Mobile DNA(2023) 14:22
Ichiyanagi et al. Mobile DNA
https://doi.org/10.1186/s13100-023-00310-9

Open Access

MEETING REPORTThe sixth Japanese meeting on biological
function and evolution through interactions
between hosts and transposable elements
Kenji Ichiyanagi1*, Yoko Ikeda2* and Kuniaki Saito3*

Abstract
The sixth Japanese meeting on host–transposon interactions, titled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements,” was held on August 24th and 25th, 2023, at the National Instituteof Genetics as well as online. This meeting was supported by the National Institute of Genetics and aimed to bring
together researchers studying the diverse roles of TEs in genome function and evolution, as well as host defense
systems against TE mobility, TE bursts during evolution, and intron mobility in mammals, insects, land plants, yeast,
protozoa, and bacteria. Here, we have presented the highlights of the discussion.
Organizers: Kenji Ichiyanagi, Yoko Ikeda, and Kuniaki Saito.
Keywords Transposon, Retrotransposon, Epigenetics, Gene innovation, Evolution
Introduction
Transposable elements (TEs) constitute a significant proportion of the eukaryotic genome and play key biologicaland evolutionary roles. The interactions between TEs andtheir hosts were discussed in a series of meetings titled“Biological Function and Evolution through Interactions
between Hosts and Transposable Elements” [1–3]. This
meeting was held at the National Institute of Genetics(NIG), Mishima, Japan, where biologists studying TEs in

different species gathered and exchanged ideas. At thesixth meeting, held on August 24th and 25th, 2023, we
invited 17 speakers to present their recent works in six
sessions chaired by Akira Kanazawa (Hokkaido University, Japan), Tetsuji Kakutani (University of Tokyo, Japan),Yoichi Shinkai (RIKEN, Japan), Mari Ohnuki (Kyoto University, Japan), Yota Murakami (Hokkaido University,
Japan), and Haruhiko Siomi (Keio University, Japan). The
meetings had approximately 50 onsite attendees and 40
online attendees.

*Correspondence:
Kenji Ichiyanagi
ichiyana@agr.nagoya-u.ac.jp
Yoko Ikeda
yikeda@okayama-u.ac.jp
Kuniaki Saito
saitok@nig.ac.jp
1
Laboratory of Genome and Epigenome Dynamics, Departmentof Animal Sciences, Graduate School of Bioagricultural Sciences, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464‑8601, Japan
2
Institute of Plant Science and Resources, Okayama University, 2‑20‑1
Chuo, Kurashiki 710‑0046, Japan
3
Invertebrate Genetics Laboratory, National Institute of Genetics, Yata
1111, Mishima, Shizuoka 411‑8540, Japan

Highlights of the talks
Epigenetic regulation of TEs

Epigenetic regulation involving DNA methylation, histone modification, and small RNA pathways is a pivotal
mechanism for silencing the transcription and mobility of TEs in eukaryotes. Taiko Kim To (Tokyo Instituteof Technology, Japan) introduced recent findings on anovel de novo DNA methylation pathway named RdDMindependent CH methylation Establishment (RiCHE) [4].
In many plant species, the silencing of heterochromatin
depends on H3K9 methylation and DNA methylation© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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Ichiyanagi et al. Mobile DNA(2023) 14:22

of CpG and non-CpG sequences (or CpH; H stands for
A/T/C). The establishment of DNA methylation in bothsequences depends on an RNAi-based pathway called
RNA-directed DNA methylation (RdDM). However,paradoxically, deficiency in the RdDM mechanism resultsin hypomethylation in euchromatin regions rather thanin heterochromatin regions. Therefore, it is unclear how
heterochromatin marks are targeted to specific regions.
Dr. To presented recent epigenomic analyses [5, 6], indicating that a novel pathway designated as RiCHE canstrongly and precisely induce de novo methylation inboth the CpH sequence and histone H3K9 in most TEs.
Interestingly, this pathway prefers the coding regions ofTEs, and its specificity has been suggested to involve histone H2A variants. Furthermore, the establishment ofRNAi-independent CpH/H3K9 methylation is dependent
on CpG methylation, indicating that crosstalk among epigenetic modifications drives heterochromatin formationin Arabidopsis. Yoko Ikeda (Okayama University, Japan)
presented a DNA methylation regulatory mechanism in
the liverwort, Marchantia Polymorpha, which is a member of a basal land plant lineage. Flowering plants have
evolved a system for maintaining stable CpG methylationin the next generation. In contrast to that in mammals,
active DNA demethylation does not occur in plant germ
cell lines. However, in Marchantia, genome-wide changes
in both CpG and non-CpG methylation were observed,
suggesting the presence of different regulatory mechanisms for DNA methylation. MpMET, an ortholog of themaintenance CpG methylation enzyme, and two copies
of orthologs of DNA demethylation enzymes were foundin the Marchantia genome [7]. Based on the disruption
mutant analysis, Dr. Ikeda and her colleagues found thatthese contribute to DNA methylation and TE regulation through unique features of Marchantia. Eriko Sasaki(Kyushu University, Japan) discussed the genetic architecture underlying DNA methylation variation (i.e., epigenetic diversity) and its role in regulating TEs in natural
populations of Arabidopsis thaliana. Although this variation is often referred to as the epigenome and is speculated to result from past environmental effects, Dr. Sasaki
and her colleagues have demonstrated that a significant
portion of this variation is influenced by genetic variation in trans modifiers, especially epigenetic regulators
including CHROMOMETHYLASE2 (CMT2), CMT3,
and NRPE1, the largest subunit of RNA polymerase V[8]. She also showed that the novel regulator JMJ26 specifically controls CHG methylation in RdDM-targeted
TEs. These alleles displayed a geographic cline and were
strikingly associated with the regional climate, suggesting a form of local adaptation. Although the specificnature of this adaptation remains unclear, the results of
population genetics provide evidence that combinations

Page 2 of 6

of trans-modifiers influence the mobilization of TEs
by controlling DNA methylation levels under natural
conditions.
In budding yeast, Saccharomyces cerevisiae, a longterminal repeat (LTR) retrotransposon named Ty1
exhibits an interesting characteristic regarding the regulation of transcription. Hiroshi Masumoto (Nagasaki
University, Japan) demonstrated the silencing effect of
Ty1, which has a sequence that can silence a transgene
inserted between gag and pol [9]. Dr. Masumoto andhis colleagues recently found that the transcription fork
originating from the 5′-LTR promoter passes throughthe insertion region, canceling the silencing effect andallowing expression of the inserted gene. In contrast, thesilencing effect is restored when the transcription fork
originating from the 5′-LTR is stopped. This sequence
region exerted a silencing effect in only one direction
and did not interfere with the generation of transcription
forks from the 5′-LTR upstream. This makes it a uniquesilencing region, in which the silencing effect can beswitched on and off by a transcription fork.
In animals, PIWI-interacting RNAs (piRNAs) play
an important role in TE silencing in germ and gonadal
somatic cells. These RNAs are generated from precursor transcripts. However, the evolutionary origin ofpiRNA source loci, called piRNA clusters, which aredispersed over TE-accumulated regions in the genome,
remains elusive. Keisuke Shoji (University of Tokyo,
Japan) presented a model for the origin of piRNA clusters in insects. His colleagues first identified a piRNA
cluster named “torimochi” in BmN4 cells from silkworm
ovaries [10]. Using an updated silkworm genome, Shojiet al. found that torimochi is a complete transposon with
LTRs. Its transposition activity in BmN4 cells, assessedusing a nanopore sequencer, was the highest among all
the TEs. When the cells were in vitro differentiated into
non-gonadal cells (i.e., adipocytes), the expression of torimochi decreased, whereas other TEs were derepressed.
Taken together, they proposed that torimochi, an unfragmented active transposon, integrates with a BmN4specific activation mechanism and serves as a model for
nascent piRNA cluster formation [11].
Anti‑TE mechanisms at the protein and DNA levels

TE activity is restricted by mechanisms other than epigenetic regulation. Kuninori Suzuki (University of Tokyo,
Japan) demonstrated that selective autophagy downregulates Ty1 retrotransposition in budding yeasts.
Autophagy is induced under starvation conditions andplays a cytoprotective role by degrading unwanted cytoplasmic components. Ty1 is the most abundant retrotransposon in S. cerevisiae and introduces mutations via
retrotransposition. Retrotransposition intermediates

Ichiyanagi et al. Mobile DNA(2023) 14:22

are found in virus-like particles (VLPs), which are localized in the cytoplasm. Interestingly, Atg19-dependent
selective autophagy eliminates Ty1 VLPs from the cytoplasm under starvation. In the absence of Atg19, the Ty1transposition frequency continually increased during
starvation [12]. This suggests that selective autophagy
safeguards genomic integrity by suppressing TE mobility
during nutrient starvation in eukaryotic cells.
Binuclear protozoans, ciliates, whose lineage is evolutionarily distant from animals or plants, also use epigenetic marks to transcriptionally silence TEs; however,they also eliminate the DNA sequences of marked TEs
from their active genome. The ciliate Tetrahymena thermophila possesses two distinct nuclei within a single
cell: the transcriptionally inactive germline micronucleus(MIC) and the transcriptionally active somatic macronucleus (MAC). During sexual reproduction, the MIC
differentiates into the MAC of sexual progeny. In thisprocess, more than 12,000 loci containing TEs and theirremnants are heterochromatinized de novo and eventually eliminated from the MAC genome. Kensuke Kataoka(National Institute for Basic Biology, Japan) studied the
dynamics of heterochromatin and its effects on DNA
elimination [13, 14]. Kataoka et al. found seven HP1-likeproteins that accumulate with TEs before their elimination, and a subset of these HP1 proteins plays a central
role in DNA elimination. These HP1 proteins physicallyinteract with each other and cooperatively recognize histone H3 methylated at K9 or K27, whose genomic locations are dictated by small RNA pathways. These findings
suggest that in addition to small RNA-directed TE recognition, the cooperative action of multiple HP1-like proteins ensures robust DNA elimination of thousands ofTE-related sequences in Tetrahymena.
TE burst in plants

In some species, certain TEs are amplified rapidly (TEburst), which provides their hosts with an opportunity tosignificantly change their phenotype. The genus Vigna is
a reservoir of diversity consisting of more than 100 species, many of which have adapted to harsh environments,
including coastal, desert, marsh, and limestone karst[15]. Given that most Vigna species have a small diploid
genome (2n = 22, 500–600 Mb in haploids), the genus
Vigna can serve as a model for comparative genomic
studies to elucidate the evolution of responses to abiotic stresses. Using a long-read sequencer, Ken Naito(National Agriculture and Food Research Organization,
Japan) determined the genomes of 12 Vigna species,
including V. marina and V. riukiuensis, that have adapted
to coastal environments and acquired salt tolerance. The
scaffolds obtained showed that the overall structure of
the genome, including gene order, was highly conserved

Page 3 of 6[16]. However, subsequent ortholog analysis identified
very few cases of copy number variations in stress-related
genes, which are often reported in species adapted to
extreme environments. GO-enrichment analysis on species-specific orthogroups revealed that V. marina and V.
riukiuensis have accumulated many copies, with V. riukiuensis having more than 1000 copies, of WUSCHELrelated homeobox (WOX) gene family. The amplified
WOX genes in the two species were mostly surroundedby identical LTR sequences at both ends, with typical target site duplications (TSDs). Although the contributionof WOX gene amplification to coastal adaptation remains
unknown, their study revealed that LTR retrotransposons
can burst rapidly and have the potential to affect gene
content in certain species.
Eigo Fukai (Niigata University, Japan) and his colleagues have been working on TEs in the model legume
Lotus japonicus. They found recent retrotransposition
events of several LTR retrotransposons in three recombinant inbred-line populations (RILs) of L. japonicus[17]. These LTR retrotransposons remained silenced for along time in the parental accessions, suggesting that they
were epigenetically activated by hybridization and subsequent selfing during the establishment of RILs. Although
the molecular mechanism of this phenomenon is still
unknown, their observations argue in favor of a hypothesis that hybridization can induce “genomic shock,” whichcauses transgenerational TE derepression and consequent TE amplification, even if the parents do not havestrong reproductive barriers.
Yuki Mizunaru (Kyushu University, Japan) discussed
the transposition of Tpn1 family transposons (Tpn)
belonging to the CACTA​ superfamily in Japanese Morning Glory (Ipomoea nil). Transposition of the Tpn1 family frequently accounts for most mutations affecting thephenotype. However, most genomic Tpn copies are nonautonomous elements that require a transposase encodedby an autonomous element for transposition [18]. TpnA2,
one of the autonomous elements, is present in only one
copy in Asian strains including Japan, and maintains thetransposase gene stably due to deletion of the 3′ terminal region [19]. Genetic experiments have shown that
TpnA2 is a major transposase source. In Japan, numerous mutants have been recorded since the middle of theeighteenth century, suggesting that such mutations arecaused by the activation of Tpn transposition. Analysis of the genomic sequences of 140 Japanese strains
revealed that, in hypermutable strains, the number of
Tpn copies has increased. Most of these strains sharemutations caused by Tpn transposition in two flower
color genes, suggesting that they were derived from asingle plant with increased Tpn transposition activity.
Taking advantage of the homogeneity of the genome,

Ichiyanagi et al. Mobile DNA(2023) 14:22

Mizunaru et al. identified candidate genes for Tpn activation in Japanese strains by comparing the genome data
between two groups showing activated and silenced Tpn
transpositions.
TE functions as a protein and DNA

TE sequences are known sources of novel protein-coding genes. So Nakagawa (Tokai University, Japan) has
discussed the diversity and evolution of de novo protein-coding genes derived from endogenous virus-likeelements (EVEs), including endogenous retroviruses(ERVs). Several protein-coding EVEs, primarily ERVs,
are present in the genomes of various mammalian species. Most of these encoded proteins are truncated owing
to nonsense and/or frameshift mutations and are epigenetically silenced. However, some are under purifying
selection, suggesting that they provide new functions
to their hosts, such as cell-cell fusion activity involvedin placental development [20]. However, it is difficult todetermine whether EVE-derived ORFs function in thehost. For example, although the ORF remains intact,
they recently found that syncytin-2, which is involvedin placental development in humans, does not exhibit
fusogenic activity in various Platyrrhini (i.e., New World
monkeys) [21]. To investigate functional protein-coding
EVE-derived genes (> 80 amino acids with viral protein
motif(s)), Dr. Nakagawa his colleagues constructed a
database named gEVE (http://​geve.​med.u-​tokai.​ac.​jp) of
19 mammalian genomes [22]. Various high-quality mammalian genomes have been sequenced. Thus, updating
the gEVE database will provide a foundation for studying the specialization and evolution of mammalian species. For example, Nakagawa and his colleagues found, inthe echidna genome, an ERV envelope-derived sequenceshowing a fusogenic activity [23]. Koichi Kitao (Nagoya
University, Japan) presented his research on the searchfor protein-coding genes derived from LTR retrotransposons, including ERVs, in the genomes of monotremes,
birds, and reptiles. His group first identified ERV-derived
protein-coding genes, designated as RTOM1, 2, and3, by comparative genomic analyses of two egg-laying
mammals: the platypus (Ornithorhynchus anatinus) andechidna (Tachyglossus aculeatus). RTOM genes encode
reverse transcriptases and are expressed specifically inthe testes of both the platypus and echidna. Such testisspecific reverse transcriptase genes have not been foundin therian mammals, but their involvement in reproductive functions unique to monotremes has been previously
discussed [24]. He also analyzed the genomes of birds andreptiles and found conservation of several protein-coding
genes derived from LTR retrotransposons. These results
indicate the diverse functions of LTR retrotransposonderived proteins in vertebrates. Kuniaki Saito (National

Page 4 of 6

Institute of Genetics, Japan) discussed the subcellular
localization of gypsy Gag and Env proteins in Drosophila
ovarian somatic cell line OSC, in which Piwi-piRNAdependent TE silencing occurs. Although the expressionof ERV-derived proteins in the ovary has been reported
previously [25], their functions and actions are not fully
understood. To elucidate this in detail, Saito et al. generated monoclonal antibodies against gypsy Gag and Env
proteins and found that Gag co-localized with Env in thecytoplasmic granules of Piwi-depleted OSCs. Env depletion dispersed Gag localization, whereas Gag depletion
did not change Env localization. This implies the cooperative action of Gag and Env proteins in Piwi-depleted
OSCs.
TEs also provide cis-regulatory elements that havealtered gene expression during evolution. Many LTR
retrotransposons in the human genome are primatespecific and introduce novel cis-regulatory elements into
the lineage. While the transcriptional activity of LTRs is
restricted by the host through various mechanisms, thenucleotide changes in LTRs associated with their activity
and expansion during primate evolution remain largely
unknown. Fumitaka Inoue (Kyoto University, Japan)
et al. found that the MER11 family is one of the youngest simian-specific ERVs and is highly enriched withactive epigenetic marks in human pluripotent stem cells.
They analyzed the phylogenetic relationship between
the MER11A/B/C subfamilies and classified them into
multiple phyletic groups, which resolved the sequenceand epigenetic heterogeneity of the MER11 subfamilies. To understand the functional divergence of MER11
sequences, massively parallel reporter assays (MPRAs)
were used to systematically analyze MER11 sequencevariants in human, chimpanzee, and macaque genomes
to determine their regulatory activities [26, 27]. They
found that transcription factors, such as ZIC and TEAD,
are responsible for the regulatory activity of evolutionarily old MER11 phyletic groups. In contrast, evolutionarily
young MER11 phyletic groups acquired POU- and SOXbinding motifs via nucleotide deletion(s) in the active
core sequence. MPRA validated the correlation between
POU and SOX motif acquisition and regulatory activity. Thus, this study delineates the functional divergenceof MER11 along with nucleotide changes in primate
genome evolution.
In Archaea, some tRNA genes have introns and sometRNA genes are split. Recently, an association between
some intron-containing tRNA genes and TEs wasreported. Akio Kanai (Keio University, Japan) et al. discovered disrupted tRNA genes in various species bycomputational analysis of their complete genomes. Notably, within the hyperthermoacidophilic archaeon Caldivirga maquilingensis, they found that ­tRNAGly(TCC) andIchiyanagi et al. Mobile DNA(2023) 14:22­tRNAGly(GCC) consist of three separate transcripts, aphenomenon termed “tri-split tRNA” [28]. Interestingly,
the leader sequences, which serve as scaffolds for interlinking tri-split tRNAs within the cell, correspond to thetRNA intron regions found in closely related archaeal
species, suggesting an evolutionary relationship between
split tRNAs and intron-containing tRNAs. Moreover,certain tRNA introns transpose from one tRNA into
multiple other tRNAs [29]. In this regard, such tRNA
introns are considered as TEs. He also reported thatthe 16S and 23S rRNA genes of Candidate Phyla Radiation (CPR), a recently discovered bacterial phylum that is
uncultured or challenging to cultivate, contain numerous
introns. Interestingly, these introns are mobile group I or
II introns, and some encode homing endonucleases [30],which shed light on the association between TEs andRNA genes in the prokaryotic world.
TE expression in development and stress response

In preimplantation development in mice, the LTR retrotransposon MERVL is transiently expressed during
zygotic gene activation (ZGA) at the 2-cell stage, whichis essential for blastocyst formation [31]. In embryonicstem cells (ESCs) derived from the inner cell mass of
blastocysts, MERVL expression is suppressed; however,
MERVL-positive cells called 2-cell-like cells (2CLCs),
spontaneously arise at a rate of approximately 0.1%. Cells
expressing MERVL have been suggested to be totipotent.
Hirotsugu Ishizu (Keio University) presented the mechanism underlying the emergence of 2CLCs through thereprogramming of mouse ESCs. By performing singlecell RNA-seq analysis of the cell population transitioning
from ESCs to 2CLCs, they found that the retinoic acidsignaling pathway plays an essential role in shaping the
totipotent program. Indeed, treating ESCs with a retinoic
acid analog activated the expression of MERVL and other
2-cell stage-specific genes, which promoted the conversion of the cells to 2CLCs. Elucidating the functions of
MERVL regulated by the retinoic acid signaling pathway
remains a task for future research.
In contrast to the expression analysis of long interspersed elements (LINEs) and LTR retrotransposons,the expression of short interspersed elements (SINEs),
which are transcribed by RNA Pol III and are typically< 500-bp long, is difficult to analyze by deep sequencing because many copies reside within other transcripts
such as mRNAs. Kenji Ichiyanagi (Nagoya University,
Japan) recently established a deep sequencing method,
melRNA-seq, to determine the RNA abundance, RNA
length, and source loci of SINEs [32, 33]. They analyzed the expression of Alu SINE in human cells which

Page 5 of 6

revealed a 9-fold increase in Alu expression after heatshock treatment. The loci expressed under heat shock
were predominantly located outside Pol II-transcribedgenes and, interestingly, often contained binding motifs
for transcription factors (TFs) involved in the immune
response to viral infection. These results suggested thatthese TFs were involved in the upregulation of Alu during heat shock and viral infection. Because Alu RNA
has been shown to regulate the activity of Pol II, thisAlu-TF association may have biological significanceand will be studied in the future.
Abbreviations
2CLC	2-cell-like cell
CPR	candidate phyla radiation
ESC	embryonic stem cell
EVE	endogenous viral-like element
ERV	endogenous retrovirus
LINE	long interspersed element
LTR	long terminal repeat
MAC	macronucleus
MIC	micronucleus
MPRA	massively parallel reporter assay
NIG	National Institute of Genetics
ORF	open reading framepiRNA	PIWI-interacting RNA
RdDM	RNA-directed DNA methylation
RiCHE	RdDM-independent CH methylation establishment
RIL	recombinant inbred line
RNAi	RNA interference
SINE	short interspersed elementTE	transposable element
TF	transcription factor
TSD	target-site duplication
VLP	virus-like particles
WOX	WUSCHEL-related homeobox
ZGA	zygotic gene activation
Acknowledgements
We acknowledge all invited speakers for their assistance with manuscript
preparation. We would like to thank Editage for English language editing.
Authors’ contributions
K.I, Y.I., and K.S. organized the meeting and wrote the manuscript.
Funding
This meeting was supported by the NIG-JOINT (grant ID: 8R2023) by National
Institute of Genetics, Japan.
Availability of data and materials
Not applicable.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent of publication
Not applicable.
Competing interests
The authors declare no competing interests.

Ichiyanagi et al. Mobile DNA(2023) 14:22

Received: 7 November 2023 Accepted: 27 November 2023

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