Molecular Function Laboratory • Kanemaki Group

Caspase-activated DNase (CAD), a DNase responsible for DNA fragmentation in apoptosis, is activated upon degradation of its inhibitor ICAD. However, it has not been known whether ICAD degradation is sufficient for induction of apoptosis. To answer this question, we established DT40 cells, in which ICAD can be degraded by addition of auxin using the auxin-inducible degron system. Upon rapid degradation of ICAD, we showed that DT40 cells were induced for apoptosis, indicating that ICDA degradation is sufficient for apoptosis. Moreover, this cell-killing system works in budding yeast cells. We suggest that the cell-killing system we established in this work might be applicable for containment measures of gene-modified organisms in the future.

This study was carried out as collaboration with Dr. Kumiko Samajima and Professor William Earnshaw in University of Edinburgh, UK. Dr. Earnshaw is also an invited professor of NIG.

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By adding auxin, ICAD was rapidly degraded, resulting induction of apoptosis (a3). In these cells, genomic DNA was fragmented (b).

　Naruya SAITOU, Professor of Division of Population Genetics, was awarded “Docteur Honoris Causa (an honorary doctorate)” from Université Toulouse III. The award presentation ceremony was held on October 8th in Toulouse, France.
　Established in 1220, Université Toulouse III is one of the oldest and prestigious Universities. The university is also known as Paul Sabatier University after Paul Sabatier who received a Nobel Prize in Chemistry with François Auguste Victor Grignard in 1912.
Three other recipients are from Belgium, the United States, and Spain.

Division of Population Genetics • Saitou Group

Pradeep Lal (Postdoctoral Researcher at Division of Molecular and Developmental Biology Laboratory) presented a poster at the 20th Japanese Medaka and Zebrafish Meeting, 20-21 September 2014 held at Tokyo, Japan and he received Best Poster Award for his presentation. The title of his poster was “Genetic dissection of neural circuits mediating fear-learning in zebrafish”.

Division of Molecular and Developmental Biology • Kawakami Group

Molecular Function Laboratory • Kanemaki Group

Budding yeast has been an important eukaryotic model organism because there have been many genetic technologies available. In the yeast research field, temperature-sensitive (ts) mutants have been generated in order to inactivate a target protein conditionally. Although ts-mutants have been useful, there are problems as well; a drastic temperature shift causes heat-shock responses and there are some cases in which a target protein is not inactivated in a short period of time. To overcome these problems, we transplanted a plant-specific degradation pathway controlled by the plant hormone auxin to budding yeast and, in such yeast cells, a protein of interest can be degraded upon addition of auxin. We named this technology as the auxin-inducible degron (AID) system (Nishimura et al, Nature Methods, 2009) and subsequently improved it by making efficient degron cassettes (Kubota et al. Molecular Cell, 2013). The AID technology is getting popular in the filed of yeast studies. In this paper, we described a detailed protocol for generation of budding yeast AID mutants by one transformation. All plasmids and yeast strains required for the experiment are available from NBRP (http://yeast.lab.nig.ac.jp/nig/index_en.html). We hope that this protocol would help your study in the future.

(A) Schematic illustration showing the mechanism of the auxin-dependent degradation of a degron-fused protein. (B) Schematic illustration showing the protocol for generation of AID mutants.

Division of Molecular Genetics • Fukagawa Group

Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, Fukagawa (Dep. Mol. Genet.) and Earnshaw (ex visiting professor of NIG, Edinburgh Univ) describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, and use of artificial chromosomes and kinetochores to study centromere function.

Molecular architecture of natural kinetochores. Centromere chromatin including is crucial for centromere specification and kinetochore assembly at natural centromeres. At the base of the structure are CENP-A containing nucleosomes, centromere specific H3 nucleosomes, and a CENP-T-W-S-X nucleosome-like structure in centromere chromatin. Centromere-specific chromatin structure is established by coordination of these components. CCAN proteins assemble on the centromeric chromatin and the microtubule-binding complex is subsequently recruited to assemble the functional kinetochore.

Biomolecular Structure Laboratory • Shirakihara Group Division of Microbial Genetics • Araki Group

The initiation of eukaryotic chromosomal DNA replication requires formation of an active replicative helicase at the replication origins of chromosomal DNA. Yeast Sld3 and its metazoan counterpart Treslin are the hub proteins mediating protein associations critical for the helicase formation. In this study we show the crystal structure of the central domains of Sld3 that is conserved in Sld3/Treslin family proteins. The domain consists of two segments with 12 helices and is sufficient to bind to Cdc45, the essential helicase component. The structural model of the Sld3-Cdc45 complex that is crucial for formation of the active helicase was proposed.

(A) Sld3 and Cdc45 form a complex that associates with origins in a mutually dependent manner,and its association to origins depends on the phosphorylation of the Mcm2-7 helicase core complex. Further protein interactions through Sld3 recruits GINS, another essential component of the active helicase. (B) Representation of the tertiary structure of the homology domain shared by Sld3 and Treslin. The proposed model of Sld3-Cdc45 complex is also shown.

Division of Brain Function • Hirata Group

Four-transmenbrane protein family M6 is concentrated on axon tips. Although in vitro studies have suggested its involvement in axon elongation, in vivo evidence has been lacking. In this study, we made double knockout mice for two family members M6a and M6b, and showed that these membrane proteins in fact control axon elongation in vivo. In the M6a/M6b double knockout brain, the corpus callosum, a long axon commissure that connects left-right brain hemispheres, was severely hypoplastic; many axons were found to stop short of reaching the midline. Furthermore, a fraction of callosal axons were misrouted subcortically. These results show that M6 proteins are indeed important regulators of axon growth and guidance in the developing brain.

Superenova system developed by the Division of Neurogenetics is used in this study.

Top: the bundle of callosal axons (green) connecting brain hemispheres (arrow) is thinner in the M6a/M6b double mutant brain (right). Bottom: callosal axon trajectories traced with Supernova system. In the double mutant brain (right), the axons are not simply shorter but also disorganized, and some are even misdirected subcortically (arrows).

Mouse Genomics Resource Laboratory (MGRL) • Koide Group

The Japanese wild-derived mouse strain MSM/Ms (MSM) retains a wide range of traits related to behavioral wildness, including high levels of emotionality and avoidance of humans. In this study, we observed that MSM showed a markedly higher level of aggression than the standard laboratory strain C57BL/6J. We identified two chromosomes, Chr 4 and Chr 15, which were involved in the heightened aggression observed in MSM. These chromosomes had different effects on aggression: whereas MSM Chr 15 increased agitation and initiation of aggressive events, MSM Chr 4 induced a maladaptive level of aggressive behavior. Expression analysis of mRNAs of serotonin related genes indicated that the expression of Tph2, an enzyme involved in serotonin synthesis, in the midbrain was increased in the Chr 4 consomic strain, as well as in MSM, and that there was a strong positive genetic correlation between aggressive behavior and Tph2 expression at the mRNA level.

Genetic mapping of escalated aggression in MSM by using consomic mouse strains

Division of Brain Function • Hirata Group

The highly evolved neocortex is a marked characteristic of mammals. It develops as layers, in which neurons are sequentially added from deep to the surface in a birth-order-dependent manner. Evolutionary processes of the layered neocortex have been a long mystery. We recently reported that the chicken brain contains neuron subtypes that are similar to deep and upper layer neurons in the mammalian neocortex . However, the comparison of only two distant animal groups was not sufficient to specify potential evolutional changes, and similar analyses of other animal groups have been awaited. In this study, we performed gene expression analyses in the softshell turtle brain, which is often referred to as an ancestral form of amniote brains. The turtle brain contains a single neuronal layer, exhibiting a clear difference from the chicken brain. Our expression analyses of orthologs of neocortical layer marker genes highlighted similarities in neuronal arrangements between the two species. Specifically, in both of the species, deep layer neurons are positioned in the medial part, whereas upper layer neurons are positioned in the lateral part. Thus, this medio-lateral neuronal arrangement appears to be an ancestral mode in sauropsids. The results support our hypothesis of an ancient origin of neocortical neuron subtypes. We assume that the generality of birth-order-dependent mechanisms for specification of neuron subtypes probably underlies the their unexpected conservation among diverse amniote species.

Evolutionary model of brains in amniotes

Notwithstanding the importance of effective approaches to analyze social interaction between animals in experimental settings, the methods that are currently available to do this rely predominantly on human observation. This makes large-scale studies of social interaction behavior difficult.

In this paper, we report the development of freeware, called DuoMouse, that enables video recording to track the movements of two mice from a movie file, analysis of behavioral states using a hidden Markov model (HMM), and visualization of the results. We used this software to compare social behavior in consomic and subconsomic strains, and to map a genetic locus responsible for differences in social interaction between the strains. We report a locus that increases social interaction only in males and that lies within a 24.6-Mb region of chromosome 6.

We propose that the method applied in the present study is very efficient and useful for large-scale genetic and pharmacological studies of social behavior in mice. All of the software is open-source, and the source code will also be provided for further development by others.
(https://zenodo.org/records/12577797)

This research was supported by the Research Organization of Information and Systems, Transdisciplinary Research Integration Center, JSPS KAKENHI Grant Numbers 23650243 and 25116527, and NIG Cooperative Research Program (2010-A40, 2012-A85).

Diagram showing the Markov transition probabilities for the two-state HMM and three-state HMM. The image showing one of the pages of the freeware, DuoMouse.

Division of Molecular and Developmental Biology • Kawakami Group

The lateral line system of teleost fish is composed of mechanosensory receptors (neuromasts), some of which are superficial whereas others are embedded in canals running under the skin. Canal diameter and size of canal neuromasts are correlated with increasing body size, thus providing a very simple system to investigate the mechanism underlying the coordination between organ growth and body size. Here, we examine the development of the trunk lateral line canal system in zebrafish. We demonstrate that trunk canals originate from scales through a bone remodeling process. We suggest that the bone remodeling process is essential for normal growth of canals and of canal neuromasts. Moreover, we show that the presence of lateral line cells is required for the formation of canals, suggesting the existence of mutual interactions between the sensory system and surrounding connective tissues.

(A) The lateral line sense organ, the neuromast, is located on the skin surface in embryonic stages. (B) When fish undergo metamorphosis, a pair of ridges extends outward from the scale. At the same time, bony elements under the neuromast gradually disappear. (C) The neuromast becomes embedded in the canal running under the scale in adult fish.

Genome Informatics Laboratory • Nakamura Group Division of Cytogenetics • Kobayashi Group

Genetics is a powerful approach to discovery of the molecular mechanisms underlying biological phenomena. However, experiments for identification of mutations by classical genetics is a time-consuming and laborious process. Modern whole-genome sequencing, coupled with bioinformatics analysis, has enabled fast and cost-effective mutation identification. However, for many experimental researchers, bioinformatics analysis is still a difficult aspect of whole-genome sequencing. To address this issue, we developed a browser-accessible and easy-to-use bioinformatics tool called Mutation discovery (Mudi; http://naoii.nig.ac.jp/mudi_top.html). Users can run Mudi analysis with ‘one-click’ operation in a web browser after uploading genome resequencing data. Mudi simplifies analysis of whole-genome sequencing and thereby expands the possibilities for systematic forward-genetic approaches in various organisms.

Workflow of “Mudi” system. Genomic DNA is prepared from pooled mutants by pooled-linkage analysis. Mudi calls prioritized mutation candidates by bioinformatics analysis using genome-resequencing data.

Mouse Genomics Resource Laboratory (MGRL) • Koide Group

Genetic mapping using congenic strains is one of the most powerful methods available to show the existence of QTLs in the genomic regions substituted into recipient strains. In this case, genetic mapping can be conducted by making a series of congenic strains that cover genomic regions that partially overlap with the adjacent congenic strains. In the analysis of the phenotype using a series of congenic strains, however, the phenotype shows a variety of levels, which indicates the existence of multiple QTLs in the mapped chromosomal regions. These results show that the precise genetic mapping of fine QTLs is extremely difficult using current methods.

Dr. Tsuyoshi Koide’s group collaborated with Drs. Shogo Kato and Satoshi Kuriki in the Institute of Statistical Mathematics and established a new method for mapping multiple QTLs using data from a series of congenic strains by applying a regression model for the analysis of total home-cage activity in mice. The results of the analysis identified four significant QTLs in a 14.5 Mb genomic region. Among these, three have negative effects but one has a positive effect on total home-cage activity. In further analysis of recombinants obtained from the congenic strains, we confirmed the existence of the QTL that has a positive effect on the activity as well as another QTL that suppresses the effect of this positive QTL. These results clarify for the first time the association of a complex genetic mechanism in the QTL cluster on chromosome 6 with the regulation of home-cage activity.

This work was supported by the Research Organization of Information and Systems, Transdisciplinary Research Integration Center, JSPS KAKENHI (Grant Numbers 23650243 and 25116527), and Yamada Science Foundation.

A cluster of four QTLs for behavior segregated by new method using a statistical regression model. The data of genomic regions carried in the congenic strains as well as the data of home-cage activity exhibited by congenic strains are analyzed with regression model. The study revealed four QTLs which are clustered in a small genomic region.

Reproductive isolation is important for speciation, because it suppresses free genetic exchange between two diverged populations and accelerates the genetic divergence. Hybrid sterility (sterility in hybrid animals), one of the reproductive isolation phenomena, is possibly caused by deleterious interactions between diverged genetic factors brought by two distinct populations. A mouse inter-subspecific X chromosome substitution strain, B6-ChrXMSM, in which X chromosome of west European subspecies-derived C57BL/6J is replaced by counterpart of Japanese subspecies-derived MSM/Ms strain, shows reproductive isolation characterized by male-specific sterility due to disruption of meiotic entry in spermatogenesis (Oka et al, Genetics, 2004, 2007, 2010). In this study, we conducted comprehensive transcriptional profiling of the testicular cells of this strain by microarray. The results clearly revealed gross downregulation of gene expression in the substituted donor X chromosome. Such misregulation occurred prior to detectable spermatogenetic impairment, suggesting that it is a primal event in reproductive isolation. Furthermore, this misregulation subsequently resulted in perturbation of global transcriptional regulation of autosomal genes, probably by cascading deleterious effects. Remarkably, this transcriptional misregulation was substantially restored by introduction of chromosome 1 from the same donor strain as the X chromosome. This finding implies that one of regulatory genes acting in trans for X-linked target genes is located on chromosome 1. This study collectively suggests that regulatory incompatibility is a major cause of reproductive isolation in the X chromosome substitution strain.

This study was done as a research project “Life Systems” of　Trans- disciplinary Research Integration Center, ROIS.

Figure 1. Misregulation of X-linked genes in the X-chromosome substitution strain (CSS). Meiotic spermatocytes were observed in testes of wild type (left upper panel), whereas they were rarely observed in testes of X-chromosome CSS (left bottom panel). Approximately 20% of expressed X-linked genes were misregulated in the X-chromosome CSS.

The adaptive nature of aggressive behavior in social animals accounts for its observation throughout the animal kingdom. However, given that aggressive behavior is no longer adaptive when it exceeds the species-typical level, mechanisms should curb excessive aggression to keep it within the adaptive range. The prefrontal cortex has been implicated in the inhibitory control of emotional outbursts, including aggression and violence. In this study, we used optogenetics to demonstrate that temporal activation of excitatory neurons in the medial prefrontal cortex (mPFC), but not the orbitofrontal cortex (OFC), inhibits inter-male aggression in mice. Activation of the mPFC suppresses aggressive bursts and reduces the intensity of aggressive behavior, but does not change the duration of the aggressive bursts. Our findings suggest that mPFC activity inhibits the initiation and execution, but not the termination, of aggressive behavior, and maintains such behavior within the adaptive range.

Optogenetic activation of the mPFC reduced the frequency of attack bites.
ON: light stimulation (blue light, 20 Hz, 5 mW), OFF: no light stimulation, gray lines: attack bites in each individual, black line: mean values.

Experimental Farm • Nonomura Group Plant Genetics Laboratory • Kurata Group

Meiosis is a pivotal biological event for breeding programs, and elucidatiing molecular mechanisms of meiosis will lead to achieve stable and efficient seed production. In this paper, we focus on MEL1 protein, which plays essential roles in progression of meiosis in rice. MEL1 is an Argonaute family protein known as an effecter of RNA silencing.

Of all MEL1-binding small RNAs (MEL1-sRNA) isolated and sequenced, 75% were 21 nt long and 80% beared a 5′-terminal cytosine. Supprisingly, MEL1-sRNA derived from more than 1,000 genomic loci, and most of the loci were mapped onto the intergenic regions. We discovered that more than 700 species of large intergenic noncoding RNAs (lincRNA) were transcribed from the MEL1-sRNA loci after the reproductive phase transition. The lincRNAs were double-stranded and processed in the 21-nt phase by tasiRNA biogenesis pathway, resulting in production of the 21-nt small RNAs including MEL1-sRNA.

This study brought us important information to consider the role of RNA silencing in plant germline development and meiosis.

Analysis of MEL1, the rice Argonaute protein with specific expression in the germline.
(A) The mel1 mutant is sterile. (B) MEL1 gene is expressed in germ cells. (C) RNA-immunoprecipitation of wt (1) and muntat panicles (2). (D) MEL1-binding small RNAs derive from >1,000 intergenic loci (blue). (E) ZEP1 doesn’t elongate between homolog pairs in mel1 meiocytes.
Panels A-B and C-E were cited from Plant Cell 19: 2583-2594 (2007) and Plant J (2014), respectively, with slight modifications.