatives. throughput sequencer (HTS) enables us to make full use of the genetic variation in a population and to conduct a fine scale inference of the demographic history. We developed a method for demographic inference based on diffusion approach under modeling variable population size with nonparametric regres- sion. This model is able to infer smooth changes in population size through time. We applied this method to the simulation data and human genome data produced by HTS.

1D MATSUMOTO, Tomotaka1, AKASHI, Hiroshi1 (1Div. 1D MATSUMOTO, Yuki1,2, GOTO, Tatsuhiko1, -08 Evolutionary Genet., Natl. Inst. Genet.) -10 NAKAOKA, Hirofumi3, NISHINO, Jo4, TANAVE, Akira1,2, MOTT, Richard F5, KOIDE, Tsuyoshi1,2 Context-dependent fitness and the evolution of near neutral- (1Mouse Genomics Resource Lab., Natl. Inst. Genet., ity 2Dept. Genet., The Grad. Univ. Adv. Stud. 3 “Near neutrality” proposes that genome evolution is caused by (SOKENDAI), Div. Human Genet., Natl. Inst. Genet., 4 the interaction of weak forces (genetic drift and very small fitness Dept. Mathematical Analysis and Statistical effects). This model, proposed by Tomoko Ohta over 40 years ago, Inference, The Inst. Statistical Mathematics, 5 is now widely supported by patterns of within and between- Wellcome Trust Centre for Human Genetics) species DNA variation. However, weak selection is highly Wild-derived heterogeneous stock mice to find the loci parameter-sensitive; nearly neutral mutations in vertebrates associated with tame behavior should be strongly selected in microbes. Strong context depend- ence, in particular “diminishing returns” in fitness, may underly Wild ancestors of domesticated animals had genetic variation for the prevalence of near neutrality. Such concave fitness functions tameness. In order to understand the genetic basis of tame (CFFs) arise in metabolic flux and protein stability theory, behavior in mice, we were conducted genome-wide association however, have not been tested in natural populations. We employ studies (GWAS) using wild-derived heterogeneous stocks (WHS) computer simulation to study epistasis in genome evolution and which are originated from eight wild-derived strain (MSM, HMI, show that CFFs lead to near neutrality under a wide parameter BLG2, PGN2, KJR, CHD, NJL, and BFM/2). First, we obtained range. We also show how patterns of sequence variation may behavioral data related to tameness and 37,714 SNPs data from allow us to distinguish context dependent and independent genotyping array (MegaMUGA) in 378 WHS mice. Second, using fitness effect and test whether CFFs are a key factor in genome these data, we implemented the linear model to map the loci evolution. associated with tame behavior. As a result, we identified several loci at genome-wide significance level. However, we could not find any causative genes that have registered around the candidate loci in MGI database. Here, we also show the ongoing project of artificial selection to detect the loci associated with tame behavior. Genes Genet. Syst. (2014) 89 315

1D TESHIMA, Kosuke1, CHAKRABORTY, Ranajit2 1E NAKAGAWA, So1 (1Dept. Molec. Life Sci., Tokai Univ. -11 (1Dept. Biol., Faclt. Sci.s, Kyushu Univ., 2Center for -01 Sch. Med.) Computational Genomics, Dept. Molecular and Medical Genetics, Univ. North Texas Health Science Comparative genomic analysis on endogenous viral elements Center) About 10% of mammalian genomes correspond to endogenous viral elements (EVEs), which are thought to be derived from Distribution of the number of unmatched Y-STR markers ancient viral infections of germ cells. We previously determined between a pair of haplotypes transcriptomic profiles of bovine trophoblast cells using a high- Y chromosome is widely used in population genetics to inves- throughput sequencer, and found that a certain proportion of tigate population structure and population history. It is also used EVEs could be expressed (Nakagawa et al., GBE 2013). Recent in forensic genetics to distinguish male lineages and to provide studies have also showed that some EVEs function in host species information about relationship between lineages. To investigate on various aspects such as placental morphogenesis and viral the relationship between a pair of Y haplotypes, it is important to infections. Here I have obtained EVEs coding more than 80 understand the haplotype matching probability. In this study, the amino-acid length from 18 mammalian species, and compared distribution of the number of mismatched loci out of L loci typed among them. Here in this conference, I will present the progress was derived. of this project including several collaboration studies. Using the derived distribution, the ability to differentiate male individuals with RM Y-STR markers was assessed. RM Y-STR markers are recently identified markers which have mutation rate above 10-2. It was demonstrated that these markers provide improved ability to differentiate closely related male lineages compare to currently used Y-STR markers. It was also shown that the proportion of the haplotype match caused by convergence of multiple mutations (identical by state) increases for distantly related pairs when typed with these markers. This means RM Y- STRs are clearly powerful markers in separating closely related males in forensic genetics but it should be cautious when applied to evolutionary studies.

1D GO, Yasuhiro1, TATSUMOTO, Shoji2, FUKUTA, 1E KITAZAWA, Moe1, ONO, Ryuichi1, TAMURA, -12 Kentaro2,NOGUCHI,Hideki3, TOMONAGA, -02 Masaru2, KANEKO-ISHINO, Tomoko3, ISHINO, 4 5 4 Masaki , HIRAI, Hirohisa , MATSUZAWA, Tetsuro , Fumitoshi1 (1Med. Res. Inst., Tokyo Medical and 6 2 1 AGATA, Kiyokazu , FUJIYAMA, Asao ( Dept. Brain Dental Univ., 2Riken BRC, 3The Sch. Health Sci., Sci.s, Center for Novel Sci. Initiatives, Natl. Institutes Tokai Univ.) of Natural Sci., 2Comparative Genomics Lab., National Inst. Genet., 3Advanced Genomics Center, Natl. Inst. Role of a eutherian-specific gene, Peg11/Rtl1, in embryos and 4 Genet., Dept. Behavioral and Brain Sci.s, Primate placentas in mice Res. Inst., Kyoto Univ., 5Dept. Cellular and Molec. Biol., Primate research Inst., Kyoto Univ., 6Lab. Uniparental duplication of the mouse distal chromosome 12 Molecular Dev. Biol., Grad. Sch. Sci., Kyoto Univ.) displays late-fetal and/or neonatal lethality with growth abnor- malities that are mainly attributed to either lack or over Direct estimation of mutation rates in a chimpanzee family expression of paternally expressed gene, Peg11/Rtl1. Maternally trio by ultra deep whole genome sequencing expressed gene, AntiPeg11 regulates Peg11 mRNA level. We Mutations generate genetic variation and are a driving force of previously produced Peg11 and AntiPeg11 knockout (KO) mice evolution. Therefore, estimating mutation rates is critical for under- and analyzed their phenotypes as models for human maternal standing the genetic basis of the diseases and the evolution of and paternal disomies 14. We reported that both Peg11 and organisms. In this study, we report the whole genome sequences of a AntiPeg11 play essential roles in mouse development. Peg11 chimpanzee family trio. Further, we used ultra-deep genome coverage protein localized exclusively in fetal capillary endothelial cells in (>150-fold) to identify the sites of Mendelian inheritance errors (MIEs) that could not have been inherited through Mendelian inheritance placenta to play a role in maintenance of the feto-maternal from either of the parents. We identified 889 MIEs and classified them interface during mid-late fetal development. into four categories: [i] de novo single nucleotide variants (SNVs), [ii] In Peg11 KO placentas, we reported that a lot of clogging in the copy number neutral inherited variants, [iii] hemizygous deletion fetal capillaries but we recently found that the labyrinth layer inherited variants, and [iv] de novo copy number variants (CNVs). We itself was 50 % thinner than wild type, and low density of fetal identified 45 de novo SNV candidates that appear only in the offspring capillaries. Thus, it is highly probable that inefficient nutrient –8 and estimated a germline de novo SNV rate of 1.71 × 10 per site per and oxygen exchange of Peg11 KO due to these double effects generation. This rate falls between those of the commonly used decreases blood flow to the fetus and then causes fetal growth phylogenetic per-site mutation rate and the pedigree-based mutation retardation and lethality. We also determined the cause of rate of the human genome. Moreover, the present study demonstrates neonatal lethality by using micro-CT. the significance of ultra-deep whole genome sequence analysis for identifying not only de novopoint mutations (de novo SNVs) but also de novostructural changes (de novo allelic conversion and de novo CNVs) through transmission from parents to offspring in a single generation. 316 Genes Genet. Syst. (2014) 89

1E INOUE, Kota1, ICHIYANAGI, Kenji1, FUKUDA, Kei1, 1E GOTO, Yuji1, TANAKA, Asami2, KAWAMOTO, -03 SHIMOSUGA, Kenichi1, SASAKI, Hiroyuki1 (1Div. -05 Yoshiyuki2, KUBOTA, Souichirou1 (1Dept. Biol., Faclt. Epigenomics and Dev., Med. Inst. Bioregulation, Sci.s, Toho Univ., 2Dept. Biomedical Sci., College of Kyushu Univ.) Life and Health Sci., Chubu univ.) Distinct retrotoransposon regulation at different develop- Epigenetic alterations of histone methylation in human mental stages of mouse male germ cells by the piRNA system melanoma development and chromatin modification Melanoma is a highly aggressive cancer without very effective During male germ cell development in mice, retrotoransposons treatments. Recently, not only genetic mutations of the oncogenes are regulated by interplay between DNA methylation and piRNA. and tumor-suppressor genes but also epigenetic mutations However, such epigenetic regulation has been examined for only including DNA methylations and histone modifications are a few retrotransposons. We thus examined DNA methylation responsible for the cancer development and its progression. Here levels retrotransposons in Pld6- and Dnmt3l-knockout germ cells, we showed that the Lysine 4 of histone H3 (H3K4), were highly which are deficient in piRNA biogenesis and de novo DNA methylated (mono- and di-methylation) in the several melanoma methylation, respectively. Whereas the Dnmt3l mutation greatly cell lines while showing low level of methylation in normal decreased DNA methylation levels of almost all retrotransposons, human melanocytes (NHEM). Opposite result was also observed the Pld6 mutation did not affect DNA methylation of most against H3K9 mono (me1)- and di-methylation (me2) in both retrotransposons expect for several elements such as L1 cells. Furthermore, ChIP assay clearly showed that LINE-1 promoters. RNA expression levels of most retrotransposons were repeats and some SINE repeats has mono- and di-methylation of only marginally increased in both mutants at postnatal day 0 (P0) H3K4 in all melanoma cell lines but not NHEM. Since H3K4 and P7, despite their low methylation levels. On the other hand, methylations are generally regarded as a euchromatic modifica- the expression of some limited families of retrotransposons was tions associated with the transcriptional activation and H3K9 highly elevated in the Pld6 mutants at P14, where some germ methylations are key modification of heterochromatin formation cells initiated meiosis. These results suggest that distinct and their maintenance, our results suggested that genome-wide regulatory systems play roles in the restriction of retrotransposon methylation of H3K4 occurred during melanoma development transcription at different developmental stages in mouse male and progression. germ cells.

1E IWAMA, Hisakazu1, FUJITA, Koji2, IMACHI, 1E MATSUNAGA, Wataru1, MASUTA, Yukari2, -04 Hitomi3, MURAO, Koji3, MASAKI, Tsutomu2 (1Life -06 MITANI, Namiki3, MA, Jian Feng3, KATO, Atsushi2, Sci. Res. Cent., Kagawa Univ., 2Dept. Gastroenterology ITO, Hidetaka2 (1Grad. Sch. Life Sci., Hokkaido Univ., and Neurology, Faclt. Med., Kagawa Univ., 3Dept. 2Faclt. Sci., Hokkaido Univ., 3Inst. Plant Sci. and Advanced Med. and Lab. Med., Faclt. Med., Kagawa Resources, Okayama Univ.) Univ.) Transcriptional activity and silencing of heat activated Period of origin of human microRNAs retrotransposon in Arabidopsis MicroRNAs (miRNAs) are short non-coding RNAs that affect A retrotransposon named ONSEN is transcriptionally activated transcriptome through posttranscriptional repression. MiRNAs subjected to heat stress in Arabidopsis. The heat activation of undergo fast processes of gene gains and losses. Genome ONSEN is controlled by a promoter in the long terminal repeats sequences of 38 mammals and five out-group species were sought (LTRs) that involves a cis-regulatory sequence. To verify the for orthologs of human miRNAs. Fifteen evolutionary periods tissue specific regulation of ONSEN, we produced a transgenic were set from the mammalian root to human by the divergences Arabidopsis that possessed an intact LTR fused with a gene for of species and/or clades including a period of ‘conserved before the green fluorescent protein. Since we expected that the transcrip- mammalian evolution’. Based on the time of the first appearances tional activity of ONSEN was maintained long term in a shoot of each ortholog, the period of origin for each of 849 human apical meristem (SAM), we performed a quantitative analysis of mature miRNAs derived from 777 loci of the human genome ONSEN mRNA in a SAM. (which were confirmed to be unambiguously expressed) was assigned to any of the 15 periods. Using multiple RNA-seq datasets, human miRNAs of more recent origins were shown to be expressed at lower levels than those of older origins. Predicted target sites of human miRNAs of recent origins tended to be less compared to those of older origins. There were differences in target functions of the human miRNAs along the period of origin of the human miRNAs. Genes Genet. Syst. (2014) 89 317

1E SATO, Mio1, MASUDA, Seiji2, MASUTA, Yukari3, 1E HABU, Yoshiki1, SAIKA, Hiroaki1, NUMA, Hisataka1 -07 KATO, Atsushi3, ITO, Hidetaka3,4 (1Sch. Sci., -09 (1Plant Genome Engineering Unit, Natl. Inst. Hokkaido Univ., 2Grad. Sch. Life Sci., Hokkaido Univ., AgroBiol. Sci.) 3Faclt. Sci., Hokkaido Univ., 4Japan Sci. and Tech. Angency, PRESTO) Analysis of a DNA-type transposon of rice that is activated during callus induction Analysis of heat-activated transposon in Brassicaceae The genomes of higher plants contain a number of transposon- like sequences. Many of these sequences are remnants of previously active transposons that have accumulated various types of alterations in their DNA sequences. Most of structurally intact transposons are also kept in inactive states by epigenetic mechanisms and can be activated by environmental stresses. In this study, we report about changes in activity and DNA methylation of a rice DNA-type transposon during callus induction.

1E KAWAGISHI, Yuki1, MASUTA, Yukari2, MASUDA, 1E KAWAI, Tsubasa1, OKUMURA, Keisuke1, -08 Seiji3, MATSUNAGA, Wataru3, KATO, Atsushi2, ITO, -10 UCHIYAMA, Ryo1, KOUNOSU, Asako2, ITOH, Hidetaka2,4 (1Div. Biol., Dept. Biol. Sci.s, Sch. Sci., Masanobu1 (1Dept. Appl. Biol. Kyoto Inst. Tech., Hokkaido Univ., 2Faclt. Sci., Hokkaido Univ., 3Grad. 2Dept. Biochem. Mol. Biol., Nippon Medical School) Sch. Life Sci., Hokkaido Univ., 4Japan Sci. and Tech. Angency, PRESTO) Specific internal deletion is associated with high trans- position rate of KP element in Drosophila melanogaster Research of a retrotransposon activated by heat stress in R. P-element (2,907bp), a DNA transposon in D. melanogaster, sativus produces incomplete derivatives with a variety of internal A retrotransposon named ONSEN is transcriptionally activated deletions. KP-element (1,154bp), one of the incomplete elements, subjected to heat stress in Arabidopsis. High frequency of has an A/T substitution at the 32nd nucleotide position (32A/T) retrotransposon was detected in the progenies of heat-stressed and an internal deletion (D808-2560). Unlike complete P- siRNA mutant plants. Recently we found that some Brassicaceae elements, KP-element cannot be autonomous because of largely species have ONSEN-like retrotransposons which are activated lost of the exon coding region. However, KP-elements are world- by a heat stress. We report here an ONSEN-like retrotransposon widely common in current wild populations. To elucidate this in R. sativus that is one of the most important Brassicaceae extraordinary predominance of KP-element, we examined trans- species for breeding in Japan. We sequenced conserved protein- position efficiency of KP-element using a new KP-element vector, coding region of ONSEN-like elements in twenty varieties of R. pWIZ-KP, and a popular P-element vector, pWIZ. As a result, sativus. Furthermore, we analyzed the transcriptional activation pWIZ-KP showed a higher transformation rate than pWIZ. of ONSEN-like retrotransposons in R. sativus that was activated Moreover, when the 3’ region of pWIZ-KP covering the junction by heat stress. of the deletion was replaced by the 3’ sequence of pWIZ, the rate decreased. On the other hand, replacing the 32T by A increased the rate. These results suggest that KP-element can transpose more efficiently than some other P-elements and that the two structural characteristics affect transposition of KP-element reversely: the 32T substitution negatively and the internal deletion positively. 318 Genes Genet. Syst. (2014) 89

2A MURAI, Koji1 (1Dept. BioSci., Fukui Pref. Univ.) 2A SUGIE, Atsushi2, HAKEDA, Satoko1, SUZUKI, -01 -03 Emiko3, TAVOSANIS, Gaia2, SUZUKI, Takashi1 (1Grad. Sch. BioSci. and BioTech., Tokyo Inst. Tech., Combining of gene network models for flowering in temper- 2DZNE, Bonn, GErmany, 3Natl. Inst. Genet.) ate cereals Molecular remodeling of the presynaptic active zone of In temperate cereals, the expression patterns of three genes play Drosophila photoreceptors via an activity-dependent feed- the important roles in flowering, namely, VERNALIZATION 1 back signal (VRN1), VRN2 and Wheat FLOWERING LOCUS T (WFT). VRN1 is an APETALA1/FRUITFULL-like MADS-box transcription Neural activity contributes to the regulation of the properties of factor, VRN2 is a protein with a zinc finger motif and a CCT synapses in sensory systems, allowing for adjustment to a domain, and WFT is florigen. My research group is presenting a changing environment. Little is known about how synaptic model for flowering, in which VRN1 is upstream of WFT and molecular components are regulated to achieve activity-depend- activates WFT expression under LD conditions. Vernalization ent synaptic plasticity in central synapses. Here, we found that down-regulates VRN2 and up-regulates VRN1 independently of the presynaptic active zone in Drosophila photoreceptors under- each other. VRN2 is also suggested to be down-regulated by WFT. goes structural and molecular remodeling, including Bruchpilot There is a conflicting model, in which VRN1 is downstream of and DLiprin-a, upon mild but prolonged light exposure. The WFT. The extra early-flowering (exe) mutants which lack a clock levels of neuronal activity of photoreceptors and of depolarization gene Wheat PHYTOCLOCK 1 do not have a functional VRN2 of their postsynaptic neurons are critical for the light-induced locus because the original einkorn strain KU104-1 carried a changes in photoreceptor active zone composition, suggesting the VRN2 deletion. Our present results that the level of VRN1 existence of a feedback signal. In search of the signal, we found expression can control earliness in exe mutants without the need that a reorganization of the microtubule meshwork acts down- for VRN2 expression indicate that it is not possible to reconcile stream of the divergent canonical Wnt pathway, potentially via our conclusion on the function of VRN1 expression in flowering the kinesin-3 Immaculate connections. These data reveal changes earliness with the expectations of the conflict model. in composition of active zones at central synapses upon natural stimuli and the molecular machinery that actively contributes to them.

2A KAMAKURA, Masaki1 (1Dept. BioTech., Faclt. Eng., 2A HOZUMI, Shunya1, MIYAMOTO, Ryosuke1, KUME, -02 Toyama Prefectural Univ., 2PRESTO, JST) -04 Shoen2, KIKUCHI, Yutaka1 (1Dept. Biol. Sci., Grad. Sch. Sci., Hiroshima Univ., 2Dept. Stem Cell Biol., HR38 regulates developmental time in honeybee and fruit fly Inst. Molec. Embryology and Genet., Kumamoto Univ.) Royalactin in royal jelly (RJ) induces caste differentiation of Transcription factors are required for induction of mesoder- honeybees. Royalactin increased body size and ovary develop- mal and endodermal gene expressions in neural cells ment and shortened developmental time in honeybee. Further- more, it also showed similar effects in fruit fly. However, the It is well known that vertebrate embryos generate three primary mechanism through which RJ regulates developmental time in germ layers, known as ectoderm, mesoderm, and endoderm, honeybees and flies has remains elusive. Here, I investigated how through the induction by various growth factors and function of RJ or royalactin decreased developmental time in honeybees and downstream transcription factors. Although the linage-specified flies. differentiated cells are though to be very stable, recent studies Ecdysone induces gene expression of various nuclear receptors. showed that the differentiated cells could be reprogrammed by Therefore, changes of gene expression of nuclear receptors in flies overexpression of several germ layer specific transcription reared with RJ during the larval period were measured. The factors, called direct reprogramming. However, it is still difficult increase of gene expression of Drosophila HR38 (DHR38) was to convert from one germ layer to another germ layer fate. We are observed in salivary gland of flies given RJ. DHR38 RNAi in now trying trans-germ layer conversion by using zebrafish. The salivary gland suppressed decrease of developmental time with- zebrafish has emerged as an important model system for the out affecting body size in flies reared with RJ. Overexpression of investigation of vertebrate cell differentiation and cell fate DHR38 in salivary gland of flies induced decrease of devel- decision. We will present our new approach by using tran- opmental time. Furthermore, suppression of honeybee HR38 with scription factors in zebrafish larvae. RNAi inhibited the decrease of developmental time induced by RJ, but did not affect changes of body size or ovary development. These results indicated that HR38 regulates developmental time in insect. Genes Genet. Syst. (2014) 89 319

2A NAKAHARA, Yoshinari1, MUTO, Akihiko1, KUME, 2A TAKAYAMA, Kazuya1, SHIMODA, Nobuyoshi2, -05 Shoen2, SAKUMA, Tetsushi3, YAMAMOTO, Takashi3, -07 TAKANAGA, Shunsuke1, HOZUMI, Shunya1, KIKUCHI, Yutaka1 (1Dept. Biol. Sci., Grad. Sch. Sci., KIKUCHI, Yutaka1 (1Dept. Biological Science, Hiroshima Univ., 2Dept. Stem Cell Biol., Inst. Hiroshima Univ., 2Dept. Regenerative Med., Natl. Embryology and Genet., Kumamoto Univ., 3Dept. Inst. for Longevity Sci.) Mathematical and Life Sci.s, Grad. Sch. Sci., Hiroshima Univ.) Analyses of the mouse dnmt3 homologs during development and fin regeneration in zebrafish Analyses of the roles of Notch signaling and its downstream DNA methylation is crucial for gene regulation. Although studies effectors in the adeonhypophysis cell specification in mammalian cells revealed that the de novo DNA methyltrans- Adenohypophysis (AH), an anterior part of the pituitary, is a ferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the regulatory organ in the endocrine system and controls various establishment of DNA methylation patterns, their roles in physiological processes such as growth, reproduction, and development and tissue regeneration are still unclear due to metabolism. AH consists of six cell types, each of which secrets the embryonic lethality of dnmt3-deficient mice. Here, we used different hormone, and their differentiation has been shown to be zebrafish as a model examined the detailed expression profiles of regulated by Notch signaling. Although a previous study in three zebrafish dnmt3 homologs, dnmt3aa, dnmt3ab and dnmt4, zebrafish revealed that Notch-mediated lateral inhibition be- by whole-mount in situ hybridization. In embryos, these genes tween adjacent cell types is important for specification of AH cell were expressed in distinct patterns, suggesting their tissue- fate, molecular mechanisms underlying the regulation remain to specific roles in development. On the other hand, in regeneration be elucidated. In this study, we identified two transcriptional fins, dnmt3aa among them was predominantly expressed in the regulators expressed in AH as downstream effectors of Notch regeneration blastema. We also found that the levels of DNA signaling and found that these factors are involved in differ- methylation at adjacent to the amputation plane was correlated entiation of the distinct AH cell types. Based on the results, we with the ability of regeneration. These results indicate that DNA will discuss the mechanisms of Notch-dependent AH cell methylation may play important roles both in embryonic develop- specification. ment and tissue regeneration.

2A SHIOMI, Taishi1, MUTO, Akihiko1, KIMURA, 2A KAWAGUCHI, Akane1, OCHI, Haruki2, SUDOU, -06 Hiroshi2, KIKUCHI, Yutaka1 (1Dept. Biol. Sci., Grad. -08 Norihiro3, OGINO, Hajime1 (1Nagahama Inst. Bio-Sci. Sch. Sci., Hiroshima Univ., 2Grad. Sch. Front. BioSci., Tech., 2Yamagata Univ. Faclt. Med., 3Tokyo Women’s Osaka Univ.) Med. Univ.) Regulatory mechanisms of the plasticity of cell fate decision The histone demethylase Jmjd3 regulates pax6 expression for during early embryonic development eye development Most animal embryos form three germ layers, the ectoderm, endoderm, and mesoderm during developmental process. All tissues and organs are produced from each germ layers. Accordingly, three germ layers formation is fundamental for early developmental process. However, it remains unknown when and how germ layers decide their cell fates and whether they have plasticity between them. Therefore, to reveal these problems, we time-specifically overexpressed endoderm inducing signals in early zebrafish embryos. We will present the plasticity of ectoderm cells that change its cell fate into endoderm cells. 320 Genes Genet. Syst. (2014) 89

2A SUGAYA, Kimihiko1, ISHIHARA, Yoshie1, INOUE, 2B OKAMOTO, Sho1, NIKI, Hironori1 (1Natl. Inst. -09 Sonoe1, HIROBE, Tomohisa1 (1Fukushima Project -01 Genet.) Headquarters, Natl. Inst. Radiological Sci.) Activation of blue light dependent transcription regulators in The effects of ionizing radiations on the regeneration of hair the dimorphic fission yeast Schizosaccharomyces japonicas follicles are carried over to the later generation Many fungi respond to light and regulate fungal development and The effects of ionizing radiations on somatic stem cells largely behavior. The first photoreceptor was found in Neurospora crass. remain to be studied. Hair follicles are self-renewing structures The photoreceptor is activated by blue light and consists of two that reconstitute themselves through the cycle comprising subunits, WC-1 and WC-2. The WC1/2 complex works as anagen (growing phase), catagen (regression phase) and telogen transcription factor to induce genes involved in the light (resting phase), suggesting the presence of their own stem cells. dependent reaction. The molecular analysis of the fungal photo- In the past study, -rays were irradiated whole body of 22~24-day- receptor has been carried out by using N. crass: WC-1 binds to old (P22~P24, the 1st telogen) C57BL/10JHir mice, and the Flavin, and both subunits have the PAS domain that contained in effects of radiations were analyzed at the 2nd anagen, P35~P37. many signaling proteins. WC-1 and WC-2 are assembled via the The decrease in the density of hair follicles and the increase in PAS domain. Recent genomic sequencing reveals that orthologs of the frequency of hypopigmented hair bulbs were observed in a WC-1 and WC-2 have been found mainly in filamentous fungi. dose-dependent manner. In this study, we analyzed the effects of However, the orthologs are identified in only 3 species of yeast. radiations on the regeneration of hair follicle at the 3rd anagen, We found that the fission yeast Schizosaccharomyces japonicus P100. The density of hair follicle was decreased and curved hair responds to blue light depending on Wcs-1 and Wcs-2, which are follicles were also found. In addition to these keratinocyte-derived members of the WC-1 and WC-2 family, respectively. When we anomalies, melanocyte-derived anomalies such as white hair and made comparison between WC1/2 and Wcs-1/2, there are several hypopigmented hair bulbs were also found. These results suggest differences in structure of the subunits. However, fundamental that the effects of -rays on the regeneration of hair follicles are functions of the blue light dependent transcription factors are carried over to the later generation. conserved between N. crass and S. japonicas.

2A SHIBUYA, Hirotoshi1, WATANABE, Ryutaro1, 2B WADA, Chieko1, UETA, Masami1, WADA, Akira1 -10 MAENO, Akiteru2, TAMURA, Masaru3, WAKANA, -02 (1Yoshida Biol. Lab. inc. Biol. Inform. Resrarch) Shigeharu3, SHIROISHI, Toshihiko2, YAMAMOTO, Hiroaki4 (1Grad. Sch. Bioscience, Nagahama Inst. Bio- Formation of 100S ribosomes in the hibernation stage is Sci. Tech., 2Mammalian Genet. Lab., Natl. Inst. inhibited in rpoS mutant of Esherichia coli 3 4 Genet., Riken BRC, Faclt. BioSci., Nagahama Inst. Escherichia coli and related bacteria accumulate sigma S Bio-Sci. Tech.) subunit (RpoS), which controls the expression of a large number of genes involved in the cellular response to stress and the Melanocytes may contribute to the structure of their transition into stationary phase. Cellular levels of ribosome extracutaneous habitats modulation factor (RMF) and hibernation promoting factor (HPF) Melanocytes originate from the vertebrate embryo-specific neural drastically increase during the stationary phase and induce to crest, migrate to and settle in various organs, including not only dimerize most 70S ribosomes to 100S ribosomes, which exhibit no the skin but also extracutaneous locations, where only indirect translational activity. This process is referred to as ‘Ribosomal light may illuminate them. How do extracutaneous melanocytes, hibernation’. We studied the relationship between ribosomal which continue to produce melanin, function in sun-protected hibernation and RpoS in stationary phase, and found that areas of the skin? Examples of extracutaneous melanocytes formation of 100S ribosomes was greatly inhibited in rpoS include choroidal melanocytes located in the uvea between the deletion mutant. RpoS positively regulated RMF and HPF, which retinal pigment epithelium and the sclera in the eye and cochlear affect the formation and stability of 100S ribosomes, while it melanocytes mainly localized in the stria vascularis. In order to negatively regulated YfiA, which dissociates 100S into 70S elucidate whether extracutaneous melanocytes contribute to the ribosomes. These results show that the decrease in 100S ribosome structure of their habitats, we used a melanocyte-deficient mouse formation in rpoS mutant can be attributed to a decrease in RMF mutant, Mitfmi-bw. The choroid and the stria vascularis normally and HPF levels and an increase in YfiA levels. It shows that RpoS develop rich capillary networks surrounded by melanocytes. Our controls translational activity by dimerization of 70S ribosome in study suggests that melanocytes are indispensable partners to Escherichia coli during stationary phase. maintain the spatial organization of those tissues/organs. Genes Genet. Syst. (2014) 89 321

2B MORIKI, Shogo1, HANAFUSA, Ken2, TENNO, 2B SHIRAKAWA, Fuminori1, ASAI, Kei1, IKEDA, -03 Takeshi3, HIROAKI, Hidekazu3, ABO, Tatsuhiko1,2 -05 Soutarou1 (1The Grad. Sch. Sci. Eng., Saitama Univ.) (1Grad. Sch. Natural Sci. Tech., Okayama Univ., 2Dept. Biol., Faclt. Sci., Okayama Univ., 3Grad. Sch. Analysis of expression mechanism and structure of the a Pharm. Sci., Nagoya Univ.) operon of Bacillus usbtilis In bacteria, ribosomal protein genes form an operon, and their Characterization of substitution mutants of Escherichia coli expression is regulated through an autogenous feedback mech- ArfA protein anism. The alpha operon of Escherichia coli consists of the genes for ribosomal proteins and alpha subunit of RNA polymeralse (S13-S11-S4-a-L17), and its expression is regulated by S4 protein. On the other hand, The alpha operon of Bacillsu subtilishas about 150 bp non-coding region in place of the gene for S4 (S13- S11-a-L17). Therefore, regulation of expression of the a operon in B. subtilis is different from that in E. coli. We analyzed the transcription unit and gene expression mechanism about the a operon of B. subtilis, indicating that L17 encoded by rplQ, which is co-transcribed with rpoAcoding for a, is indispensable,, and there is no promoter in the proximal non-coding region. Growth of the strain, in which rpoA-rplQ was under control of Pspac promoter without the non-coding region, was retarded. In addition, colony size and spore formation of this strain were also affected. These suggest that the non-coding region located upstream of rpoAis involved in regulation of rpoA expression.

2B OKUDA, Rikumi1 (1Dept. Appl. Chem., Natl. Defense 2B TONE, Takahiro1, TAKEUCHI, Ari1, MAKINO, -04 Acad.) -06 Osamu2 (1Fac. of Sci. and Tech., Sophia Univ., 2Dept. Materials and Life Sci.s, Fac. Sci. and Tech., Sophia Identification of the Novel Sigma-E Dependent Promoter of Univ.) the rpoD Gene in Escherichia coli Searching for host factors involved in f29 DNA replication The sigma-E is the extra-cytoplasmic stress sigma factor of RNA polymerase holo enzymes in Escherichia coli. The sigma-E Bacillus subtilis phage f29 has a linear double-stranded DNA signaling pathway is known to monitor the cell-envelope stresses genome with a terminal protein covalently linked to each 5’ end, induced by a variety of signals, such as high temperatures, high and has been studied extensively as a model system of the pH, and toxic compounds. The two-plasmid system, which protein-priming DNA replication in which a viral-encoded protein consists of a sigma-E expression plasmid under the arabinose- provides the hydroxyl group needed by viral DNA polymerase to inducible promoter and a plasmid bank with various kinds of E. initiate the DNA synthesis. f29 gene 1 mutants which do not coligenomic DNA fragments fused to the b-galactosidase gene, produce functional gene 1 product (gp1) can replicate their was used to identify promoters recognized by RNA polymerase genome at 30˚C but can not at 42˚C, indicating that gp1 is containing the sigma-E. By monitoring of the b-galactosidase required for f29 DNA replication in a growth-temperature activities of the cells, we have identified such thirty sigma-E dependent fashion. Although several characteristics of gp1 have dependent promoters, which include eleven previously known been reported, its role in viral DNA replication is still unclear. By sigma-E dependent promoters, such as htrG, ftsZ and rybB isolating temperature-resistant revertants with spontaneous (encoding a small non-cording RNAs) promoters, and nineteen mutations arose from f29 gene 1 mutants, we have identified new members of promoters. They included promoters for the several second-site mutations that suppressed the defect of the chaA, dxs, and dcm genes, and a novel promoter for the rpoD gene 1 mutations at 42˚C. These suppressor mutations were gene encoding sigma-70 subunit of RNA polymerase. We located in gene 3 that encodes gp3 protein acting as a protein determined the fine structure of the novel sigma-E dependent primer and as a replication origin in initiation of viral DNA promoter of the rpoD by the footprinting analysis etc. replication. Although we have investigated function of the suppressors in in vitro f29 DNA replication, no differences were found between the wild type and the suppressors. We think other factors may be involved in the f29 DNA replication. To identify the unknown factors, we have trying to find gp1-associated proteins. 322 Genes Genet. Syst. (2014) 89

2B MATSUOKA, Satoshi1, NOBE, Kaori2, MATSUMOTO, 2C UNOZAWA, Eri1,2, KOBAYASHI, Takehiko1,2 (1Dept. -07 Kouji1, HARA, Hiroshi1 (1Grad. Sch. Sci. Eng., -01 Genet., Sch. Life Sci., The Grad. Univ. Adv. Stud., Saitama Univ., 2Dept. Biochem. Mol. Biol., Faclt. Sci., SOKENDAI, 2Kobayashi Lab., Dept. Cytogenetics, Saitama Univ.) The Natl. Inst. Genet., NIG) Influence of glucolipids on regulation of SigI in Bacillus The nuclear pore-associated proteins are involved in the subtilis maintenance of rDNA stability In Bacillus subtilis, glucolipids are synthesized by transfer The ribosomal RNA gene repeats (rDNA) in budding yeast has a glucose from UDP-glucose to diacylglycerol. This reaction is gene amplification recombination system in which the repeat catalyzed by UgtP and ugtP-disrupted cells were bent and number is recovered through the DSB repair pathway with sister distended. In these cells, activation of mreBH expression which chromatid recombination. In this system, at the replication fork is controlled by SigI, was observed. SigI have reported as a heat barrier (RFB) site which is associated with Fob1, the replication shock response sigma factor, and tethered by their cognate anti- fork is arrested, DNA double-strand break (DSB) is induced, the sigma factor RsgI to the membrane in non-stressed conditions. DSB is repaired by homologous recombination with sister- Promoter analysis by lacZ fusion revealed that sigI expression chromatid, some copies are replicated twice and the copy number was activated in ugtP disruptant. Therefore the glucolipids are increases. Once DSB recombines with improper copies, deletional considered to have a significant role in the activation of SigI. recombination of the repeat occur, and the rDNA becomes the There is a possibility that the regulation mechanism of anti- most unstable region in the genome. Therefore, the rDNA is sigma factor RsgI senses the change by the lack of glucolipids. expected to have a system in which recombination is properly Then we have introduced sigI-rsgI operon of B. subtilis into regulated. To reveal system, we performed ChIP assay and Escherichia coli cells and analyzed the activities of sigI w/o ugtP investigated the localization of DSB in the amplification expression by PsigI-lacZ as an indicator. As a result, sigI activity recombination. DSB in the rDNA is surely located around the was repressed by induction of ugtP in E. coli cells. It suggests that nuclear pore. The pore localization is decreased in the fob1 glucolipids may regulate SigI activity via membrane protein mutant in which DSB is not induced. We identified genes that are RsgI. required for the localization. We speculate that DSB in the rDNA is isolated at the nuclear pore and improper recombination is avoided.

2B HIRAI, Hiroyuki1, NAKAGAWA, Yukihiko1, 2C TAN, Kang Way1, PHAM, Tuan Minh1, FURUKOHRI, -08 FURUKAWA, Takehito1, CHE, Fang-Sik1 (1Grad. -02 Asako1, MAKI, Hisaji1, AKIYAMA, Masahiro T1 Sch. Bioscience, Nagahama Inst. Bio-Science Tech.) (1Grad. Sch. Biol. Sci., Nara Inst. Sci. Tech.) Specific recognition mechanism of flagellin from Acidovorax Reduced speed of DNA replication fork in the SOS response of avenue by rice Escherichia coli cells The flagellin from Acidovorax avenae rice avirulent N1141 strain induced several rice immune responses, while the flagellin from rice virulent K1 strain did not. Because recombinant N1141 and K1 flagellins equally induced the immune responses, post- translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analysis using glycosyltransferase-deficient mutants showed that 1,600 Da and 2,150 Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin acquired the immune induction activity, suggesting that epitope site of K1 flagellin is masked by the glycan. Structure analysis showed that although both glycans of N1141 and K1 flagellins were composed of trisaccarides, structure of non-reducing terminal sugar was different. Therefore, non-reducing terminal sugar-deleted N1141 and K1 flagellins were generated. The non- reducing terminal sugar-deleted K1 flagellin induced the immune responses, suggesting that the non-reducing terminal sugar of glycan in K1 flagellin is involved in the masking of the epitope site. Genes Genet. Syst. (2014) 89 323

2C KAWAKAMI, Hironori1, OHASHI, Eiji2, KAWANISHI, 2C NOGUCHI, Yasunori1, SAKIYAMA, Yukari1, -03 Tomohito1, TSURIMOTO, Toshiki2, KATAYAMA, -05 KATAYAMA, Tsutomu1 (1Mol. Biol., Pharm Sci., Tsutomu1 (1Dept. Molec. Biol., Grad. Sch. of Kyushu Univ.) Phamaceutical Sci.s, Kyushu Univ., 2Dept. Biol., Faclt. Sci.s, Kyushu Univ.) Analysis of DnaA complex organization on the E. coli replication origin Regulation of ORC: structure-function relationship of Orc1- In E. coli, ATP-DnaA binds to specific DNA sequences called Orc6 interaction DnaA boxes within the chromosomal replication origin (oriC), The origin recognition complex (ORC), a highly conserved, which results in formation of initiation complexes, thereby eukaryotic heterohexameric complex, recruits the helicase core initiating replication. In initiation complexes, the DnaA arginine MCM to chromosomal replication origins. Based on biochemical finger motif in AAA+ domain is thought to recognize ATP bound and single-particle EM analyzes, we previously found binary to an adjacent DnaA, which leads to formation of head-to-tail interaction between Orc1 and Orc6 subunits and proposed DnaA multimers. oriC bears a DnaA assembly region (DAR) dramatic structural changes within Orc1 and Orc6 in the process including two high affinity DnaA boxes, R1 and R4 boxes, and ten of MCM loading (Structure 2012; NSMB 2013). Recent progress low affinity DnaA boxes. DnaA boxes R1 and R4 reside on the left in the interaction between the two ORC subunits will be and the right ends of DAR, respectively, and these sequences are presented. mutually oppositely oriented. The R1 and R4 boxes have been considered to be assembly cores of DnaA multimers on oriC.In the present study, we constructed a novel DnaA mutant for analyzing organization of DnaA multimers bound to oriC.

2C KASHO, Kazutoshi1,FUJIMITSU,Kazuyuki2, 2C YOSHIYAMA, Kaoru Okamoto1, KIMURA, Seisuke1 -04 KATAYAMA, Tsutomu1 (1Dept. Molec. Biol., Grad. -06 (1Life Sci.s, Kyoto Sangyo Univ., 2JSPS) Sch. Pharm. Sci.s, Kyushu Univ., 2UCL Cancer Inst., University College London) The regulatory mechanism of SOG1, a plant-specific tran- scription factor of DNA damage response Biochemical search for novel regulatory factors of a non- As the genome contains all the information requires for develop- coding DNA element DARS2 ment and maintenance of an organism, it is very important to In Escherichia coli, the initiation of chromosomal DNA repli- protect the genome from DNA damage caused by the action of cation is regulated to occur only once per cell cycle, in a timely exogenous or endogenous sources. In response to DNA damage, manner. This regulation is achieved by multiple regulatory eukaryotic cells activate elaborate signaling pathways called systems for DnaA and the replication origin. DnaA activity is DNA damage response (DDR), which stimulates DNA repair, cell regulated by its bound nucleotides. ATP-bound DnaA (ATP- cycle checkpoints, and eventually apoptosis to remove or to DnaA) is active for initiation whereas ADP-DnaA is inactive. The tolerate lesions in their genetic material. We previously reported cellular ATP-DnaA level temporally increases only before that Arabidopsis SOG1 is a plant-specific transcription factor initiation. Immediately after initiation, DnaA-ATP is hydrolyzed that governs DDR. Furthermore, we showed that SOG1 is by the complex of Hda protein and the DNA-loaded clamp subunit regulated by AtATM through post-translational phosphorylation. of DNA polymerase III holoenzyme. This inactivation system is The C-terminal region of SOG1 has five SQ motifs, and one or required for repressing extra initiations. Recently, we revealed more of the SQ motifs were targets for the phosphorylaton. It is that a specific DnaA binding locus, datA, stimulated DnaA-ATP currently unknown which SQ motif(s) are actually phosphory- hydrolysis in a RIDA-independent manner. We termed this novel lated, and how SQ phosphorylation contributes to SOG1 mechanism DDAH (datA-dependent DnaA-ATP hydrolysis). functions. These points are currently being addresses using (Kasho and Katayama, 2013 PNAS). The resultant ADP-DnaA transgenic plants carrying SOG1 mutants, which encodes serine- can be reactivated by another DnaA binding locus DARS2 (DnaA to-alanine substitutions at four out of the five SQ motifs. We reactivating sequence 2) which stimulates nucleotide exchange of would also like to discuss when plants acquire the SOG1 during DnaA. In this meeting, we will report that two nucleoid proteins their evolution. are identified as DARS2 activators and propose a model in regulatory mechanisms for timely initiation of chromosomal replication. We are now searching regulatory factors for DARS2-activator binding. 324 Genes Genet. Syst. (2014) 89

2C YOSHIDA, Asami1, KEYAMURA, Kenji1, HISHIDA, 2C HIGASHIDE, Mika1, SHINOHARA, Miki1 (1Lab. -07 Takashi1 (1Dept. of Life Sci., Grad. Sch. Sci., -09 Genome and Chromosome Functions, Inst. Prot. Res., Gakushuin Univ.) Univ. Osaka) A genetic screen for histone H3 and H4 mutants affecting Gmc2, a novel synaptonemal complex component, regulates DNA damage response in Saccharomyces cerevisiae the number of crossovers between homologous chromosomes in meiosis Genomic DNA is constantly threatened by various DNA-damag- ing agents. Therefore, eukaryotic organisms have evolved several Crossover (CO) formation between homologous chromosomes DNA damage response mechanisms that repair and tolerate DNA ensures proper segregations of homologous chromosomes via damage to maintain genome stability. In eukaryotic nuclei, DNA chiasma formation in meiosis I. Then, at least one CO must be is wrapped around histone proteins and is packaged into higher assured on each homolog pair. Meiosis-specific chromosome order levels of chromatin structure. It has been suggested that structure synaptonemal complex (SC) component ZMM com- the histone-dependent chromatin dynamics play an important plexes play an important role for CO regulation. However, the role in the DNA damage response. However, how they affect function of SC elongation in CO control is still unknown. Recently repair and tolerance mechanisms remain largely unclear. In this it was reported that a novel SC component, Gmc2-Ecm11 study, we constructed a series of double mutants combined a complex, facilitates SC elongation via Zip1 polymerization. Then, histone H3- or H4-substituted mutation with deletion of RAD18, we focused on gmc2 or ecm11 mutations as a convenient tool to and screened for histone alleles that affect the sensitivity of examine the function of SC elongation. In our study, to rad18 deletion cells to DNA-damaging agents. Rad18 plays investigate the role of SC elongation in CO regulation, we central roles in DNA damage tolerance pathway. Thereby, examined CO frequency in gmc2 deleted cells using genetic histone alleles involved in the DNA damage response can be analysis. Then, while we observed CO reduction in shorter detected with high sensitivity by using the rad18 deletion chromosomes, we observed CO elevation in longer chromosome. background. As a result of screening, we identified novel histone This result indicated the presence of a mechanism that gathers alleles and characterized roles of those. Based on these findings, COs to shorter chromosomes dependent on SC elongation in wild- we will discuss novel functions of histone in the DNA damage type cells. Then, I carried out cytological analysis to investigate response. progression of CO and SC formation. Here, I would like to introduce and discuss our work in detail.

2C TSUTSUI, Yasuhiro1, ITOH, Kentaro1, KUROKAWA, -08 Yumiko2, IWASAKI, Hiroshi1 (1Grad. Sch. BioSci. and BioTech., Tokyo Inst. Tech., 2Education Academy of Computational Life Sci., Tokyo Inst. Tech.) Regulation of Rad51 recombinase by Fbh1 in fission yeast In mitotic cells, homologous recombination (HR) is required for repairing DNA double-strand breaks (DSBs), which are induced by endogenous factors such as collapse of DNA replication fork or by exogenous factors such as DNA damaging agents. DSBs are prone to cause chromosome rearrangement, cell death, or tumorigenesis in mammals, if improperly processed. Therefore, HR is tightly regulated at several steps. Specifically, DNA helicases have been shown to be important for suppression of inappropriate recombination events. In this study, we analyzed one such DNA helicase, fission yeast F-box DNA helicase, Fbh1. We found that Fbh1 inhibits the strand-exchange reaction in the absence of Swi5-Sfr1, but stimulates the reaction after it starts in vitro. Furthermore, we found that SCFFbh1 has ubiquitin-ligase activity toward Rad51 in vitro and that Fbh1 regulates the protein level of Rad51 in the stationary phase. Based on these results, we propose that Fbh1 regulates Rad51-mediated HR by its two activities, DNA helicase/translocase and ubiquitin ligase. Genes Genet. Syst. (2014) 89 325

2D YUUDAI, Tsujino1, KAWABE, Akira1, YOSHIDA, 2D SUGINO, Erika1, TAKASAKI, Shintarou1, HARADA, -01 Takanori1, FURIHATA, Hazuka Y1 (1Faclt. Life Sci., -03 Yu1, SUN, Hui2, NEVO, Eviatar3, ASADA, Nobuhiko1 Kyoto Sangyo Univ.) (1Dept. Zool., Fac. Sci., Okayama Univ., Sci., 2School Life Sci., Northeast Norm. Univ., Changchun, 3Inst. Integration pattern of chloroplast DNA fragment to nuclear Evolution, Fac. Sci. Edu., Univ., Haifa) genome in Arabidopsis species Molecular evolution of mtDNA COI in Drosophila mela- In eukaryotes, organelles and nucleus hold their own DNA. nogaster at the Evolution Canyon Organelles DNA fragments were often transferred to the nucleus. However, most of them were lost. We analyzed the existence Fine-scale genetic variation of mitochondrial cytochrome oxidase pattern of nuclear plastid DNAs fragment (NUPT) in Arabidopsis subunit I DNA (mtCOI) was analyzed in Drosophila melanogast- thaliana and its closely related species Arabidopsis lyrata. er. 2-1 and 6-1 strains were used collected at the south-facing Compared with A. thaliana, A. lyrata have many young NUPTs. slope (warmer) and 6-1 strain from the north facing slope (cooler), A. thaliana might have efficient removal mechanism of NUPTs respectively, at the “Evolution Canyon”, Haifa, Israel, respec- than A. lyrata. In addition, we analyzed the pattern of mutations tively. Oregon-R strain was used as the control. DNA was in NUPTs. In both A. thaliana and A. lyrata, young NUPTs have prepared from adult flies, subjected to amplify by PCR, and higher number of C to T and G to A transitions than T to C and A analyzed using MEGA software (version 6). Alignment analyses to G transitions. In contrast, this bias was not found in the old showed significantly higher exchange rate of DNA and amino NUPTs. We also analyzed patterns of mutations in detail by acid in 6-1 strain than in 2-1 strain. Higher ratio of non- using closely related species A. lyrata for NUPTs found in both A. synonymous to synonymous substitution rates (dN/dS) within lyrata and A. thaliana. The mutation biases toward GC to AT species suggested as balancing selection with microclimatic stress were found just after integration of NUPTs, but this mutation and the life history difference. The enigma of genetic diversity bias might became lower during time. and molecular evolution in nature has been fruitfully explored by using molecular analysis. Biodiversity evolution, even in small- scale population, is primarily driven the effect of free gene flow, random genetic drift, and/or stochasticity at the “Evolution Canyon”.

2D KUSUMI, Junko1,2, TACHIDA, Hidenori2 (1Dept. 2D NAKAMURA, Yo1, TORII, Yuji1, ISOBE, Kotoha1, -02 Environmental Changes, Faclt. Social and Cultural -04 SATOMURA, Kazuhiro1, TAMURA, Koichiro1 (1Dept. Studies, Kyushu Univ., 2Dept. Biol., Faclt. Sci.s, Biol. Sci.s, Tokyo Metro. Univ.) Kyushu Univ.) Population genetic analysis of cold tolerance candidate genes Evolutionary rate in conifers (Cupressaceae) in Drosophila albomicans The genomes of conifers are characterized by large size, slow Drosophila albomicans has expanded the distribution range from evolutionary rates, and accumulations of a large amount of tropical to temperate zones during the last three decades. In our noncoding DNAs. Such genomic characteristics of conifers have previous studies, we found that temperate populations had lately attracted considerable attention as to what makes the stronger cold tolerance after cold acclimation than tropical differences in these genomic characteristics, what kind of evolu- populations, and that up-regulation of sdr was involved in the tionary features does the conifer genome have. Here, we effect of cold acclimation. In this study, determining and estimated evolutionary rates in two conifer species, Taxodium comparing nucleotide sequences of sdr between populations, we distichum and Cryptomeria japonica to analyze their molecular examined the trace of natural selection on the sdr. We also evolutionary features by using hundreds of genes. The mutation investigated sequence variations of pepck,whichwasalso rates based on dS of two species were estimated to be 0.67×10-9 suggested as a candidate gene. As the results, we found a and 0.59×10-9 /site/year, respectively, which are 15-25 times tendency that the upstream of pepck was differentiated between lower than those in angiosperm species. In addition, the means of populations. Comparing phylogenetic trees reconstructed for each of two species are almost the same, suggesting the level of 500 bp segment between the initiation codon and its 2.5 kb functional constraint is constant among the lineages on average. upstream, we found that the -1 to -500 upstream sequences have We found a significant positive correlation between dS and GC3. been conserved in the temperate populations. This may suggest This result could be explained by local and/or temporal mutation that the regulation of pepck expression was a target of natural bias but more precise estimation of dS is necessary to know selection during the distribution expansion of D. albomicans.In biological implications of these observations. contrast, neither upstream nor protein coding region of sdr did not show such tendency. 326 Genes Genet. Syst. (2014) 89

2D MIYAGI, Ryutaro1, OSADA, Noki2, TAKAHASHI, 2D ISOBE, Miyu1, NUNOME, Mitsuo2, SUZUKI, Hitoshi1 -05 Aya1,3 (1Dept. Biol. Sci.s, Tokyo Metro. Univ., 2Div. -07 (1Grad. Sch. of Env. Science Div. Biosphere Sci. Evolutionary Genet., Natl. Inst. Genet., 3Res. Cent. for Hokkaido Univ., 2Grad. Sch. Bioagricultural Sci., Genomics and Bioinform., Tokyo Metropolitan Univ.) Nagoya Univ.) Cis-regulated expression variation of the melanin biosyn- Evolutionary dynamics of 5S ribosomal DNA in the house thesis pathway genes in a population of Drosophila mela- mice (Mus musculus) nogaster We conducted three experiments to better understand of the Variation of D. melanogaster pigmentation in cuticle color, which evolutionary patterns of the 5S ribosomal DNA (5S) in the mouse, is likely to be an adaptive trait, is mainly caused by changes in Mus musculus. First, we examined copy numbers of 5S in eight cis-regulated gene expression level of a melanin biosynthesis laboratory strains with the pulsed-field-gel electrophoresis gene, ebony. Yet little is known about the effect of other melanin method and found that the estimated copy numbers ranged from biosynthesis genes such as tan and yellow, which are known as 130-170. Second, we assessed the trend of acceleration of meiotic responsible genes for the divergence of body pigmentation among recombination across the 5S array, examining the natural Drosophila species. In this study, a detailed analysis on the population of northern Japan where two subspecies linages are melanin biosynthesis genes was conducted to investigate the mingled. We found that the 5S array did not accelerate the effects of their cis-regulatory variations on pigmentation traits. meiotic recombination processes at an accelerated rate. Third, we We quantified cis-regulated allelic expression ratios (cAERs) for estimated the nucleotide diversity in six loci lying adjacent to the the sampled alleles from the wild-derived Drosophila Genomic 5S array in each subspecies group. A trend of a selective sweep Resource Panel by crossing each strain to a standard reference was visible in some subspecies groups. These results may suggest strain. Correlation analysis between cAERs and pigmentation that the sequence similarity of the 5S copies within a genome and scores indicated that cAERs in ebony and tan were significantly within a population are attributed to different systems of certain correlated with the overall body pigmentation and the abdominal mechanisms that operate a single 5S array and natural selection stripe patterns, respectively. The results suggested that changes on 5S arrays, respectively. in cis elements of those genes are responsible for the pigmenta- tion variation in this species.

2D SATOMURA, Kazuhiro1 (1Dept. Biol. Sci.s, Tokyo 2D IWABE, Naoyuki1 (1Dept. Biophys., Grad. Sch. Sci., -06 Metro. Univ., 2Res. Cent. for Genomics and Bioinform., -08 Kyoto Univ.) Tokyo Metropolitan Univ.) Molecular evolutionary rates and functional constraints on The influence of recombination on neo-Y chromosome multidomain proteins evolution of Drosophila albomicans Based on molecular evolutionary analyses of protein kinase (PK) Sex chromosomes have evolved independently in various eukary- and immunoglobulin family members, Kuma et al. (1995) otic taxa. After recombination suppressed between X and Y proposed that functional constraint on proteins were separated chromosomes, the sex chromosomes differentiates radically. into two different components, local and global ones: Local Absence of recombination impairs the efficacy of natural selection constraint is related to functional and structural features and decreases genetic diversity. To clarify the influence of the characteristic of individual molecules, while global constraint is meiotic recombination suppression between X and Y chromo- derived from higher levels like tissue or organs. In this study, somes on the Y chromosome evolution, young Y chromosome is synonymous and nonsynonymous substitutions of multidomain required. members of PK and protein tyrosine phosphatase (PTP) families A fruit fly species, Drosophila albomicans has neo-X and neo-Y were calculated between human and mouse data, and then the chromosomes originated by a fusion of X and an autosome and a nonsynonymous values of orthologs were compared between fusion of Y and the autosome, respectively. The neo-X and the different genes or between different domains. The results were neo-Y chromosomes shares so many genes derived from the as follows: 1) Some PTP domains evolved 50 to 100 times more autosome. Therefore, comparing genetic diversity between the rapidly than the slowest evolving one. 2) Very slowly evolving neo-X and the neo-Y chromosomes, we investigated the influence genes were expressed ubiquitously, or in many cell types, while of meiotic recombination on early stage of Y chromosome rapidly evolving ones tended to be expressed in specific cell types, evolution. such as hematopoietic cells. 3) Many multidomain members of PK The genetic diversity of the neo-Y genes was similar to that of the and PTP families evolved under the effect of global constraint neo-X genes. The result suggests recombination between the neo- that simultaneously affected mutational changes of different X and the neo-Y chromosomes in ancestral population. Further- domains in a protein. more, low genetic diversity of the neo-Y genes was observed in Japan/Taiwan population. It may be caused by hitchhiking effect. Genes Genet. Syst. (2014) 89 327

2D HAYAKAWA, Toshiyuki1, ANGATA, Takashi2 (1Faclt. 2E TAKADA, Toyoyuki1, KONDO, Shinji2,3, ABE, -09 Arts and Sci., Kyushu Univ., 2Inst. Biological -01 Takashi4, KIYOSAWA, Hidenori5, TOYODA, Atsushi6, Chemistry, Academia Sinica) FUJIYAMA, Asao6,SHIROISHI,Toshihiko1 (1Mammalian Genet. Lab., Natl. Inst. Genet., Human-specific Siglec paired receptors in the brain 2Transdisciplinary Res. Integration Cent., Res. 3 Sialic acids are a family of nine-carbon sugars that are found at Organization of Information and Systems, National 4 the terminal end of glycan chains on cell surface. Siglec-11 is a Inst. Polar Res., Dept. Inform. Eng., Niigata Univ., 5 sialic-acid receptor that gained expression on brain microglia Dept. Environmental Medicine, Kochi Univ., 6 uniquely in the human lineage, and shows a neuroprotective Comparative Genomics Lab., Natl. Inst. Genet.) function in brain immunity. A mutation that conferred the brain Allele-specific gene expression analysis by mouse inter- expression of Siglec-11 occurred about one million years ago in a subspecific genome divergence gene conversion by Siglec-16 gene. Interestingly, Siglec-16 gained microglial expression uniquely in the human lineage as well as An inbred strain MSM/Ms (MSM) is derived from Japanese wild Siglec-11. Siglec-11 functions as an inhibitory receptor, but mice, Mus musculus molossinus. Since fine genome sequence of Siglec-16 is an activating receptor. To know the role of Siglec- C57BL/6J (B6) and SNP information of MSM are available, it is 11 and Siglec-16 in the human brain, we examined sialic acid becoming realistic to explore the genome functions based on recognition properties of these Siglecs. statistical linking of phenotypes and gene expression levels of The recombinant proteins of human Siglec-11 revealed a glycan these two strains. Now, we are developing a platform to measure binding preference similar to that of human Siglec-16. In allele-specific gene expression using RNA-sequencing analysis addition, both Siglec proteins bind to oligo sialic acids [(Neu5- with various tissues of (B6 x MSM) F1 mice. In this meeting, we

Ac2-8)2] which are enriched in the brain. Human Siglec-11 and will show our trial to analyze allele-specific gene expression in Siglec-16 are therefore regarded as paired receptors and contrib- liver of the F1 mice. ute to the fine-tuning of immune responses of brain microglia.

2D FUJITO, Naoko T1, HAYAKAWA, Toshiyuki2, HANE, 2E AIZAWA, Yasunori1, FUKUDA, Makiha2, ITO, -10 Masaya3, KITAJIMA, Ken3, SATO, Chihiro3, SATTA, -02 Tomoya2, KITANO, Shohei2, OHNO, Tomoyuki2, Yoko1 (1Sch. Adv. Sci., Grad. Univ. Adv. Stud., 2Faclt. KUBO, Natsuki2, MATSUSHITA, Ei2 (1Center for Arts and Sci., Kyushu Univ., 3BioSci. and BioSci. Biol. Resources and Inform., Tokyo Inst. Tech., 2Grad. Center, Nagoya Univ.) Sch. BioSci. and BioTech., Tokyo Inst. Tech.) Adaptive evolution of the promoter region of the Sialyltrans- Functional studies of human protein-gene candidates ferase 8B (STX) gene STX is an enzyme responsible for the synthesis of polysialic acid (PSA) in a human brain. PSA is a ligand for Siglec-11 and -16, showing human-specific expression in the brain, and believed to play an important role for higher order of the function in the human. Variants of the STX have shown association with various mental disorders, such as bipolar disorder and autism. Among them, we focused on the association between polymorphisms in the STX promoter region and schizophrenia. “Risk” and “protec- tive” SNPs in the region have been reported. Interestingly, one of human “risk” SNPs has been fixed in apes and “protective” type is likely to have been derived in the human lineage. The promoter activity of the risk types is significantly higher than that of the protective type. We determined haplotype sequences within the 10 kb surrounding the SNPs, using genomic DNA of 63 human individuals from a wide range of ethnic groups. Phylogenetic and population genetic analysis based on the entire sequences showed that the protective type emerged in Africa about 0.8 MYA. The protective type showed the prevalence in Asian, suggesting some local selection on it or linked some other loci. 328 Genes Genet. Syst. (2014) 89

2E SUZUKI, Motoo1, IMAOHJI, Haruyuki1, KUWAHARA, 2E YAHARA, Koji1, DIDELOT, Xavier3, ANSARI, M -03 Tomomi1 (1Dept. MicroBiol., Faclt. Med., Kagawa -05 Azim4, SHEPPARD, Samuel K2, FALUSH, Daniel5 Univ.) (1Biostatistics Center, Kurume Univ., 2Swansea Univ, 3Imperial College London, 4Univ. Oxford, 5Max Planck Spore purification of mouse gut derived non-culturable Institute) bacterium Efficient inference of recombination hot regions in bacterial Segmented filamentous bacteria (SFBs) are clostridia-related genomes Gram-positive bacteria with unique morphology. SFBs are non- culturable spore-forming bacteria, tightly attaching to the Surveys of recombination rates in eukaryotes have shown intestinal epithelial cells. SFBs play crucial roles in the variation at multiple genomic scales and the presence of maturation of gut immune function, especially the induction of “hotspots” of highly elevated recombination, although studies of Th17 lymphocytes. However, we found the phenomenon that recombination rate variation are less developed in bacteria. Here Th17-induction of SFBs was not detected by repeating fecal we present a computationally efficient algorithm for identifying passages in germ-free mice. recombination “hot regions” of the genome where DNA is We suspected that Th17-uninducible SFB strain was selected transferred frequently between isolates. Using simulated data, from multiple clones, since we observed many single?nucleotide we show that hot regions have consistently higher deviations polymorphisms in DNA sample obtained for whole-genome from the genome wide average than normal regions. We applied sequencing analysis. To clarify the effect of each SFB strain on our approach to previously analysed Escherichia coli genomes, Th-17 induction, we attempted to isolate single-clone SFB. In this and revealed that the new method is highly correlated with the report, we have established the method for purifying spores from number of recombination events affecting each site. Furthermore, feces of SFB-gnotobiotic mice. The SFB spore induced Th17 by we analysed recombination hot regions in Campylobacter jejuni inoculating into germ-free BALB/c mice when spores were by using 200 genomes. We identified three recombination hot purified from dried feces used for genome sequencing. However, regions which are enriched for genes related to membrane the SFB spores purified after repeating fecal passage were Th17- proteins. Our approach and its implementation, which is down- uninducible. These results suggest possibility of single-clone SFB loadable from https://github.com/bioprojects/orderedPainting, isolation from original dried feces containing multiple SFB will help to develop a new phase of population genomic studies clones. of recombination in prokaryotes (Yahara et al, 2014, MBE).

2E ABE, Takashi1, NAKATA, Toshiyoshi2, KUMAGAI, 2E MIAWA, Kazuharu1 (1Advanced Center for -04 Yosuke1, SATO, Shusei3, HIRAKAWA, Hideki3, -06 Computation and Communication) KONDO, Akihiro2, SUGIMOTO, Chihiro4, IKEMURA, Toshimichi5, MATSUI, Kazuhiko2 (1Niigata Univ., A new homology search based on MAFFT 2 3 4 Hiyoshi Corp., Kazusa DNA Res. Inst., Hokkaido In 2009, real-time sequencing from single polymerase molecule 5 Univ., Nagahama Inst. Bio-Sci. & Tech.) was developed (Eid et al. 2009, Science 323: 133-). The DNA sequencers which can obtain real-time sequencing from single Metagenomic studies of activate sludge to search for genes polymerase molecule are known as the third-generation se- and genomes with environmental and industrial importance quencers. The third-generation sequencers enable us to sequence Metagenome analyses, which directly sequence mixed genomes of reads of several kilobases. Unfortunately, the raw data generated uncultured environmental microorganisms, can provide detail of from the third-generation sequencer is known as error-prone the microbial community structure and genetic potential of (Quail et al. 2012, BMC Genomics 13: 341-). Because of environmental samples such as sea water and activate sludge. sequencing errors, it was difficult to find genes which are We have previously developed a Batch Learning Self-Organizing homologous to the reads obtained by the third-generation Map (BLSOM) (Genome Res. 13, 693-702, 2003), which can sequencers. In this study, a new method for homology search is cluster genomic fragment sequences according to phylotypes developed. This method is based on the MAFFT algorithm (Katoh solely dependent on oligonucleotide composition, and applied to et al. 2009, Nucleic Acids Res 30: 3059-). Like MAFFT, several metagenome studies (DNA Res. 12, 281-290, 2005; Nature homologous regions are rapidly identified by this method based Biotech. 23, 88-93, 2005; ISME J. 7, 1003-1015, 2013). The on the fast Fourier transform (FFT). Thanks to FFT, the new present BLSOM study has analyzed metagenomic sequences method can find global homology rather than local homology. The from 8 activated sludge samples with different activities and new method can find sequence homology even when the error predicted microbial diversities in each sample. In order to rate in sequencing is high. The new method will boost application understand genetic potential in the activated sludge, we have of the third-generation sequencer. performed functional annotations of gene sequences with se- quence similarity searches and compared candidate genes with environmental and industrial importance between samples. The present metagenome study including BLSOM analysis can provide a systematic strategy for understanding microbial diversities and functional details of microbial ecosystems. Genes Genet. Syst. (2014) 89 329

2E FUJIMOTO, Akihiro1 (1RIKEN Center for Integrative 3B TSUJI, Masaya1, HIRAI, Noriyuki1, OZAWA, -07 Medical Sciences) -01 Chikako1, NARA, Atsuki1 (1Nagahama Inst. Bio-Sci. Tech.) Detection of mutations in microsatellite region from next- generation sequencing data MLN64 with lipid binding region controls the cellular uptake of low density lipoprotein Microsatellite instability (MSI) is a genetic hypermutability that results from impaired DNA Mismatch Repair. MSI is one of the We are interested in the unknown communication mechanism most important features of the mutation signature in cancer. between the endosomes and mitochondria. We research and However, MSI identification is difficult by next generation believe that protein MLN64 is the key. sequencer (NGS), and only a few studies have been carried out In the present study, the late endosomal membrane protein to identify MSI in cancer. We have developed a method to identify MLN64 was found in fractions containing early endosomes in mutations in microsatellite regions and applied the method to lipid deficient conditions. Furthermore, MLN64 stable knock- liver and colorectal cancer (CRC) samples. down cells were not imported low density lipoprotein, and the We first estimated the error rate for different patterns of receptor had accumulated in the cells. In the future, I identify microsatellite, such as mononucleotide, dinucleotide, and trinu- this accumulated partition whether it is early endosomes. In cleotide repeats. Then, we calculated likelihood for each micro- order to unravel the mechanism of the cholesterol transport satellite region based on the estimated error rate. Using the between endosome and mitochondria, and is planning an in vitro likelihood, we identified mutations in microsatellite regions. production system construction of pregnenolone synthesized from Experimental validation suggested that the false positive rate of cholesterol in mitochondria. the method is about 7 %. We analyzed 11 liver cancer and three CRC samples. The numbers of MS mutations in the most liver cancers and CRCs were small, but a liver cancer and a MSI+ CRC had a large number of MS mutations. Mutation rates of these samples were estimated to be 6% (liver cancer) and 3% (MSI+ CRC).

2E SATO, Yukuto1, KOJIMA, Kaname1, NARIAI, Naoki1, 3B TAKANO, Akira1, SHIBA, Yasuhiro1, MIYAGAWA, -08 YAMAGUCHI-KABATA, Yumi1, KAWAI, Yosuke1, -02 Hiroyoshi1, UMEKAWA, Mitsuru1, MATSUMOTO, NAGASAKI, Masao1 (1Dept. Integrative Genomics, Kouji1, MATSUOKA, Satoshi1, HARA, Hiroshi1 Tohoku Medical Megabank Organization, Tohoku (1Dept. Biochem. and Molecular Biol., Grad. Sch. Sci. Univ.) and Eng., Saitama Univ.) SUGAR: graphical user interface-based high-resolution data Analysis of interaction between RcsF and periplasmic region cleaning tool for high-throughput sequencing data of RcsC of the Rcs phosphorelay signal transduction system of Echerichia coli To obtain valuable insights from high-throughput sequencing data, it is essential to discard low-quality reads or nucleotides The Rcs phosphorelay signal transduction system controls genes affected by technical errors such as air bubbles in sequencing for capsule production and many other envelope-related functions fluids. We developed a software SUGAR (subtile-based GUI- and is implicated in biofilm formation. Environmental stimuli are assisted refiner) that handles full data of Illumina HiSeq and sensed by a minor outer membrane lipoprotein RcsF and MiSeq machines with GUI interface and interactive analysis transmitted to RcsC histidine kinase in the inner membrane. capability. The SUGAR generates high-resolution quality heat- Rcs system was activated when RcsF was mislocalized to the maps of the flowcell (~100x100 per tile), enabling users to find inner membrane or to the periplasm. Expression of the signals of technical errors during sequencing. In addition, the periplasmic region of RcsC (RcsCperi) fused to maltose-binding sequence reads/nucleotides generated from error-affected regions protein (MBP) in the periplasm repressed activation of the Rcs of a flowcell can be removed by automated analysis or GUI- system. Thus we suppose RcsF directly interacts with RcsCperi. assisted operations. We show that the automated cleaning We expressed RcsF as a periplasmic protein directed by the signal operation applied to a public human genome sequencing data sequence of MBP and deleted for proline-rich region (PRR). improved its overall mapping quality (MapQ) (29.5 to 30.1; an Deletion of PRR in the N-terminal region of RcsF increased average MapQ of the discarded reads was 25.0). Such a data activity of the Rcs system. We also expressed MBP-RcsCperi cleaning enabled by the SUGAR would improve subsequent data fusion protein in the same cell. By formaldehyde cross-linking analysis for which the high-quality base calls and mapping and Western blotting we detected several high molecular weight results are required. Consequently, this software will be complexes containing RcsF?PRR and MBP-RcsCperi. These especially useful for the analyses of cancer genomes, somatic complexes seem to contain MBP-RcsCperi and multiple copies mutations, mitochondrial heteroplasmy, etc. of RcsF?PRR. Or else, they may contain another protein mediating the interaction of RcsF and RcsCperi. 330 Genes Genet. Syst. (2014) 89

3B SEKI, Takahiro1,MINESHIMA,Ryota1, 3B AIHARA, Reiko1, HIRASAWA, Yukei2, MERA, -03 HASHIMOTO, Michihiro1, MATSUOKA, Satoshi1, -05 Hanaka2, EZAKI, Kazune2, HIRATSU, Keiichiro3, MATSUMOTO, Kouji1, HARA, Hiroshi1 (1Grad. Sch. NUNOSHIBA, Tatsuo1,2 (1Grad. Sch. Arts and Sci., Sci. Eng., Saitama Univ.) International Christian Univ., 2College of Liberal Arts, International Christian Univ., 3Dept. Appl. Chem., The role of glucolipids in the regulation of ECF sigma factors Natl. Defense Acad.) in Bacillus subtilis Analysis of Genomic Integrity in Thermus thermophilus-The Bacillus subtilis has membrane glucolipids, which are synthe- Roles of AP endonuclease - sized by UgtP. The ugtP mutant cells lacking glucolipids show abnormal morphology and the activation of three extracytoplas- High temperature increases DNA damages such as AP sites. mic function (ECF) sigma factors, sM, sV,and sX. In non-stressed Therefore, Thermus thermophilus, which grows at around 70?C, conditions the ECF sigma factors are sequestered by their is a useful organism for understanding mechanism of genome cognate transmembrane anti-s factors, from which they are stability. As the homolog of E. coli 5’ AP endonuclease nfo, which released under stress conditions. The Escherichia coli membrane repairs AP sites, TTC0482 has been confirmed inT. thermophilus. does not contain glucolipids. When the genes for sM, sV, and sX To clarify the roles of AP endonuclease TTC0482 on genome and their cognate anti-s factors were introduced into E. coli cells, stability under high temperature, we have analyzed both rate of expression of lacZ fused to the ECF s-regulated promoters homologous recombination (HR) in nfo deficient strain and the indicated ECF s factor activity. Additional expression of the ugtP nfo gene expression. gene in these E. coli cells led to synthesis of small amounts of In our HR detecting system with vector pMO, when HR occurs glucolipids and repression of the activities of sM and sV to about over the homologous regions inside tet gene, the tet gene recovers 60% and 40%, respectively, but sX was unaffected. These results and converts the colony from sensitive to resistant against suggest that glucolipids directly influence anti-sM and anti-sV tetracycline. We adopted this phenotypic shift as the marker for factors by inducing conformations that sequester the respective HR. In gene expression analysis, crtB, which encodes phytoene ECF s factors. synthase, was used as a reporter, and its final product carotenoid was extracted, and measured OD454 for quantification of gene expression. From these results, the roles of AP endonuclease on genome integrity will be discussed.

3B OZAWA, Yoshiki1, SHINJOU, Yu1, HASEGAWA, 3B HATAMOTO, Yuki1, KUTSUKAKE, Kazuhiro1 -04 Toshio1, ASAI, Kei1 (1The Grad. Sch. Sci. Eng., -06 (1Grad. Sch. Natural Sci. Tech. and Dept. Biol., Faclt. Saitama Univ.) Sci., Okayama Univ.) Analysis of mechanism of vetiver extracts-induced stress Expression control of the flagellar regulon by SdiA in response in Bacillus subtilis Escherichia coli The plant extracts such as the essential oil have been used in SdiA is a LuxR family protein believed to be involved in quorum various purposes including medicine and incense for many years, sensing in E. coli. SdiA has been known to negatively regulate and it has been thought that they possess some kind of motility and flagellar gene expression, though its molecular bioactivity. The extract from the root of Vetiveria zizanioides mechanism remains unknown. SdiA has been also shown to (vetiver) has striking antimicrobial activity against B. subtilis activate the transcription of the ydiV gene, which encodes an which is representative of the Gram positive bacterium. We found anti-activator protein specific for FlhD4C2, the master regulator that one of the extract, sesquiterpene compound designated of the flagellar regulon. Taken together, these facts suggested a Khusimol, had antimicrobial activity. We analyzed the effect of possibility that SdiA regulates flagellar gene expression through Khusimol on stress response of B. subtilis. The sensitivity of the its positive regulation of the ydiV gene. This work was carried out B. subtilis cells, in which some transcriptional regulators so- to test this possibility. As expected, overproduction of the SdiA in called ECF sigma factors were deleted, to the vetiver extract the wild-type strain inhibited the transcription of the ydiV gene increased comparing with that of wild type cells. The suppressor and the production of the YdiV protein. However, it was found mutants, which restored ability of tolerance to vetiver extract, that the overexpressed SdiA still inhibited motility and flagellar were obtained from the ECF sigma-deleted strain exposed to gene expression even in the absence of YdiV. This suggests that vetiver extract. We have been examined what kind of stress B. SdiA can regulate E. coli motility through at least two subtilis responded to when the cells were exposed to vetiver independent pathways: one is via its positive regulation of the extract by means of reporter genes for oxidative stress and DNA ydiV gene and the other is via direct or indirect, but YdiV- damage. independent, negative regulation of the flagellar genes. Genes Genet. Syst. (2014) 89 331

3B KATSURAGI, Yuya1, MORIMOTO, Takumi1, 3B KISHIMOTO, Naoki1 (1Natl. Inst. AgroBiol. Sci.) -07 KATAYAMA, Takara1, MURAKAMI, Takahiko1, -09 TAKAI, Ryota1, CHE, Fang-Sik1 (1Grad. Sch. Bio-Sci, Nagahama Inst. Bio-Sci. Tech.) Preliminary studies on plant sirtuins (SIRTs) and SIRT4 ortholog mutants of rice, Oryza sativa Rice recognition system of flagellin derived from phytopatho- My blast results with human SIRTs (SIRT1∽SIRT7) suggested genic bacteria gymnosperms had SIRT4, SIRT5 and SIRT6 orthologs, as moss Flagellin from rice-avirulent strain of phytopathogenic bacteria (Physcomitrella) and fern (Selaginella) do, but not as Angio- Acidovorax avenae induces several immune responses in rice. sperms do (SIRT4 and SIRT6 orthologs only). Arabidopsis recognizes the most conserved N-terminal domain of Two rice mutants of SIRT4 ortholog were found in Tos17-inserted flagellin that consists of a 22-amino acid peptide (flg22) using lines (cultivar ‘Nipponbare’). The homozygous mutants (mu) FLS2 receptor. By contrast, rice did not recognize flg22, and the showed normal growth but produced no seeds though the wild flagellin C-terminal domain induced the rice immune responses. types (wt) and heterozygotes bore normal seeds. Agilent 44K Gene expression profiling during flagellin treatment showed that array analyses with leaf RNAs of the mu and the wt, both of expression of several genes encoding the receptor kinase were which grew in a greenhouse keeping relatively hot and humid, increased. Transient expression and knock out of the identified identified 33 and 100 activated genes in the mu and the wt, genes showed that Flagellin-induced Receptor Kinase 2 (FliRK2) respectively. For these genes, I compared the expression levels in is involved in the recognition of flagellin C-terminal domain. The the mu and the wt with those in dataset of RiceXPro, a binding experiment using ELISA demonstrated that FliRK2 transcriptome database based on the same array and cultivar. interacts with the flagellin C-terminal domain. Furthermore, the For ca. 2/3 of the 133 genes, the expression levels in paddy-grown flagellin recognition ability in flirk2 was recovered with FliRK2 plants of RiceXPro appeared like those in the mu than those in expression, but not kinase domain deleted FliRK2. Phosphor- the wt, suggesting the mu could not properly respond to abiotic ylation of MAPKs was observed after the flagellin C-terminal environments in the greenhouse. Intriguingly the level of a domain treatment. These results indicate that this flagellin glyoxylate cycle gene (scarce in leaf, very high in endosperm; by recognition system has functional homogeny with mammalian RiceXPro) was low in the mu and very high in the wt. innate immunity mediated by TLR.

3B KAMIMURA, Mayu1, HAN, Yulong2, CHISAKA, 3B KAJITANI, Takuya1,2,KATO,Hiroaki3, -08 Mami1, KITO, Nobuki2, CHE, Fang-Sik1,2 (1The Dept. -10 CHIKASHIGE, Yuji4,HIRAOKA,Yasushi4,5, BioSci., Nagahama Inst. Bio-Sci. Tech., 2Grad. Sch. of KIMURA, Hiroshi5, OHKAWA, Yasuyuki6, Bio-Sci., Nagahama Inst. Bio-Sci. Tech.) HERMAND, Damien7, MURAKAMI, Yota1 (1Grad. Sch. Sci., Hokkaido Univ., 2Grad. Sch. Med., Kyoto Transduction pathway of pathogen recognition signal medi- Univ., 3Grad. Sch. of Medical Research, Shimane ated by calcium-dependent protein kinase12 in rice Univ., 4Advanced ICT Res. Inst., NICT, 5Grad. Sch. 6 The generation of reactive oxygen species (ROS) was observed Frontier BioScience, Osaka Univ., Gradiate Sch. 7 when the flagellin, the main component of the bacterial Medical Sci., Namur Research College, The Univ. flagellum, treated to rice plants. Genome-wide analyses revealed Namur) that several calcium-dependent protein kinases (OsCPKs) are Phosphorylation at Ser7 of RNAPII-CTD ensures on-chro- present in rice. Among OsCPKs, OsCPK12 gene was expressed matin retention of nascent ncRNAs, triggering RNAi-depend- after the flagellin treatment. OsCPK12 knock-down mutant or ent heterochromatin formation knock-out mutants lost ability of the ROS generation after flagellin treatment. To identify the OsCPK12 substrates, Phosphorylation of RNAPII-CTD coordinates co-transcriptional OsCPK12 interacting proteins were investigated using Escher- events such as active histone modification and RNA processing. ichia coli-screening system based on BiFC technology. Since Although Ser7 phosphorylation (Ser7P) is well conserved mod- interaction between OsCPK12 and OsSYP13b/OsrbohA were ification from yeast to human and found across the genomes, the intimate roles have remained elusive. confirmed in rice protoplasts, OsSYP13b and OsrbohA were To explore this issue, we analyzed in fission yeast using aAlanine identified as the interacting proteins of OsCPK12. substitution mutant (ctdS7A) of fission yeast RNAPII-CTD. Surprisingly, ctdS7A exhibited loss of peri-centromeric siRNAs and H3K9me deposition, which indicated impairment of RNAi- dependent heterochromatin formation at peri-centromere. Fur- ther studies indicated ctdS7A remarkably reduced the amount of on-chromatin retention of heterochromatic non-coding RNAs, which is required for efficient siRNA production by RNAi factors. This chromatin retention of ncRNA was cooperatively stabilized by Ser7P and RNA binding activity of Chromodomain protein, Chp1. Moreover, Chp1 physically associated with RNAPII in Ser7 dependent manner. Our finding highlights a novel function of Ser7P, which ensures on-chromatin retention of ncRNAs, triggering RNAi-dependent heterochromatin formation. 332 Genes Genet. Syst. (2014) 89

3B TANAKA, Naoko1, TAI, Akiko1, MUKAI, Yukio1 3B WATANABE, Nao1, OKU, Shota1, HAYASHI, Hiroki1, -11 (1Grad. Sch. Bioscience, Nagahama Inst. Bio-Sci. -13 KISHI, Tsutomu1 (1Faclt. Eng., Nihon Univ.) Tech.) CDK is involved in the control of the spindle checkpoint The yeast Cyc8p-Tup1p complex activates transcription of Ubiquitin-dependent degradation is important for various cellu- tryptophan permease genes, TAT1and TAT2, by functioning lar regulatory mechanisms. The SCFCdc4 complex is an ubiquitin as a coactivator ligase complex that acts as a regulator of cell cycle, signal The Cyc8p-Tup1p complex is recruited to promoter regions transduction and transcription. We have previously reported that through interaction with a DNA-binding protein to repress the SCFCdc4 complex degrades Swi5, the transcription factor transcription of many gene families. CYC8-deleted cells grow required for transcription of SIC1, the S phase Cdk inhibitor. We more slowly than TUP1-deleted and wild-type cells. We isolated also reported that such degradation is important for the initiation three tryptophan-related genes, TAT1, TAT2 and TRP1, that, of S phase. Here we describe that degradation of Swi5 is also when overexpressed by multicopy plasmid, complemented the involved in the control of the chromosome segregation. We growth defect of Dcyc8 cells. TAT1 and TAT2 encode a tryptophan arrested cells at S phase by HU and expressed the stabilized permeases and TRP1 encodes a phosphoribosylanthranilate version of Swi5. When released into medium that did not contain isomerase in tryptophan biosynthetic pathway. Supplementation HU, degradation of securin and therefore chromosome segrega- with excess tryptophan partially restored cell growth of Dcyc8. tion was inhibited. Chromosome segregation was recovered by Transcript levels of TAT1 and TAT2 genes were reduced in Dcyc8 the deletion of MAD2 that is essential to the control the spindle and Dtup1. We found nucleotide sequences that are recognized by assembly checkpoint. Inhibition of chromosome segregation was DNA-binding transcriptional activator Fhl1p in upstream of due to the overexpression of SIC1 that resulted in the decrease in TAT1 and TAT2. Transcript levels of TAT1 and TAT2 were the CDK activity. These results suggest that CDK is involved in decreased in Dfhl1/+ cells. These suggest that Cyc8p-Tup1p the control of chromosome segregation via the spindle assembly complex is recruited to TAT1 and TAT2 promoters through checkpoint. interaction with Fhl1p and activates transcription by functioning as a coactivator.

3B HAYASHI, Hiroki1, KISHI, Tsutomu1 (1Dept. Eng., -12 Nihon Univ.) SCFCdc4-dependent degradation of Swi5 is essential for viability A large number of transcription factors undergo degradation through the ubiquitin/proteasome system. Such degradation regulates gene expression at the transcription level. We pre- viously reported that Swi5, a transcription factor that is required for expression of genes that function in G1 phase, is targeted for degradation by the SCFCdc4 (Skp1, Cullin/Cdc53, and the F-box protein Cdc4) ubiquitin ligase complex in Saccharomyces cer- evisiae. Here we report that degradation of Swi5 is triggered by phosphorylation via the protein kinase Pho85/Cdk5. Swi5 was highly stabilized in pho85 mutants. A mutant form of Swi5; Swi5- NLST12A, in which twelve CDK consensus sequences were substituted with alanine residues was highly stabilized. Fur- thermore, we demonstrated that expression of SWI5-NLST12A under the control of its own promoter impaired colony formation. These results suggest that SCFCdc4-dependent degradation of Swi5 is essential for cell cycle progression. Our results also suggest the involvement of another protein kinase(s) in the degradation of Swi5, because deletion of PHO85 extremely slowed the growth but did not cause lethality. Genes Genet. Syst. (2014) 89 333

3C MAKINO, Shigeru1, ZHULYN, Olena2, MURATA, 3C KOZAWA, Yasuyo1, HIROTA, Kazuyuki1, TOKI, -01 Takuya1, FUKUMURA, Ryutaro1, ISHITSUKA, -03 Hideaki1, WAKANA, Shigeharu1, TAMURA, Masaru1 Yuichi1, KOTAKI, Hayato1, HUI, Chi-Chung2, (1RIKEN BioRes. Cent.) GONDO, Yoichi1 (1Mutagenesis and Genomics Team, RIKEN BioRes. Cent., Japan, 2University of Toronto, Functional analysis of Dnase1l2 gene using the exhaustive The Hospital for Sick Children, Canada) mouse phenotyping pipeline Deoxyribonuclease 1 Like 2 (DNASE1L2) belongs to DNASE1 Regulation of Gli transcription factors based on Sufu family, which consists of four genes, DNASE1, DNASE1L1, mutations in the mouse DNASE1L2 and DNASE1L3. Many lines of evidence suggested Hedgehog (Hh) signaling plays critical roles in organogenesis that mutation of DNASE1 or DNASE1L3 causes Systemic Lupus processes, e.g. limb development. Sufu is a cytoplasmic compo- Erythematosus (SLE). Moreover, DNASE1 is located on telomeric nent of Hh signaling. Recent studies in Sufu knockout mutants end of short arm of human chromosome 16, 16p13.3, and it is have indicated that Sufu regulates two primary transcription thought that the haploinsufficiency of DNASE1 is partially factors, Gli2 and Gli3, at multiple steps. However, little is known involved in human chromosomal disorder called 16p13.3 micro about the precise roles of Sufu in regulation specific to Gli2 and/or deletion syndrome or Rubinstein-Taybi syndrome, which shows Gli3. moderate to severe degree of learning difficulty, short stature, Previously, we established 16 mutant mouse lines in Sufu from thumb anomaly, microcephaly, typical facial dysmorphology and/ the RIKEN ENU gene-driven mutagenesis system. In this study, or congenital heart disease. we found that homozygous embryos of SufuT396I, one of the DNASE1L2 is also mapped to 16p13.3, as is case of DNASE1. missense alleles, exhibited severe polydactyly. Concomitantly, However, correlation between DNASE1L2 mutation and human significant quantitative reductions of unprocessed Gli3 and disease remains unclear. In this study, we carried out detailed proteolytically processed Gli3 were seen. SufuT396I also qualita- phenotyping of Dnase1l2 knockout mice using the exhaustive tively reduced the processing of Gli3. In contrast, SufuT396I mouse phenotyping pipeline to elucidate the involvement of exhibited any mutational effects on Gli2 regulation. Taken DNASE1L2 to the human disease. Based on those results, we will together, this study showed that Sufu regulated Gli3, but not discuss the Dnase1l2 function related to the human disease. Gli2, through a mechanism dependent on the Thr396 residue of Sufu, demonstrating that Sufu played a central role in demarcat- ing its regulatory actions of Gli2 and Gli3.

3C SUGIMOTO, Michihiko1, TAMURA, Masaru2, ABE, 3C YONEKAWA, Hiromichi1, MATSUOKA, Kunie2, -02 Kuniya1 (1Tech. and Dev. Team for Mammalian -04 KIKKAWA, Yoshiaki2, SHITARA, Hiroshi1, KOHNO, Genome Dynamics, RIKEN Biores. Cent., 2Tech. and Kenji3, TAYA, Choji1 (1Lab. for Transgenic Tech., Dev. Team for Mouse Phenotype Analysis, RIKEN Tokyo Metropolitan Inst. Med. Sci., 2Mammalian BioRes. Cent.) Genet. Project, Tokyo Metro. Inst. Med. Sci., 3Molecular and Cell Genet., Nara Inst. Sci. Tech.) The role of Vps52 and related genes on mouse embryogenesis Generation of model mice for human diseases by TRECK tw5 mutation occurring in a t-haplotype of the mouse causes (Toxin Receptor Mediated Cell Knockout) method embryonic death at gastrulation stage. We identified Vps52 as the responsible gene for tw5. Vps52 is a mouse homolog of yeast Basophils and eosinophils play important roles in various host VPS52, one of the core components of Golgi-associated retrograde defense mechanisms but also act as harmful effectors in allergic protein (GARP) complex involved in intracellular vesicle trans- disorders. We generated novel basophil- and eosinophil-depletion port. However, Vps52 function in mammalian development has mouse models by introducing the human diphtheria toxin (DT) been totally unknown. We revealed that Vps52 promotes growth receptor gene under the control of the mouse CD203c and the and differentiation of pluripotential cells in embryos and in ES eosinophil peroxidase promoter, respectively. In the basophil- cell-derived embryoid bodies via cell-cell signaling, suggesting depletion model, DT administration attenuated a drop in body developmental roles of the GARP complex. However, homozygous temperature in IgG-mediated systemic anaphylaxis in a dose- mutant of one othter member of the complex showed totally dependent manner and almost completely abolished the develop- different lethal phenotype compared to the Vps52 mutant. We ment of ear swelling in IgE-mediated chronic allergic inflamma- will describe the role of Vps52 and its related genes during mouse tion (IgE-CAI), a typical skin swelling with massive eosinophil embryogenesis revealed by analyses of those mutant embryos. infiltration. In contrast, in the eosinophil-depletion model, DT administration ameliorated the ear swelling in IgE-CAI whether DT was administered before, simultaneously, or after, antigen challenge, with significantly lower numbers of eosinophils infiltrating into the swelling site. These results confirm that basophils and eosinophils act as the initiator and the effector, respectively, in IgE-CAI. Thus, these models are useful and powerful tools for studying the in vivo roles of basophils and eosinophils. 334 Genes Genet. Syst. (2014) 89

3C SEKI, Yuta1, SUZUKI, Sari1, MIYASAKA, Yuki1, 3C UEYAMA, Morio1, ISHIGURO, Taro2, FUJIKAKE, -05 MATSUOKA, Kunie1,KIKKAWA,Yoshiaki1 -07 Nobuhiro1, KONNO, Takuya3, KOYAMA, Akihide3, (1Mammalian Genet. Project, Tokyo Metro. Inst. Med. ONODERA, Osamu3, WADA, Keiji1, NAGAI, Sci.) Yoshitaka1 (1Dept. Degenerative Neurological Diseases, Natl. Inst. NeuroSci., Natl. Cent. of Mechanisms underlying hearing impairment associated with Neurology and Psychiatry, 2Dept. Neurology and heterozygosity of the Myosin VI mutant allele in mice Neurological Sci., Grad. Sch., Tokyo Medical and 3 Mouse mutants of the myosin VI gene (Myo6), Myo6ksv and Dental Univ., Dept. of Neurology, Brain Res. Inst., Myo6rsv, show abnormal behavior and deafness caused by the Niigata Univ.) fusion of the stereocilia (SC) of the inner ear hair cells. Although Establishment of a novel animal model of ALS expressing both heterozygous mice have normal balance, their mutants show GGGGCC repeat RNA in Drosophila early-onset progressive hearing loss (EPHL). However, the hearing phenotypes of the Myo6 mutants differed; the EPHL of Amyotrophic lateral sclerosis (ALS) is a devastating disease with the ksv/+ was more severe compared with that of rsv/+. To clarify movement disorder characterized by degeneration and loss of the mechanisms underlying the severe hearing defects of ksv/+, motor neurons. Recently, an abnormal expansion of GGGGCC we performed phenotypic and expression analyses. By these repeat (>20) in the untranslated region of C9ORF72 gene has analyses, we found fused SC and detected mislocalization of been found to be responsible for familial ALS. Although an MYO6 to the fused SC in the hair cells of ksv/+. Since the ksv expanded repeat RNA transcribed from this repeat expansion is mutation causes abnormal splicing, we speculated that the thought to be involved in the neurodegeneration, its molecular mislocalized MYO6 in ksv/+ may be associated with the splicing mechanism leading to ALS pathogenesis remains unclear. To variants. Next, we quantified the MYO6 expression level. The elucidate the pathogenic mechanisms of ALS caused by an MYO6 expression levels were approximately 40% less in ksv/+ expanded repeat RNA, we tried to establish a novel Drosophila than in wild-type. These findings suggested that the severe model of ALS expressing the GGGGCC repeat RNA by using the EPHL observed in ksv/+ is caused by dominant effects of GAL4-UAS system and characterized its phenotypes. We found abnormal splicing variants and haploinsufficiency of MYO6. that flies expressing the expanded repeat RNA in neurons exhibited a reduction in eclosion rate, motor dysfunction, and reduced life-span compared with that of flies expressing the normal repeat RNA. Moreover, expression of the expanded repeat RNA in the eye resulted in a rough eye phenotype. Our results indicate a successful establishment of a novel animal model for ALS, which is a useful tool for genetic analyses to explore the pathogenic mechanisms of ALS.

3C KIKKAWA, Yoshiaki1, MIYASAKA, Yuki1, WADA, 3C MIYAJI, Masahiro1, ZHANG-AKIYAMA, Qiu-Mei1 -06 Kent1,2, YONEKAWA, Hiromichi3, SHITARA, -08 (1Stress Response Biol., Div. Biol. Sci., Grad. Sch. Sci., Hiroshi3, MATSUOKA, Kunie1 (1Mammalian Genet. Kyoto Univ.) Project, Tokyo Metropolitan Inst. Med. Sci., 2Dept. Bioproduction, Tokyo Univ. Agr., 3Lab. for Transgenic The Analysis of Function of C. elegans Oxidation Resistance 1 Tech., Tokyo Metro. Inst. Med. Sci.) (OXR1) Gene OXR1 was identified as a novel gene that contributes to the Identification of cell-specific functional genes using a mouse defense against oxidative stress. The OXR1 gene is present from model for hearing impairment by the selective ablation of yeast to human, but its function still remains uncertain. In this inner ear sensory cells in vivo study, we identified the homolog of human OXR1 (CeOXR1)in The inner ear is the organ for mechano-electrical transduction the nematode Caenorhabditis elegans. The lifespan of CeOXR1 (MET), in which sound impulses are converted into neural mutant was significantly reduced compared to that of the wild- impulses and transmitted to the brain. The organ of Corti of type strain N2. We also examined the effect of deletion in the inner ear is responsible for auditory transduction and is CeOXR1 on lifespan in daf-2 mutant. The daf-2 mutant is known composed of inner hair cells (IHCs) and outer hair cells (OHCs). to show longer lifespan and increased oxidative stress resistance Although both hair cell types have important roles in MET, the in a DAF-16 dependent manner. We presumed that daf-2; functional genes involved in their development and maintenance CeOXR1 mutant showed reduced lifespan compared to daf-2 have not been identified. In this study, we established a novel mutant. However, in fact, the lifespan of daf-2;CeOXR1 mutant mouse model, OHC-TRECK, for hearing impairment by introduc- was slightly extended compared to that of daf-2 mutant. The ing the human diphtheria toxin (DT) receptor gene under the additional deletion of daf-16 completely lost the life lengthening control of the OHC-specific promoter of the prestin gene. DT effect of deletion in CeOXR1 on daf-2 mutant. This result indicate administration to OHC-TRECK mice resulted in the specific that CeOXR1 controlled DAF-16 function negatively, and there- depletion of OHCs without any effects on IHCs or vestibular hair fore affect lifespan, on the daf-2-defective genetic background. cells. We performed a differential expression analysis for DT- administered wild-type and OHC-TRECK mice using micro- arrays. In this analysis, we found some down-regulated genes in DT-administered OHC-TRECK mice. Moreover, we also identified OHC-specific genes expressed in the inner ear. These results suggest that the OHC-TRECK mouse is a useful model to screen functional genes in OHCs. Genes Genet. Syst. (2014) 89 335

3C UEDA, Naoko1, MATSUO, Takuya3, HIROTSU, 3C UDA, Misato1, UJISAWA, Tomoyo1, KIMURA, Mai1, -09 Takaaki2 (1The Dept. Biol., Faclt. Sci., Kyushu Univ., -11 MIURA, Toru1, IOROI, Makoto1, TAKAGAKI, 2The Dept. Biol., Grad. Sch. Sci.s, Kyushu Univ., Natsune1, OHTA, Akane1, KUHARA, Atsushi1 3Grad. Sch. Systems Life Sci.s, Kyushu Univ.) (1Konan Univ., Faclt. Sci. Eng., & Inst. Integrative NeuroBiol.) The neural circuit mechanisms in the olfactory preference change after odor experience Temperature habituation through trimeric G-protein and CREB dependent temperature memory in C. elegans Animals show preference to odorants to escape from danger and to obtain food. The olfactory preference changes according to Animals can respond and habituate to the changes in ambient conditions. To analyze the olfactory preference change, we use C. temperature. However, the molecular mechanism underlying elegans which has only 302 neurons and the whole neural circuit temperature habituation remains largely unclear. We are inves- has been completely described. tigating about the molecular physiology of temperature habitu- Recently, we revealed that C. elegans shows preference change ation and memory in C. elegans. 15˚C-cultivated animals can after odor experience. In this study, to identify the neural circuit survive at 2˚C, whereas 20˚C- or 25˚C-cultivated animals can not underlying the preference change, we analyzed the functions of survive after cold shock. Mutants impaired with ASJ sensor- interneurons which have direct connections with olfactory yneuron, including trimeric G protein and cGMP-gated channel, neurons. By using UNC-103(gf) which can inhibit the neural showed abnormal cold tolerance. We found that three trimeric G activity, we disrupted the activation of interneurons respectively. protein redundantly function in temperature signaling ASJ As a result, inhibition of AIA or AIY interneurons caused severe sensoryneuron, which are important thermosensory neuron in defects in the preference change. Then, we directly monitored the cold habituation. We recently established temperature shift assay activity of interneurons by live imaging technique. We observed for studying temperature memory in cold habituation. We found the activity of both AIA and AIY interneurons was changed that temperature memory is replaced within only three hours, depending on odor experience. This result suggests that changes and this temperature memory is regulated by CREB, which is of the activity of these interneurons occur in the olfactory important transcriptional factor in mammalian brain. preference change. Now, we try to analyze the function of neuropeptides and their receptors in regulation of the activity of interneurons.

3C UJISAWA, Tomoyo1, SONODA, Satoru1, OHTA, 3C SONODA, Satoru1, TANAKA, Saki1, OHTA, Akane1, -10 Akane2, KUHARA, Atsushi2 (1Dept. of life and -12 KUHARA, Atsushi1 (1Dept. Biol., Konan Univ.) function sci., Grad. Sch. of Nat. Sci., Konan Univ., 2Dept. of Bio., Grad. Sch. of Nat. Sci., Konan Univ.) Interaction between neuron and sperm in cold habituation of C. elegans Molecular neural systems for temperature habituation in C. Temperature is critical stimulus and cause biochemical changes. elegans We are studying habituation mechanism to environmental Animals can respond and habituate to ambient environmental temperature changes in C. elgans. 20˚C-cultivated animals were temperature. C. elegans has a memory-based temperature destroyed by cold stimuli, while 15 ˚C cultivated animals can habituation, which is a useful model for studying temperature survive by cold stimuli. We recently found that this cold response and memory. We have been studying molecular habituation is regulated by ASJ sensoryneuron, which release physiological mechanisms of the temperature habituation. 15 insulin that is received by intestine and neuron. In this study, we degree-cultivated animals can survive at 2 degree, whereas 20 found that 18 mutants isolated from DNA microarray analysis degree-cultivated animals can not survive at 2 degree. We found showed abnormal cold habituation, including mutants impaired that a pair of sensoryneurons and insulin signaling regulate cold with sperm genes such as gsp-3, 4 and fem-3. To investigate how habituation. This sensoryneuron was previously known as light sperm affects cold habituation, we analyzed double mutants and pheromone-sensing neuron (ASJ). Ca2+ imaging analysis defective in sperm and known-signaling in cold habituation such determined that ASJ sensoryneuron directly senses temperature. as ASJ temperature signaling pathway and insulin pathway. Unexpectedly similar molecular pathway is used between light Genetic epistasis analysis suggest that sperm affects cold and temperature signaling in ASJ. Molecular physiological habituation in downstream of insulin signaling of intestine. We analysis indicated that insulin released from ASJ is received by unexpectedly found that abnormal cold habituation in gsp-4 was intestine and neurons, and regulates gene expression for cold strongly suppressed by mutations in the temperature signaling in habituation. Thus, we describe a novel molecular physiological ASJ. This hypothesized that sperm affects temperature signaling system for cold habituation, which is thought to be a useful in ASJ by using any feedback system such as secretly signaling. paradigm for temepature habituation (1). (1)Ohta, Ujisawa, et al., Nature commun, 2014, in press 336 Genes Genet. Syst. (2014) 89

3C YUKARI, Kinoshita1, AKANE, Ohta1, KUHARA, 3D KATOH, Takehiro K1, WATADA, Masayoshi1 -13 Atsushi1 (1Lab. Molecular and Cellular Regulation -01 (1Guraduate Sch. Sci. Eng., Ehime Univ.) Dept. Biol. Faclt. Sci. Eng., Konan Univ) Molecular phylogenetic study of the subgenus Sophophora Artificial evolution and genetic analysis of temperature (Diptera: Drosophilidae) based on 31 nuclear loci experience-dependent cold habituation of C. elegans The subgenus Sophophora is the one of largest group in the genus Temperature response is important for animal, and animals have Drosophila and includes model species, Drosophila melanogaster. habituation mechanisms to environmental temperature changes. To reveal the phylogenetic relationships within this subgenus, 31 We are utilizing temperature experience-dependent cold toler- species belonging to the subgenus Sophophora were analyzed ance of nematode, C. elegans. After cultivation at 25˚C, wild-type using the sequences data of 31 nuclear loci. The molecular can’t survive at 2˚C. In contrast, after cultivation at 15˚C, most of phylogenetic tree showed the high node support values, all four animals can survive at 2˚C. To isolate genes involved in the cold species-groups were monophyletic. For the relationships within tolerance, we are using two approaches, (1) Artificial evolution the melanogaster species-group, only the suzukii species-sub- and (2) EMS-mutagenesis. group was polyphyletic. Among the species ascribed the suzukii (1) C. elegans has strong advantage for artificial evolution subgroup, D. oshimai first branched off the other members and analysis. We are maintaining C. elegans at 15 or 23 degrees for did not belong to any species-subgroup. Drosophila mimetica gradually accumulation of mutations, and frozen-stocks of clearly belonged to the takahshii species-subgroup, and D. animals were made at every generation. We so far obtained lucipennis and D. nyinyii formed a monophyletic clade together about 190 generations animals, and phenotypes have been with the elegans and the rhopaloa subgroup. These results gradually changed. We are planning to decode the genomes by suggest that the D. lucipennis plus D. nyinyii clade and the using deep DNA sequencer. rhopaloa subgroup are integrated into the elegans subgroup, (2) We isolated several mutants defective in cold tolerance from which was the oldest among them. two thousands genomes screening by EMS. KHR1 showed significant phenotype of cold habituation. Responsible gene in KHR1 strain was mapped on chromosome X -3.6 cM~-1.14 cM. We are now analyzing detailed-mapping position by using deep DNA sequencer.

3C OKAHATA, Misaki1, OHTA, Akane1, KUHARA, 3D IWASAKI, Yuma1, TAMURA, Koichiro1,2 (1Tokyo -14 Atsushi1 (1Konan Univ., Faclt. Sci. Eng., & Institute -02 Metropolitan Univ., 2Res. Cent. for Genomics and for Integrative NeuroBiol.) Bioinform., Tokyo Metropolitan Universiry) Natural variation of genes for temperature responses and A multi-gene phylogenetic analysis of subgenus Drosophila memory in C. elegans by next generation sequencing technique Animals have adapted to environment temperature by accumu- Phylogenetic relationships among Drosophila species have been lation of natural mutations. We are studying natural variation of well studied as a model organism, D. melanogaster, is involved. genes for temperature habituation in C.elegans. Most of 25 Currently, the genus Drosophila harbors 1193 species, many of degree-cultivated wild-type N2 can not survive at 2 degree, while which belong to the subgenera Drosophila (421 species) and 15 or 17 degree-cultivated N2 can survive at 2 degree. We found Sophophora (348 species). In the subgenus Drosophila, many that various wild-type strains showed different cold habituation, species belong to the quinaria section. However, the phylogenetic in which CB4854 showed the strongest decrement of cold relations of these species have not been well analyzed. In this habituation. To identify the genes involved in the natural research, therefore, we analyzed the phylogenetic relationships variations between N2 and CB4854, we utilized deep DNA among representative species of the quinaria section. We sequencer. Responsible gene polymorphism for the cold habitu- determined cDNA sequences of 6 species, D. funebris, D. ation was mapped onto -1.6~ -1.5cM region on ChX, where six angulalis, D. orientacea, D. sternopleuralis, D. tripunctata, D. genes are predicted. We therefore measured cold habituation of albomicans using a Roche 454 Jr sequencer, and, together with these mutants, and we found that vps-52 mutant showed 12 genome sequences already published, we analyzed phyloge- abnormal cold habituation. vps-52 gene encodes Golgi-associated netic relations among 18 species using 160 orthologous genes. As retrograde protein (GARP). CB4854 has a nucleotide insertion in a result, we estimated phylogenetic trees with high bootstrap intron near splicing site. confidences to newly find a close relationship of D. sternopleuralis Recently, we are studying temperature memory by using temper- to the immigrans species group, although there were a few ature shift assay in cold habituation. We found that CB4853 topological discrepancies between nucleotide and amino acid memorizes new temperature slowly. We are using deep DNA trees. sequencer to identify the responsible gene for this memory variations. Genes Genet. Syst. (2014) 89 337

3D SETO, Yosuke1, TODA, Masanori2, TAMURA, 3D SAWAMURA, Kyoichi1, MAEHARA, Kazunori2, -03 Koichiro1,3 (1Dept. Biol. Sci.s, Tokyo Metro. Univ., -05 KEIRA, Yoko3,ISHIKAWA,HiroyukiO.3, 2The Hokkaido Univ. Museum, 3Res. Cent. for SASAMURA, Takeshi4, YAMAKAWA, Tomoko4, Genomics and Bioinform., Tokyo Metropolitan Univ.) MATSUNO, Kenji4 (1Faclt. Life and Env. Sci., Univ. Tsukuba, 2Grad. Sch. Life and Env. Sci., Univ. Molecular phylogenetic analysis of the Drosophila immigrans Tsukuba, 3Grad. Sch. Sci., Chiba Univ., 4Grad. Sch. species group using big sequence data Sci., Osaka Univ.) The subgenus Drosophila is a major subgenus in the genus Interspecific introgression of nucleoporin genes in Drosophila Drosophila. Within this subgenus, the immigrans species group is a large species group consisting of species morphologically In interspecific hybrids between Drosophila melanogaster and characterized by a row of spinules on the inner side of fore femur. Drosophila simulans, Nup96sim and Nup160sim can cause However, the phylogenetic relationships within the species group recessive lethality. Double introgression of Nup96sim and are still controversial, some molecular phylogenetic analyses Nup160sim did not lead to lethality when one was heterozygous even suggesting polyphyly. In this study, therefore, we conducted and the other homozygous (hemizygous). Therefore, introgression the RNA-seq to determine nucleotide sequences of a large number of additional autosomal D. simulans genes is necessary to cause of genes for several species belonging to the immigrans species lethality and that the effect of the introgression is dominant to D. group and conducted a phylogenetic analysis together with the 12 melanogaster alleles. Although Nup160sim can cause recessive Drosophila species genomes data. As the result, we found that D. female sterility in the D. melanogaster genetic background, annulipes and D. curveceps of the immigrans species group were Nup96sim did not. Because double introgression carrying homo- not involved in the major cluster of this species group but closely zygous Nup96sim and hemizygous Nup160sim resulted in lethal- related to Hawaiian Drosophila species, D. grimshawi, and ity, Nup96sim and Nup160sim seem to be two components of the Scaptomyza graminum and S. flava. This result strongly same incompatibility. Interestingly, the dominance of the supported the polyphyly of the immigrans species group and introgression was affected by the genetic background. suggested the necessity of revising the classification of the species belonging to the species group.

3D AKIYAMA, Noriyoshi1, MIYAGI, Ryutaro1, 3D ARAYE, Quenta1, SAWAMURA, Kyoichi2 (1Grad. Sch. -04 TAKAHASHI, Aya1,2 (1Dept. Biol. Sci.s, Tokyo Metro. -06 Life and Env. Sci., Univ. Tsukuba, 2Faclt. Life and Univ., 2Res. Cent. for Genomics and Bioinform., Tokyo Env. Sci.s, Univ. Tsukuba) Metropolitan Univ.) Population size and inbreeding depression in Drosophila Desiccation and cold resistance associated with pigmentation melanogaster lab strains variation in Drosophila melanogaster Drosophila melanogaster lab strains are generally small popula- In Drosophila melanogaster, body color is a polymorphic trait. tions. Are there any differences on genetic structure between Several studies have shown that there are correlations between domesticated (small) populations and natural (large) popula- variations in body melanisation and altitude or latitude, tions? Here we investigated genetic load in standard (wild type) suggesting that it is an adaptive trait. We focused on ebony, lab strains, Oregon-R (OR) and Canton-S (CS). We extracted which is a gene involved in body color formation in D. second chromosomes each from OR and CS via the Cy/Pm melanogaster. The purpose of this study was to investigate the method. The frequency of recessive lethal chromosomes was 0.11 relationships between its expression level and cold and desic- and 0.14 (cf. 0.16-0.41 in natural populations) and the allelism cation resistance by genetic manipulation. We first measured cold rate was 0.5 and 0.2, respectively. Under the assumption that resistance, defined as survival rate after 24h exposure to 1˚C, of 4 mutation rate is 10-5/locus/generation, effective population size wild-derived strains in The Drosophila Genetic Reference Panel was estimated to be 62 and 243, respectively. We analyzed (DGRP), which had shown differences in the ebony expression. inbreeding depression of the lab strains by comparing viability The result suggested a strong influence of the genetic back- distribution of the chromosomes, where genetic load was ground. Therefore, we tried RNAi knockdown of this gene in separated into two components: (1) genetic load caused by fixed whole fly by utilizing GAL4-UAS system. Cold resistance deleterious mutations and (2) that caused by polymorphic increased significantly in the knockdown flies compared to the deleterious mutations. We detected component (1); viability of control. We also measured desiccation resistance, defined as the lab strains was decreased. On the contrary, we hardly survival time in ~2% Rh, of the same strains in DGRP. detected component (2); homozygous load was smaller and low Desiccation resistance of the darkest strain was significantly viability chromosomes (viability < 0.75) were fewer. lower than the others, which might be due to a higher dehydration rate. 338 Genes Genet. Syst. (2014) 89

3D TOMARU, Masatoshi1 (1Drosophila Genetic Resource 3D YUKUHIRO, Kenji1, KOMOTO, Natuo1, TOMITA, -07 Cent., Kyoto Inst. Tech.) -09 Shuichior1, SEZUTSU, Hideki1, AKIDUKI, Gaku1, ITOH, Masanobu2, NAKAJIMA, Yumiko3 (1Natl. Inst. Search for factors affecting female mate discrimination in AgroBiol. Sci., 2Kyoto Inst. Tech., 3Ryukyu Univ.) Drosophila sechellia COI sequences of three island populations of Japanese Females of Drosophila sechellia discriminate against D. mela- Bombyx mandarina are classified into the clade different nogaster males, but hybrid females of these species accept D. from those of the mainland populations melanogaster males, suggesting that the discrimination against D. melanogaster males is recessive (Tomaru et al. GGS 79: 145- We analyzed nucleotide sequence variation of mtCOI gene from 150, 2004). To explore the factors affecting the discrimination, I three Japanese island populations of Tsushima, Yakushima and performed deficiency screen for the third chromosome responsible Tanegashima of wild mulberry silkmoth Bombyx mandarina, and for female mate discrimination. I used 84 strains from the then found that the three populations shared remarkably similar DrosDel deficiency strain kit of D. melanogaster. Females from sequences. These COI sequences carried five or more nucleotide each DrosDel strain were crossed with D. sechellia males to substitutions when comparing to those living in Japanese main obtain hybrid females hemizygous for the D. sechellia chromo- lands. Note that 12 substitutions were detected comparing B. some corresponding to the deficiency. Hybrid females were mandarina inhabiting in Japanese mainland and Bombyx mori. courted by D. melanogaster males and about 70% of courting This result clearly indicates separation of the three populations males attempted to copulate with females, suggesting that hybrid occurred after that of common ancestor of Japanese B. mandar- females are attractive for D. melanogaster males. Successful inahad separated from Chinese one. copulations were observed in most deficiencies with copulation attempts. However, hybrid females from a few deficiencies did not copulated. The genomic regions of D. sechellia corresponding to these deficiencies are candidates for female mate discrimination.

3D YOSHIDA, Aya1, ITOH, Masanobu1, TAKANO, 3D SUZUKI, Hitoshi1, BAWM, Saw2, THWE, Thida Lay3, -08 Toshiyuki2 (1Dept. Appl. Biol., Kyoto Inst. Tech., -10 KATAKURA, Ken4, TSUCHIYA, Kimiyuki5 (1Grad. 2Drosophila Genetic Resource Cent., Kyoto Inst. Tech.) Sch. Env. Earth Sci., Hokkaido Univ., 2Univ. Veterinary Medicine, 3Dept of Zoology, Yangon Univ., Genetic basis underlying hybrid sterility between two 4Grad. Sch. of Veterinary Medicine, Hokkaido Univ., Lepidptera, Bombyx mori and B. mandarina, with a ZZ/ZW 5OOYO SEIBUTSU Inc) sex determination system Speciation patterns of the subgenus Mus in Myanmar When one hybrid sex is sterile or inviable, it is usually the heterogametic sex. While a major reason for this Haldane’s rule is The speciation process of the members of the subgenus Mus that genes responsible for hybrid incompatibility tend to act remain still largely unknown. Here we conducted to reconstruct recessively, hybrid sterility is also caused by other factors. the evolutionary history of the subgenus in their main homeland Indeed, in Drosophila, autosomal hybrid male sterility also areas of India and Southeast Asia, focusing on the spatial and evolves much faster than female sterility, suggesting the faster temporal speciation patterns in Myanmar. The presence of two male evolution. The Haldane’s rule holds true for animals with a species endemic to Myanmar, M. nitidulus and M. lepidoides, ZZ/ZW sex determination system, but it remains untested if the simply implies that the lowland area surrounded by the two faster male evolution is also applicable to these species. Here we mountainous ranges acted to have fostered the two species. The tested this using Bombyx mori and B. mandarina. F1 hybrids mitochondrial and nuclear gene sequence analyses showed the between B. mori females and B. mandarina males showed normal close relationship of M. nitidulus with Indian species of M. fertility, although hybrid females produced slightly fewer eggs booduga and M. terricolor, suggesting migration of an ancestral than females of both parental species. By contrast, when the F1 lineage of M. nitidulus to Myanmar from the Indian subcontinent hybrid females were backcrossed to B. mori males, the F2 females at some ancient time. The phylogeographic patterns of the local showed significantly reduced fertility. More interestingly, the lineages of M. caroli suggest a possibility of ancient habitation in fertility of the F2 males widely varied from 0 to 100%. These eastern part of Southeast Asia and recent colonization to results may imply that genes involved in male reproduction are Myanmar. Further studies with nuclear gene markers are needed evolving rapidly also in Lepidoptera with male homogamety (ZZ). to better understand the roles of the geographic area of Myanmar in the lineage differentiation of the subgenus Mus. Genes Genet. Syst. (2014) 89 339

3D ISHISHITA, Satoshi1, TSUBOI, Kazuma1, OHISHI, 3E KAMEI, Yuka1, YAMAMOTO, Kaori1, DAKEYAMA, -11 Namiko2, TSUCHIYA, Kimiyuki3, MATSUDA, Yoichi1 -01 Shota1, INOUE, Yamato1, TAI, Akiko1, MUKAI, (1Grad. Sch. Bioagricultural Sci., Nagoya Univ., 2Grad. Yukio1 (1Nagahama Inst. Bio-Sci. Tech.) Sch. Sci., Hokkaido Univ., 3OOYO-SEIBUTSU INC.) Identification of transcription factor genes essential for cell Abnormal synapsis and premature dissociation of X and Y growth and replicative lifespan in budding yeast chromosomes during meiosis in interspecific hybrids of The budding yeast Saccharomyces cerevisiae is a powerful model Phodopus organism for studying cellular lifespan. Numerous lifespan genes

F1 hybrid males between the Campbell’s hamster (Phodopus have been identified using haploid yeast knockout mutants, campbelli) and the Djungarian hamster (P. sungorus) are known however, genes that are essential for cell growth have not been to exhibit sterility with impaired spermatogenesis; however, the investigated on lifespan. In this study, we examined ten yeast meiotic phenotype of the hybrid has not been well described. In transcription factor genes that are essential for cell growth and this study, we revealed that spermatocytes were accumulated in required for replicative lifespan (the number of daughter cells seminiferous tubules of the hybrids. Apoptotic spermatocyte-like produced by a mother cell before dying). Firstly, we constructed cells were observed frequently in seminiferous epithelia of the haploid strains having two copies of each of transcription factor hybrids. Asynapsis of X and Y chromosomes was observed in genes to measure their replicative lifespan. Higher expressions of pachytene-like spermatocytes of the hybrids at a high frequency, these transcription factor genes were confirmed, but no change of whereas both abnormal synapsis and unrepaired DNA double- replicative lifespan was observed between strains with one and strand breaks were observed in autosomes at a low frequency. In two copies of the genes. Next, we examined hetero-knockout spermatocytes of the hybrids, dissociation of X and Y chromo- diploid strains for the essential transcription factor genes. somes at MI stage and degeneration of MI-like nuclei were Heterozygous deletion of the FHL1, RAP1, and REB1 genes observed at a high frequency. Most of epididymal sperms of the shortened replicative lifespan. 1H-NMR analysis showed the hybrids exhibited morphological abnormalities. X and Y chromo- FHL1 and REB1 hetero-knockout strains had characteristic somes-biased asynapsis and premature dissociation are also metabolic profiles. observed in hybrid males of Mus. These findings suggest that pairing of X and Y chromosomes are more adversely affected in F1 hybrids of rodents.

3D UNO, Yoshinobu1, SASAKI, Chisato1, TSUBOI, 3E KAMEI, Yuka1, MUKAI, Yukio1 (1Grad. Sch. -12 Kazuma1, OISHI, Namiko2, TSUCHIYA, Kimiyuki3, -02 Bioscience, Nagahama Inst. Bio-Sci. Tech.) MATSUDA, Yoichi1 (1Grad. Sch. of Bioagricultural Sci., Nagoya Univ., 2Grad. Sch. Sci., Hokkaido Univ., A novel longevity gene, SNZ1, encoding pyridoxal 5’- 3Appl. Biol. Co., Ltd.) phosphate synthase in budding yeast The budding yeast Saccharomyces cerevisiae is a powerful model Epigenetic incompatibilities responsible for hybrid dysgene- for research on cellular aging and lifespan, especially to identify sis in interspecific mating between Phodopus campbelli and genes that regulate lifespan. From transcriptional analyses for P. sungorus aging cells, we found that mRNA level of the SNZ1 gene, which Interspecific F1 hybrids between two different species of the encodes pyridoxal 5’-phosphate synthase, greatly increased in old dwarf hamster, Phodopus campbelli (CAM) and P. sungorus cells, but the SNO1 gene, which encodes glutamine amidotrans- (SUN), exhibit the disturbance of parent-of-origin effects on ferase and forms a complex with Snz1p, was not upregulated. The growth and development. Cross-mating of CAM females with age-dependent induction of SNZ1 was mediated through a SUN males yields growth-retarded conceptuses, whereas the transcription factor Adr1p. The SNZ1-deleted strain did not reciprocal cross results in dysmorphic conceptuses or overgrown grow on the synthetic medium without pyridoxine, but the SNO1- embryos with the hypertrophy of the placentas. Here, we deleted strain grew, although slowly. Deletion of SNZ1, but not examined expression patterns of 13 imprinted genes using SNO1, shortened replicative lifespan on YPD medium. Addition embryos and placentas of the F1 hybrids. Eight genes (Gatm, of excess pyridoxine to YPD medium restored replicative lifespan H19, Meg1, Meg3, Peg1, Peg3, Peg9 and Usp29) exhibited bi- of snz1 cells to wild-type levels. The vitamin B6 transporter gene, allelic expression in placentas and/or embryos of (SUN x CAM) F1 TPN1, was downregulated in aging cells, suggesting intracellular hybrids, whereas only Gatmand Meg3 genes were bi-allelic in vitamin B6 level decreased with age. These results indicate that (CAN x SUN) F1 hybrids. The DNA methylation at the promoters SNZ1 is a novel longevity gene and vitamin B6 synthesis is of Meg1 and Peg3 in (SUN x CAM) F1 hybrids were clearly required for replicative lifespan. different patterns from (CAM x SUN) F1 hybrids without bi- allelic expression. These results lead us to propose a correlation between the parent-of-origin effects of embryonic growth followed by abnormal parturition and the imprinting perturbation in interspecific F1 hybrids of Phodopus. 340 Genes Genet. Syst. (2014) 89

3E ENDO, Mitsuyoshi1, TANAKA, Syuuitsu1, 3E MUTO, Akihiko1, IKEDA, Shingo1, LOPEZ-BURKS, -03 HATAKEYAMA, Shin1 (1Genet. Lab., Dept. -05 Martha E2, KIKUCHI, Yutaka1, CALOF, Anne L3, Regulatory Biol., Faclt. Sci., Saitama Univ.) LANDER, Arthur D2, SCHILLING, Thomas F2 (1Dept. Biol. Sci., Grad. Sch. Sci., Hiroshima Univ., 2Dept. Analysis of the natural death mutant of Neurospora crassa Developmental & Cell Biol., Univ. California, 3Dept. The nd (natural death) strain of Neurospora was originally Anatomy & NeuroBiol., Univ. California) isolated which showed serious growth defect even on complete Mechanisms of gene regulation mediated by long-range medium and restrictive temperature. This mutant ceases hyphal chromosomal interactions during vertebrate limb develop- growth approximately two weeks despite that of wild type is over ment two years, and causes a large deletion of mitochondrial DNA. In this study, we clarified another phenotype of the ndstrain such as Long-range enhancer-promoter interactions play pivotal roles in showing obvious MMS sensitivity at 37˚C and camptothecin tissue- and stage-specific regulation of gene expression during resistance. For attempt to determine the responsible gene of vertebrate development. Although several distinct factors includ- ndmutation, we made detail genetic map by crossing the ndand ing cohesin and the Mediator complex have been shown to be KO strains, which produced by introducing the drug resistant involved in the long-range interactions between DNA elements, it gene (hph) into the ORF, thought to be closed to ndmutation, and is still unclear how they contribute to tissue/stage-specific gene resultant offspring were analyzed. We explored one candidate expression. We found by using zebrafish as a model that gene which gene product contains mitochondrial localization expression of key genes involved in limb (pectoral fin) develop- signal gene, and some mutations were found in this gene of ment, including multiple hox genes, was altered in a similar ndmutant. Knock out of this gene showed quite similar manner by depletion of either Nipbl, a regulator of cohesin phenotypes with ndmutant, and these were partially comple- functions, or Med12, a subunit of Mediator, leading to morpho- mented by DNA fragment of this gene. Gene replacement of logical defects in fins. We also found that chromosomal mutant with wild-type gene following back-cross with wild-type organization around the hoxda cluster was disrupted in Nipbl- strain resulted in showing completely identical phenotypes to or Med12-deficient fin buds, further supporting the hypothesis wild type. The function of this responsible gene will be discussed. that long-range chromosomal interactions required for limb- specific hox gene expression are mediated by Nipbl and Mediator. These findings provide insights into both the etiology of limb defects in CdLS, a developmental disorder caused by NIPBL mutations, and the mechanisms by which Nipbl and Mediator influence gene expression.

3E ISHIKAWA, Masakazu1,SHIMIZU,Hiroshi1, 3E MATSUMURA, Kenya1,NISHIYAMA,Eri2, -04 NOZAWA, Masafumi1, IKEO, Kazuho1, GOJOBORI, -06 OHSHIMA, Kazuhiko1,2 (1Grad. Sch. Biol. Sci., Takashi1 (1DNA Data Analysis Lab., Natl. Inst. Nagahama Inst. Bio-Sci. Tech., 2Dept. Biol. Sci.s, Genet.) Nagahama Inst. Bio-Sci. Tech.) Molecular basis of endosymbiosis between hydra and alga by Identification of regulatory sequences involved in transcrip- comparing transcriptome analysis tional regulation of a young retrogene ? Symbiotic associations between two or more unrelated organisms PIPSL is a primate-specific retrogene, which was created by a are found throughout all ecosystems. It is widely known that new combination of functional domains and simultaneous gene many cnidarians, such as corals and sea anemones, show duplication. PIPSL is significantly transcribed in human and endosymbiotic associations with algae. Some kinds of Hydra, chimpanzee testes. To elucidate how this novel gene has gained fresh water cnidarian animals, also show endosymbiotic associ- the regulatory mechanism for transcription, we performed ations with green algae. In addition, those Hydra are also able to reporter assays with DNA fragments around the PIPSL, survive without symbiotic algae (aposymbiotic state). identifying a core region which enhanced the reporter gene However, the mechanisms of this endosymbiotic association at expression. Moreover, we examined upstream sequence of PIPSL the molecular level are largely unexplored. and retrogenes that are significantly transcribed in human testis. For uncovering this molecular interplay, we have examined the In many cases, we found DNA-binding sites of transcription differences of gene expression in symbiotic versus aposymbiotic factors associated with testicular differentiation, e.g., SRY, Hydra using whole transcriptome shotgun sequencing (RNA-seq). SOX9, and WT1. These results raised the possibility that some In this presentation, we discuss what kinds of genes are of these sites add tissue specificity to the PIPSL core promoter. responsible for maintenance or regulation of the endosymbiosis using differentially expressed genes between symbiotic and aposymbiotic Hydra. In addition, we also discuss what kinds of substances they exchange. Genes Genet. Syst. (2014) 89 341

3E ARAKI, Masatake1, NAKAHARA, Mai1, NAKAGATA, 3E SHOUCHI, Takamasa1, TATEYAMA, Hiroki2, -07 Naomi1, YAMAMURA, Ken-ichi1, YOSHINOBU, -09 TAKAHASHI, Satoru3, YAMAMURA, Ken-ichi2, Kumiko1, ARAKI, Kimi1 (1Inst. Resource Dev. and ARAKI, Kimi2 (1Div. Dev. Genet., Grad. Sch. Pharm. Analysis, Kumamoto Univ.) Sci., Kumamoto Univ., 2Div. Dev. Genet., Inst. Resource Dev. and Analysis, Kumamoto Univ., 3Dept. Pathway and gene ontology analysis of exchangeable gene Anatomy and Embryology, Faclt. Med., Univ. trap clone mouse lines Tsukuba) Gene trapping in embryonic stem (ES) cells is a proven method Production and Analysis of c-Maf transgenic MSM/Ms mice for large-scale random insertional mutagenesis in the mouse with Mol/MSM-1, embryonic stem cell derived from MSM/Ms genome. We have established an ezchangeable gene trap system, blastcysts in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox-mediated recombination. We MSM/Ms is an inbred strain established from Japanese wild developed the Database for the Exchangeable Gene Trap Clones mice, Mus musculus molossinus, and displays unique pheno- (EGTC) (http://egtc.jp). types, including low incidence of tumor development. Thus, Because we used a promoter trap strategy, all trapped genes were MSM/Ms strain is considered to be a good tool for analysis of rxpressed in ES cells. To understand the general characteristics genetic background involving in carcinogenesis. of the trapped genes in the EGTC library, we used Kyoto We have established a germline-competent ES cell line from Encyclopedia of Genes and Genome (KEGG) for pathway analysis MSM/Ms strain. In order to compare the mechanism of carcino- and found that the EGTC ES clones covered a broad range of genesis between MSM/Ms and C57BL/6, we produced transgenic pathways. We also used Gene Ontology (GO) classification data MSM/Ms lines carrying the c-Maf gene. Morito et al. reported provided by Mouse Genome Informatics (MGI) to compare the that overexpression of the c-Maf gene driven by the human CD2 functional distribution of genes in each GO term between trapped promoter induced T-cell lymphoma using C57BL/6 line (Cancer genes in the EGTC mouse lines and total genes annotated in Res., 66: 812, 2006). By using the same transgene, we success- MGI. We found the functional distributions for the trapped genes fully produced three transgenic MSM/Ms lines overexpressing in the EGTC mouse lines and for the RefSeq genes for the whole the c-Maf gene in the thymus. Interestingly, all these MSM/Ms mouse genome were similar, indicating that the EGTC mouse transgenic lines showed lower incidence of mortality than the lines had trapped a wide range of mouse genes. C57BL/6 Tg line. The Cyclin D2 and Nuak1, known as c-Maf target genes, were up regulated in the C57BL/6 Tg line, while the expression of these genes were not changed in all these MSM/Ms transgenic lines. These results suggest difference in developing rate of tumor between MSM/Ms and C57BL/6.

3E SATO, Shigeru1, YAJIMA, Hiroshi1, KAWAKAMI, 3E NIIMI, Yuki1, SUZUKI, Atsushi2 (1Grad. Sch. Eng., -08 Kiyoshi1 (1Div. Biol., Center for Molecular Medicine, -10 Yokohama National Univ., 2Faclt. Engneering, Jichi Med. Univ.) Yokohama Natl. Univ.) The function and regulation of Six1 in vertebrate sensory Functional analysis of mouse Dead end1 in germ cell organogenesis development In vertebrates, Six1 is expressed in sensory lineages from very Dead end is an evolutionarily conserved RNA-binding protein early stages. Mice deficient for Six1 are characterized by the loss/ implicated in germ cell development among vertebrates. Re- abnormal development of several cranial sensory organs, such as cently, mouse Dead end1 (Dnd1) was reported to be essential for the inner ear, olfactory epithelium and the taste papillae. The maintenance of the PGCs because a null mutation of Dnd1 causes goal of our study is to identify the gene cascade that governs a drastic loss of the migrating PGCs. However, the expression sensory organogenesis in order to understand the developmental profile and function of DND1 in the PGCs after migrating stage and the evolution/diversity of vertebrate sensory organs. In this are entirely unrevealing. Here, we find that DND1 is up- study, we characterized the mechanism required for the activa- regulated specifically in male gonocytes after colonization to tion of Six1-8, one of the nine Six1 enhancers, that drives embryonic gonads, while female gonocytes down-regulate it transcription in the cranial placodes/ganglia and dorsal root accompanied by the initiation of meiosis. We also find that ganglia. Tissue-specific activity of Six1-8 is controlled positively conditional deletion of DND1 (Dnd1-cKO) in male gonocytes show by nuclear receptors, Wnt and Bmp signaling pathways. In pleiotropic phenotypes; down-regulation of male-type genes, addition, using a Six1-8-Cre transgenic mouse line we showed progression of cell cycle, and meiotic initiation. In addition, that Six1-8 activates transcription in a subset of cells in the Dnd1-cKO male gonocytes transform to testicular teratoma at a olfactory placode and those cells contributed to olfactory pioneer certain rate. These results represent a first evidence of DND1- neurons. mediated sexual differentiation in mouse male germ cell develop- ment. 342 Genes Genet. Syst. (2014) 89

3E HIRAI, Kazuyuki1, SHIMA, Yukio2, MINAKUCHI, 3E KUSAMA, Sakiku1, HIRAI, Kazuyuki2, MATSUURA, -11 Yohei3, TOYODA, Atsushi3, MATSUDA, Muneo1 -13 Etsuko T.3 (1Dept. Biol., Faclt. Sci., Ochanomizu (1Dept. Biol., Kyorin Univ. Sch. Med., 2Dept. Medical Univ., 2Dept. Biol., Kyorin Univ. Sch. Med., 3The Tech., Faclt. Health Sci., Kyorin Univ., 3Center for Grad. Sch. Humanities Sci., Ochanomizu Univ.) Information Biol., Natl. Inst. Genet.) Sub-cellular localization of SIRT2 during spermatogenesis in De novo centrosome synthesis and diploidization in partheo- Drosophila genetic embryos of Drosophila ananassae SIRT2 is a member of Sirtuin family, which comprises highly In animal reproduction, diploid organisms are usually generated conserved NAD-dependent protein deacetylases. D. melanogaster by fusion of two haploid nuclei from both parents. In some has been reported to have five Sirtuins. For SIRT2, we previously species, however, parthenogenetic development that requires no examined the effects of decreased expression by RNAi on paternal contribution can be induced to occur. In the present development, male fertility, and adult life span, and the study we cytologically examined how diploidy is restored in expression profile during development. The amount of SIRT2 parthenogenetic embryos of Drosophila ananassae. We found mRNA was higher in testes than in the other regions in adults that asters that are scattered peculiarly in parthenogenetic eggs and the strong signals by in situ hybridization were detected in have essential role in generating a diploid nucleus by combining testes. To further investigate SIRT2 function, the sub-cellular two haploid cleavage nuclei. These free asters were formed localization of SIRT2 during spermatogenesis was examined by around the centrosome markers Asterless and Centrosomin, thus immunostaining using an antibody raised by peptide immuniza- de novo synthesized centrosome-like structures. One of the asters tion in the present study. SIRT2 was primarily found in the can serve as a single mitotic pole that connects two poles from cytoplasm in growing primary spermatocytes and then abundant separate bipolar spindles, while the remaining two poles were throughout those cells at metaphase I and elongating spermatids. completely separated. A diploid nucleus is produced at the However, in sperm, SIRT2 signals were restricted to a part of connected pole by integrating two haploid chromosome sets at each nucleus. The stage-specific sub-cellular localization of SIRT2 telophase, while two haploid nuclei are also produced at the other as well as its expression through spermatogenesis suggests that poles. The diploid nucleus with the centrosome may have SIRT2 may be involved in the morphogenesis of sperm and also in substantial advantage in proliferation, eventually generating a histone-to-protamine transition. diploid embryo.

3E TATSUNO, Hisashi1, SAWADA, Akiko1, NIIMI, Nao2, -12 ITOH, Masanobu1 (1Dept. Appl. Biol., Kyoto Inst. Tech., 2Dept. Appl. Biol., Kyoto Inst. Tech.) Effects of spermathecae on longevity and fertility in NK14 mutation of Drosophila melanogaster Females of a spontaneous mutation, NK14, have three sperma- thecae for sperm storage, whereas wild-type females have two. Spermathecae of NK14 are frequently accompanied with black pigments like melanoma. Extra spermatheca and melanoma-like tissue are likely involved in a mutation of en, in which a 15-nt deletion occurred in the first exon, thus presumably enNK14.To characterize this mutation in detail, we firstly investigated their longevity. Day of 50% adult death (DD50) of NK14 was significantly shorter than those of wild-type for both sexes (40.0 vs. 71.0 for female and 37.6 vs. 60.2 for male). Mating experience and melanoma-like tissue did not directly affect their longevity. Secondly, we counted the sperms in spermathecae and ventral receptacles of females after copulating with protamine-eGFP males, of which eGFP expresses at their sperm heads. As a result, one hour after the mating, NK14 was shown to keep totally less sperms in the three spermathecae than in the two of wild-type. Unexpectedly, extra spermatheca cannot increase the number of sperms stored, but decreased in NK14 females.