Regulatory mechanism of heat-active retrotransposons by the SET domain protein SUVH2.

New transposon insertions are deleterious to genome stability. The RNA-directed DNA methylation (RdDM) pathway evolved to regulate transposon activity via DNA methylation. However, current studies have not yet clearly described the transposition regulation. ONSEN is a heat-activated retrotransposon that is activated at 37°C. The plant-specific SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) family proteins function downstream of the RdDM pathway. The SUVH protein families are linked to TE silencing by two pathways, one through DNA methylation and the other through chromatin remodeling. In this study, we analyzed the regulation of ONSEN activity by SUVH2. We observed that ONSEN transcripts were increased; however, there was no transpositional activity in Arabidopsis suvh2 mutant. The suvh2 mutant produced siRNAs from the ONSEN locus under heat stress, suggesting that siRNAs are involved in suppressing transposition. These results provide new insights into the regulatory mechanisms of retrotransposons that involve siRNA in the RdDM pathway.

Epigenetic regulation of ecotype-specific expression of the heat-activated transposon ONSEN.

Transposable elements are present in a wide variety of organisms; however, our understanding of the diversity of mechanisms involved in their activation is incomplete. In this study, we analyzed the transcriptional activation of the ONSEN retrotransposon, which is activated by high-temperature stress in Arabidopsis thaliana. We found that its transcription is significantly higher in the Japanese ecotype Kyoto. Considering that transposons are epigenetically regulated, DNA methylation levels were analyzed, revealing that CHH methylation was reduced in Kyoto compared to the standard ecotype, Col-0. A mutation was also detected in the Kyoto CMT2 gene, encoding a CHH methyltransferase, suggesting that it may be responsible for increased expression of ONSEN. CHH methylation is controlled by histone modifications through a self-reinforcing loop between DNA methyltransferase and histone methyltransferase. Analysis of these modifications revealed that the level of H3K9me2, a repressive histone marker for gene expression, was lower in Kyoto than in Col-0. The level of another repressive histone marker, H3K27me1, was decreased in Kyoto; however, it was not impacted in a Col-0 cmt2 mutant. Therefore, in addition to the CMT2 mutation, other factors may reduce repressive histone modifications in Kyoto.

Regulatory mechanism of a heat-activated retrotransposon by DDR complex in Arabidopsis thaliana.

The RNA-directed DNA methylation (RdDM) pathway plays an essential role in the transposon silencing mechanism; the DDR complex, consisting of DRD1, DMS3, and RDM1, is an essential component of the RdDM pathway. ONSEN, identified in Arabidopsis, is a retrotransposon activated by heat stress at 37°C; however, studies on the regulation of ONSEN are limited. In this study, we analyzed the regulation of ONSEN activity by the DDR complex in Arabidopsis. We elucidated that loss of any component of the DDR complex increased ONSEN transcript levels. Transgenerational transposition of ONSEN was observed in the DDR-complex mutants treated with heat stress for 48 h. Furthermore, the DDR complex components DRD1, DMS3, and RDM1 played independent roles in suppressing ONSEN transcription and transposition. Moreover, we found that the duration of heat stress affects ONSEN activity. Therefore, the results of this study provide new insights into the retrotransposon regulatory mechanisms of the DDR complex in the RdDM pathway.

Efforts to Enhance Long-Stay Demand in Public Kokumin Shukusha with a Spa ─Considerations and Analysis Based on Interview Survey for the Association and Field Work─ / 日本温泉気候物理医学会雑誌

  The author has researched and reported on “Efforts to increase the demand for long-term stays in spa resorts” over three reports up until now. The target areas were spa health resorts and national park resort villages with spas. There, I pointed out the necessity to create programs for stays that combine the natural environments, historical and cultural environments, artificial environments and facilities, different environments by moving around and night-time hours that exist around each stay facility.  In this repot, I investigated the creation of programs for public Kokumin Shukusha, which are dormitories for the people who can stay with peace of mind, even with their families, at a low cost. I investigated whether or not efforts are being made to create programs for staying, such as setting walking trails that make use of external environments around the public Kokumin Shukusha.   As a result, although efforts were made, the number and diversity of course settings were insufficient. In order to increase the demand for long-term stays, it will be necessary to utilize the external environment that make the most of the nearness to existing villages. In addition, it became clear that the Onsen Stay policy promoted by the Ministry of the Environment has not been publicized, and it is necessary to promote the publicity of this policy in order to revitalize spa resorts nationwide.

Efforts to Enhance Long-Stay Demand in Public Kokumin Shukusha with a Spa—Considerations and Analysis Based on Interview Survey for the Association and Field Work— / 日本温泉気候物理医学会雑誌

  The author has researched and reported on “Efforts to increase the demand for long-term stays in spa resorts” over three reports up until now. The target areas were spa health resorts and national park resort villages with spas. There, I pointed out the necessity to create programs for stays that combine the natural environments, historical and cultural environments, artificial environments and facilities, different environments by moving around and night-time hours that exist around each stay facility.  In this repot, I investigated the creation of programs for public Kokumin Shukusha, which are dormitories for the people who can stay with peace of mind, even with their families, at a low cost. I investigated whether or not efforts are being made to create programs for staying, such as setting walking trails that make use of external environments around the public Kokumin Shukusha.   As a result, although efforts were made, the number and diversity of course settings were insufficient. In order to increase the demand for long-term stays, it will be necessary to utilize the external environment that make the most of the nearness to existing villages. In addition, it became clear that the Onsen Stay policy promoted by the Ministry of the Environment has not been publicized, and it is necessary to promote the publicity of this policy in order to revitalize spa resorts nationwide.

DRD1, a SWI/SNF-like chromatin remodeling protein, regulates a heat-activated transposon in Arabidopsis thaliana.

ONSEN is a heat-activated LTR retrotransposon in Arabidopsis thaliana. Screens to identify transcriptional regulatory factors of ONSEN revealed a SWI/SNF-like chromatin remodeling protein, DRD1, which cooperates with plant-specific RNA polymerase and is involved in RNA-directed DNA methylation. ONSEN transcript level was increased in the drd1 mutant relative to wild-type under heat stress, indicating that DRD1 plays a significant role in the silencing of activated ONSEN under the stress condition. The transcript level of HsfA2, which is directly involved in transcriptional activation of ONSEN, was not higher in the drd1 mutant than in the wild-type. Interestingly, no transgenerational transposition of ONSEN was observed in the drd1 mutant, even though DNA methylation levels were significantly reduced and expression levels were increased compared to the wild-type. These results suggest that other factors are involved in the regulation of ONSEN transposition in addition to the transcript level of ONSEN.

Determination of TE Insertion Positions Using Transposon Display.

Mapping the genomic location to which transposons jumped is of greatest interest to transposon biologists. Transposon display (TD) is the technique of choice that is easy and fast in determining the neo-insertion positions of a target transposon. Essentially, tagging of transposon is performed by digesting genomic DNA, ligating adaptors to digested DNA ends and PCR amplifying genomic regions flanking the transposon of interest. In this chapter, the experimental procedure of TD is described using Onsen retrotransposon of Arabidopsis as an example.

How to Activate Heat-Responsible Retrotransposon ONSEN in Brassicaceae Species.

A number of transposable elements are activated by environmental stress. A Ty1/copia-type retrotransposon named ONSEN is activated by heat stress in Brassicaceae species. A synthetic activation of the transposon is effective for the molecular breeding without genetic modification. Here, we described the detail procedure of heat treatment to activate ONSEN in Brassicaceae species.

ONSEN shows different transposition activities in RdDM pathway mutants.

Most transposable elements (TEs) are tightly regulated by epigenetic mechanisms such as DNA methylation. RNA-directed DNA methylation (RdDM) is a major control mechanism of TE silencing in plants. We analyzed the transposition activity of a heat-responsive retrotransposon, ONSEN, in Arabidopsis thaliana. Transgenerational transposition was observed in RdDM pathway-deficient mutants upon heat stress. The transposition frequency was higher in the mutants of the upstream processes, but lower in the mutants of the downstream steps, of RdDM. The transposition frequency was not associated with the number of extrachromosomal ONSEN copies. Constitutive heterochromatin of interphase nuclei was dispersed upon heat stress. The degree of decondensation was higher in the RdDM mutants than in wild-type plants subjected to heat stress. We discuss the possible role of RdDM in the regulation of ONSEN transposition upon heat stress.

The effect of zebularine on the heat-activated retrotransposon ONSEN in Arabidopsis thaliana and Vigna angularis.

The Ty1/copia-like retrotransposon ONSEN is conserved among Brassica species, as well as in beans, including adzuki bean (Vigna angularis (Willd.) Ohwi & Ohashi), which is one of the economically important crops in Japan. ONSEN has acquired a heat-responsive element that is recognized by plant heat stress defense factors, resulting in its transcription and the production of full-length extrachromosomal DNA under conditions with elevated temperatures. DNA methylation plays an important role in regulating the activation of this transposon in plants. Therefore, chemical inhibition of DNA methyltransferases has been utilized to study the effect of DNA methylation on transposon activation. To understand the effect of DNA methylation on ONSEN activation, Arabidopsis thaliana and adzuki bean seedlings were treated with zebularine, which is known to be an effective chemical demethylation agent. The results showed that ONSEN transcription levels were upregulated in zebularine-treated plants. Extrachromosomal DNA of ONSEN also accumulated in the treated plants.

What Is Necessary to Enhance Long-Stay Demand in Spa Health Resort？ ～From Considerations Based on Field Work～ / 日本温泉気候物理医学会雑誌

  The Ministry of the Environment is promoting an Onsen Stay policy, is suggesting long stays at spa health resorts and is working on activation in a spa from 2017. What kind of program can be carried out to accept long stay guests at the spa which fills the capacity of the health resort? Programs targeted for Gero Spa, the Osaka hot spring village (Yuya Spa, Shimojima Spa and Nigorigo Spa), Yuwaku Spa, the Hakusan hot spring village (Tedori Spa, new Iwama Spa, Ichirino Spa and Chugu Spa) and Yunokuchi Spa were investigated in this survey.  The necessity of creating a space of relaxation suitable for a stay was shown to add new space value and experience value when the area resources including the ambient surroundings in a spa were utilized for long stays in a thesis of Shimomura, who studied the spatial form of the spa and the spatial structure, and in an Onsen Stay promotion plan of the Ministry of the Environment.  In the above-named 10 spas the extent to which space value and experience value was added was investigated by listening to the guests concerned and by a local inspection of whether such a program was carried out. Since the program’s implementation in Gero Spa, the Osaka hot spring village and Ichirino Spa, as a result, I found that the long-stay program was feasible. But I also found that there are almost no long-stay guests who utilized the program because needs weren’t taken into consideration in the utilization. In high plains areas in the mountains, I also found that eco-tourism is considered as a stay plan and that town walks utilizing cultural facilities in downtown, walks and day trips to the surrounding scenic and recreation spots are considered as a stay plan in the newly-formed spa towns.  I suggest that an investigation in spas where long-stay programs have been put into effect is the next necessary step in this research.

What Is Necessary to Enhance Long-Stay Demand in Spa Health Resort？ ～From Considerations Based on Field Work～ / 日本温泉気候物理医学会雑誌

  The Ministry of the Environment is promoting an Onsen Stay policy, is suggesting long stays at spa health resorts and is working on activation in a spa from 2017. What kind of program can be carried out to accept long stay guests at the spa which fills the capacity of the health resort? Programs targeted for Gero Spa, the Osaka hot spring village (Yuya Spa, Shimojima Spa and Nigorigo Spa), Yuwaku Spa, the Hakusan hot spring village (Tedori Spa, new Iwama Spa, Ichirino Spa and Chugu Spa) and Yunokuchi Spa were investigated in this survey.  The necessity of creating a space of relaxation suitable for a stay was shown to add new space value and experience value when the area resources including the ambient surroundings in a spa were utilized for long stays in a thesis of Shimomura, who studied the spatial form of the spa and the spatial structure, and in an Onsen Stay promotion plan of the Ministry of the Environment.  In the above-named 10 spas the extent to which space value and experience value was added was investigated by listening to the guests concerned and by a local inspection of whether such a program was carried out. Since the program’s implementation in Gero Spa, the Osaka hot spring village and Ichirino Spa, as a result, I found that the long-stay program was feasible. But I also found that there are almost no long-stay guests who utilized the program because needs weren’t taken into consideration in the utilization. In high plains areas in the mountains, I also found that eco-tourism is considered as a stay plan and that town walks utilizing cultural facilities in downtown, walks and day trips to the surrounding scenic and recreation spots are considered as a stay plan in the newly-formed spa towns.  I suggest that an investigation in spas where long-stay programs have been put into effect is the next necessary step in this research.

Characterization of a heat-activated retrotransposon in Vigna angularis.

In plants, several transposable elements are conserved across species. We found a homolog of ONSEN, which is a heat-activated retrotransposon originally isolated from Arabidopsis thaliana, in Vigna. The ONSEN-like elements (VaONS) were detected in all the analyzed Japanese accessions of Vigna angularis (adzuki bean) by Southern blot analysis. However, VaONS sequences were observed to be polymorphic in the different accessions. Interestingly, extrachromosomal DNA (ecDNA) was detected in some accessions of adzuki bean, indicating the conserved heat-activation of VaONS. Furthermore, we successfully induced retrotransposition of VaONS in adzuki plant regenerated through callus. Findings of our study should provide a new tool for molecular breeding of adzuki bean.

Characterization of a heat-activated retrotransposon in natural accessions of Arabidopsis thaliana.

Natural accessions are used for studying intraspecies genetic variation in the model plant Arabidopsis thaliana in order to address fundamental questions of evolution. Transposable elements are responsible for a wide range of mutations and play significant roles in shaping a genome over evolutionary time. In the present study, we aimed to characterize ONSEN, a heat-activated long terminal repeat (LTR) retrotransposon, in natural A. thaliana accessions. Southern blot analysis demonstrated that ONSEN was present in all the studied accessions, but the copy number was diverse. Olympia-1 contained a single ONSEN copy, located in the centromere of Chromosome 3. A premature stop codon in Olympia-1 ONSEN presumably abolishes integrase activity, which in turn presumably renders the retrotransposon non-functional. Hybridization of Col-0 with Olympia-1 showed that several ONSEN copies in Col-0 were activated by heat stress and maintained their transpositional activity in the progeny.

Inducible Transposition of a Heat-Activated Retrotransposon in Tissue Culture.

A transposition of a heat-activated retrotransposon named ONSEN required compromise of a small RNA-mediated epigenetic regulation that includes RNA-directed DNA methylation (RdDM) machinery after heat treatment. In the current study, we analyzed the transcriptional and transpositional activation of ONSEN to better understand the underlying molecular mechanism involved in the maintenance and/or induction of transposon activation in plant tissue culture. We found the transposition of heat-primed ONSEN during tissue culture independently of RdDM mutation. The heat activation of ONSEN transcripts was not significantly up-regulated in tissue culture compared with that in heat-stressed seedlings, indicating that the transposition of ONSEN was regulated independently of the transcript level. RdDM-related genes were up-regulated by heat stress in both tissue culture and seedlings. The level of DNA methylation of ONSEN did not show any change in tissue culture, and the amount of ONSEN-derived small RNAs was not affected by heat stress. The results indicated that the transposition of ONSEN was regulated by an alternative mechanism in addition to the RdDM-mediated epigenetic regulation in tissue culture. We applied the tissue culture-induced transposition of ONSEN to Japanese radish, an important breeding species of the family Brassicaceae. Several new insertions were detected in a regenerated plant derived from heat-stressed tissues and its self-fertilized progeny, revealing the possibility of molecular breeding without genetic modification.

Recurrent evolution of heat-responsiveness in Brassicaceae COPIA elements.

BACKGROUND: The mobilization of transposable elements (TEs) is suppressed by host genome defense mechanisms. Recent studies showed that the cis-regulatory region of Arabidopsis thaliana COPIA78/ONSEN retrotransposons contains heat-responsive elements (HREs), which cause their activation during heat stress. However, it remains unknown whether this is a common and potentially conserved trait and how it has evolved. RESULTS: We show that ONSEN, COPIA37, TERESTRA, and ROMANIAT5 are the major families of heat-responsive TEs in A. lyrata and A. thaliana. Heat-responsiveness of COPIA families is correlated with the presence of putative high affinity heat shock factor binding HREs within their long terminal repeats in seven Brassicaceae species. The strong HRE of ONSEN is conserved over millions of years and has evolved by duplication of a proto-HRE sequence, which was already present early in the evolution of the Brassicaceae. However, HREs of most families are species-specific, and in Boechera stricta, the ONSEN HRE accumulated mutations and lost heat-responsiveness. CONCLUSIONS: Gain of HREs does not always provide an ultimate selective advantage for TEs, but may increase the probability of their long-term survival during the co-evolution of hosts and genomic parasites.

A small RNA mediated regulation of a stress-activated retrotransposon and the tissue specific transposition during the reproductive period in Arabidopsis.

Transposable elements (TEs) are key elements that facilitate genome evolution of the host organism. A number of studies have assessed the functions of TEs, which change gene expression in the host genome. Activation of TEs is controlled by epigenetic modifications such as DNA methylation and histone modifications. Several recent studies have reported that TEs can also be activated by biotic or abiotic stress in some plants. We focused on a Ty1/copia retrotransposon, ONSEN, that is activated by heat stress (HS) in Arabidopsis. We found that transcriptional activation of ONSEN was regulated by a small interfering RNA (siRNA)-related pathway, and the activation could also be induced by oxidative stress. Mutants deficient in siRNA biogenesis that were exposed to HS at the initial stages of vegetative growth showed transgenerational transposition. The transposition was also detected in the progeny, which originated from tissue that had differentiated after exposure to the HS. The results indicated that in some undifferentiated cells, transpositional activity could be maintained quite long after exposure to the HS.

An Examination of the Current State of the Specialist Accreditation System for Onsen-Ryoho-Specialist / 日本温泉気候物理医学会雑誌

  To evaluate the current state of the Onsen-Ryoho-Specialist (Broad Certified Fellow in Balneology, Climatology and Physical Medicine) training system, we surveyed the training facilities designated by the Japanese Society of Balneology, Climatology and Physical medicine (BCPM). Of the 24 facilities targeted by the survey, 21 responded (88%). Currently, the training curriculum for Onsen-Ryoho-Specialists consists of 8 units on diseases and 8 units on therapy methods. As shown in Figs. 1 and 2, it is difficult for a single facility on effectively cover all of these units. The most pressing need is to establish and implement a standardized curriculum across all facilities. Until now, each related academic society has selected training facilities based on its own criteria. Moving forwards, the review/accreditation body of the Japanese Medical Specialist Broad will make site visits to establish and review Onsen-Ryoho-Specialist training facilities. These efforts should lead to the development of fully qualified Onsen-Ryoho-Specialist training facilities.

An Examination of the Current State of the Specialist Accreditation System for Onsen-Ryoho-Specialist / 日本温泉気候物理医学会雑誌

  To evaluate the current state of the Onsen-Ryoho-Specialist (Broad Certified Fellow in Balneology, Climatology and Physical Medicine) training system, we surveyed the training facilities designated by the Japanese Society of Balneology, Climatology and Physical medicine (BCPM). Of the 24 facilities targeted by the survey, 21 responded (88%). Currently, the training curriculum for Onsen-Ryoho-Specialists consists of 8 units on diseases and 8 units on therapy methods. As shown in Figs. 1 and 2, it is difficult for a single facility on effectively cover all of these units. The most pressing need is to establish and implement a standardized curriculum across all facilities. Until now, each related academic society has selected training facilities based on its own criteria. Moving forwards, the review/accreditation body of the Japanese Medical Specialist Broad will make site visits to establish and review Onsen-Ryoho-Specialist training facilities. These efforts should lead to the development of fully qualified Onsen-Ryoho-Specialist training facilities.

Health Effects of Seasonal Bathing in Hot Water, Seasonal Utilization of Hot Spring Facilities, and High Green Tea Consumption / 日本温泉気候物理医学会雑誌

  Among lots of lifestyle factors thought to be related to the Japanese health status, bathing in hot water, utilizing onsen (hot spring) facilities, and drinking green tea are very special among the Japanese. In our previous study, we investigated the frequency of bathing, the utilization of onsen facilities, and the consumption of green tea among the Japanese and found that these activities improved subjective health to a similar degree as balanced diet, exercise, no tobacco use, and quality sleep. In the present study, to ascertain healthy activities that should be encouraged throughout the year, we included survey items about seasonal variations in taking baths, taking showers, and utilizing onsen facilities, as well as investigated the difference between consumption of brewed green tea and of canned and bottled green tea. A cross-sectional questionnaire survey was conducted of 6,000 residents in Shizuoka prefecture aged ≥20 years in 2012. Using unconditional logistic models, odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated for several factors that were considered to affect self-rated health (SRH; subjective health, sufficient sleep and rest, and subjective happiness). This time, in a more detailed investigation of bathing habits, onsen utilization, and green tea consumption, we found that daily bathing all year round (subjective health: OR=1.27, 95%CI 1.05-1.52; p=0.012, sufficient sleep and rest: OR=1.41, 95%CI 1.13-1.77; p=0.003, subjective happiness: OR=1.35, 95%CI=1.15-1.58; p0.021), and daily consumption of at least 500 mL of brewed green tea (subjective health: OR=1.24, 95%CI 1.01-1.53; p=0.039) are health-improving habits that should be proactively encouraged in daily living. We believe that the inclusion of a formal recommendation for daily green tea consumption and daily bathing and the establishment of reference values for these habits in the Health Promotion Policy will foster health and longevity in Japanese citizens.