Citation: Palazzo, A.; Caizzi, R.;
Moschetti, R.; Marsano, R.M. What
Have We Learned in 30 Years of
Investigations on Bari Transposons?.
Cells 2022,11, 583. https://
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Received: 12 January 2022
Accepted: 7 February 2022
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What Have We Learned in 30 Years of Investigations on
Antonio Palazzo , Ruggiero Caizzi, Roberta Moschetti and RenéMassimiliano Marsano *
Dipartimento di Biologia, Universitàdi Bari, 70125 Bari, Italy; firstname.lastname@example.org (A.P.);
email@example.com (R.C.); firstname.lastname@example.org (R.M.)
Transposable elements (TEs) have been historically depicted as detrimental genetic entities
that selﬁshly aim at perpetuating themselves, invading genomes, and destroying genes. Scientists
often co-opt “special” TEs to develop new and powerful genetic tools, that will hopefully aid in
changing the future of the human being. However, many TEs are gentle, rarely unleash themselves to
harm the genome, and bashfully contribute to generating diversity and novelty in the genomes they
have colonized, yet they offer the opportunity to develop new molecular tools. In this review we
summarize 30 years of research focused on the Bari transposons. Bari is a “normal” transposon family
that has colonized the genomes of several Drosophila species and introduced genomic novelties in
the melanogaster species. We discuss how these results have contributed to advance the ﬁeld of TE
research and what future studies can still add to the current knowledge.
Bari transposons; Drosophila; regulation; transposon tandem repeat; horizontal transfer;
blurry promoter; heterochromatin
Transposable elements are fundamental genetic units in the genomes of virtually
all living organisms. TEs could be depicted as the characters of a happy ending fairytale.
Initially regarded as “junk” and “useless”, TEs turned out to be considered as evolutionarily
ﬂagships after reconsidering the role they have had and still have in shaping genomes
and their functioning. Moreover, the characterization of many transposition systems has
led to the development of efﬁcient DNA integration tools [
] as well as powerful genome
engineering systems [
], and to the implementation of TE control regions into efﬁcient
expression systems .
It is a matter of fact that the hallmark of all TEs, i.e., the ability to integrate into
chromosomes, is the most interesting aspect of TEs for many biologists, due to their many
possible applications in a large group of ﬁelds in Life Sciences.
Efﬁcient genome integration tools are indeed desirable to disrupt genes, either in a
random or targeted way, and to introduce exogenous DNA into the preferred cellular or
animal model. A special application of the latter practice is gene therapy, which consists
in the introduction of the non-pathological allele in the affected cell type of a patient that
suffers from a genetic-based disease, with the aim to rescue the illness phenotype.
In the past 40 years many transposition systems have been studied in detail with the
aim to set up new and efﬁcient DNA integration tools.
Historically, the Drosophila P-element was the ﬁrst transposon-based transposition
tool to be employed in functional genomics [
]. Unfortunately, its main limitation is the
narrow host range of transposition [
], which makes it useless for much noble applications,
such as gene therapy.
Elements of the Tc1/mariner are more tractable for this kind of application. Tc1/mariner
elements belong to the Class II of the eukaryotic transposons and are widely distributed,
Cells 2022,11, 583. https://doi.org/10.3390/cells11030583 https://www.mdpi.com/journal/cells
Cells 2022,11, 583 2 of 17
from bacteria  to higher eukaryotes , with few exceptions. Their wide distribution in
living organisms allowed the foundation of the IS630/Tc1/mariner superfamily.
More in general, the so-called “cut and paste” DNA transposons are the best can-
didates to develop molecular tools for transgenesis because of their simple mechanism
of transposition and their poor requirement of host factor [
]. Two of these elements
stepped into the limelight in the past decade, the Sleeping Beauty (SB) and the piggyBac (PB)
]. SB is undoubtedly the most sophisticated transposon-based system in the
context of therapeutic setup. The CARAMBA clinical trial (https://www.caramba-cart.eu;
accessed 15 December 2021) currently uses an advanced SB-based transposon technology
for therapeutic gene delivery .
However, not all the known transposition systems support this kind of application.
Most known TEs have limited transposition performances (i.e., low transposition rate,
narrow host speciﬁcity) or they have low ﬂexibility (i.e., they are too large and complex
or display low cargo capability) to allow the development of efﬁcient genome integration
systems. Nevertheless, many TEs are studied for their role in shaping genome structure [
and gene expression  or to develop new and alternative technologies [3,4,14,15].
Among the Tc1/mariner superfamily of TEs the Bari family was discovered 30 years
ago in the former Institute of Genetics at the University of Bari (Italy). Such discovery led
to the foundation of a new research line, which is still currently under investigation, in a
laboratory up to that time devoted to the study of glutamine synthetase [16–18].
In this review, we comprehensively summarize the results of 30 years of research that
concern the Drosophila Bari elements, and frame these results in a comprehensive view
in the ﬁeld of TE research. We also provide more extensive hypotheses on the role of Bari
transposons in the genome of Drosophilidae species.
The Discovery of the Bari1 Transposon: An Historical Overview
The discovery of the Bari transposons occurred during the characterization of the h39 re-
gion of the mitotic chromosomes. This is a complex repetitive locus in Drosophila melanogaster,
adjacent to the second chromosome’s centromere [
]. It was known from previous studies
that mutant ﬂies carrying the deletion of the h39 region showed a semi-lethal phenotype
and low ﬁtness [
]. The phenotype was associated with the deletion of the Responder
satellite , the main satellite mapped in the h39 until then.
The team headed by Prof. Caizzi and Prof. Pimpinelli hypothesized that additional
genetic and molecular entities could map in the h39 region, which could also account for the
phenotype associated with the region deletion. In the main effort of characterizing the h39
region at the molecular level, they identiﬁed a novel repetitive sequence, uniquely mapping
to this region. Originally, differential hybridization technique was used to identify, isolate,
and subsequently clone h39-speciﬁc sequences. The existence of extraordinary genetic
toolkits, such as precisely mapped chromosome rearrangements (the most effective was
(R16) deletion [
]) undoubtedly aided the genetic mapping in a heterochro-
matic region. Furthermore, the availability of molecular tools, such as the possibility to
construct strain-speciﬁc genomic libraries, but most of all acrylamide gel electrophoresis to
read Sanger sequencing reactions, strongly contributed to the characterization of a previ-
ously unknown sequence isolated from single copies dispersed in the euchromatin that
was soon classiﬁed as a new transposon of the Tc1/mariner superfamily. It was named Bari1
after its discovery in the Italian city of Bari, where the laboratory was based, and making
the wish (which would later come true!) that other Bari elements might be discovered to
complete the series.
Bari1 is a DNA transposon belonging to the IS639/Tc1/mariner superfamily with 26 bp
long inverted repeats (IRs) and three direct repeated sequences (DRs) [
], serving as the
transposase binding sites (Figure 1A). As noted earlier in comparative studies, the 3xDRs
structure is almost peculiar [
], since there are few known TEs with similar organization
of the terminal sequences, including Paris , S, minos , and SB .
Cells 2022,11, 583 3 of 17
If the identiﬁcation of a new transposon in the early 1990s was per se a great advance-
ment in the ﬁeld of genome structure and evolution, the characterization of the peculiar
arrangement of Bari1 in a heterochromatic locus was breathtaking. The precise head-to-tail
organization of roughly 80 Bari1 copies in the h39 region of the mitotic chromosomes of
D. melanogaster was featured by the systematic deletion of the very ﬁrst two nucleotides
in each copy. To our knowledge, this enigmatic organization still has no comparable de-
scribed examples in the ﬁeld of TEs. While other Drosophila species contain Bari-like
transposons (see the Section “The Bari Family Grows Up”), the heterochromatic Bari1 clus-
ter seems to be speciﬁc to the melanogaster species. This organization suggests a recent-and
species-speciﬁc evolutionary origin since it is speciﬁc to a single species. It also allows
the discrimination of two sibling species (i.e., the melanogaster and simulans species) at
the molecular level. Moreover, a second minor Bari1 cluster has been recently described,
which maps on the X chromosome of D. melanogaster [
]. This is a small cluster that
is composed of six copies of Bari1. Surprisingly, both the main and the small clusters
share the same di-nucleotide deletion at the 5
end of each element, and the head-to-tail
organization. In addition, both clusters share a heterochromatic localization, although on
different chromosomes. In a retrospective view, it is worth noting that the presence of
an additional Bari1 tandem repeat was also evident since the 1993 paper. Indeed, in one
of the Southern blot hybridization analyses presented in the paper, it was evident that
background still contains an additional Bari1 sequence block arranged in a
tandem repeat conﬁguration (see Figure 4 in Caizzi et al., 1993 [
]. A similar pattern can
be observed in Caggese et al., 1995 [
] (see Figure 2 therein), an observation that allows
excluding technical artifacts. Taken together, these ﬁndings suggest a marked instability of
the Bari1 element in the melanogaster species, associated with an error-prone transposition
that has occurred at least twice during the evolution [
]. An aberrant transposase activity
that generated long concatemers form a circular transposon, in a way similar to the rolling
circle replication mechanism, has been proposed to explain the origin of both the Bari1
]. However, the structural and functional role of these clusters (if any) remains
Among the possible role of the Bari clusters, two scenarios can be envisioned. The
simplest hypothesis is that it could have a regulatory function. The second hypothesis,
which we are currently testing using transgenic strains, is that the Bari cluster could be
involved in the structural organization of h39 domains, possibly aided by other repetitive
sequences mapping in the same region [35–37].
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Cells 2022, 11, x FOR PEER REVIEW 4 of 16
Figure 1. (A) Structure and TIRs organization of the three Bari sub-families. The length of the TIRs
is indicated by colored arrowheads. Bari1 elements may have two different TIR lengths (represented
by solid and dashed green arrowheads, respectively). The three DRs (outer (red), middle (violet),
and inner (yellow)) are shown as DNA logos  obtained by comparing at least 15 different Dro-
sophila species. The elements represented in the picture (TIRs, ORFs) are not drawn to scale. (B)
Distribution of Bari-like elements in the Drosophilidae (adapted from ). Symbols are explained
in the figure legend and have the same color-code as in panel A (i.e., Bari1-like green; Bari2-like blue;
2. The Bari Family Grows Up
2.1. Introducing Bari2 and Bari3
Studies aimed at the determination of the diffusion of the Bari transposons in Dro-
sophila genus and in other Diptera species demonstrated that Bari transposons are widely
represented in the Drosophila genus and that they can be subdivided into three distinct
Former studies, mainly performed using Southern blot hybridization techniques,
demonstrated the presence of homologous Bari1 sequences in species closely related to D.
melanogaster . Weak or very faint hybridization signals suggested the presence of Bari
elements that were divergent in sequence in distant species, belonging to the Sophophora
and Drosophila sub-genera, and to the Zaprionus genus. Indeed, cloning and sequencing
hybridizing fragments (showing weak hybridization signals) from D. erecta and D.
diplacantha demonstrated the presence of another type of Bari element, which was named
Bari2, in the genome of many species in which the Bari1 element was also found. Bari2
differs from Bari1 in structure, since it is characterized by long TIRs (nearly identical 253
bp sequences) and no coding potential, due to numerous invalidating indels and
frameshift mutations that disrupt its ability to encode a transposase (Figure 1A). Not a
single active Bari2 element has been found to date, making Bari2 a non-autonomous sub-
) Structure and TIRs organization of the three Bari sub-families. The length of the TIRs is
indicated by colored arrowheads. Bari1 elements may have two different TIR lengths (represented by
solid and dashed green arrowheads, respectively). The three DRs (outer (red), middle (violet), and
inner (yellow)) are shown as DNA logos [
] obtained by comparing at least 15 different Drosophila
species. The elements represented in the picture (TIRs, ORFs) are not drawn to scale. (
of Bari-like elements in the Drosophilidae (adapted from [
]). Symbols are explained in the ﬁgure
legend and have the same color-code as in panel A (i.e., Bari1-like green; Bari2-like blue; Bari3-
2. The Bari Family Grows Up
2.1. Introducing Bari2 and Bari3
Studies aimed at the determination of the diffusion of the Bari transposons in Drosophila
genus and in other Diptera species demonstrated that Bari transposons are widely rep-
resented in the Drosophila genus and that they can be subdivided into three distinct
Former studies, mainly performed using Southern blot hybridization techniques,
demonstrated the presence of homologous Bari1 sequences in species closely related to
D. melanogaster [
]. Weak or very faint hybridization signals suggested the presence
of Bari elements that were divergent in sequence in distant species, belonging to the
Sophophora and Drosophila sub-genera, and to the Zaprionus genus. Indeed, cloning and
sequencing hybridizing fragments (showing weak hybridization signals) from D. erecta and
D. diplacantha demonstrated the presence of another type of Bari element, which was named
Bari2, in the genome of many species in which the Bari1 element was also found. Bari2
differs from Bari1 in structure, since it is characterized by long TIRs (nearly identical 253 bp
sequences) and no coding potential, due to numerous invalidating indels and frameshift
mutations that disrupt its ability to encode a transposase (Figure 1A). Not a single active
Cells 2022,11, 583 5 of 17
Bari2 element has been found to date, making Bari2 a non-autonomous sub-family. Notably,
the distribution of Bari2 in the genome of D. erecta is almost heterochromatic, suggesting
either an insertion preference of the ancestral (active) form of Bari2 or a recent elimination
of the euchromatic copies.
The evolutionary link between the potentially active Bari1 and the evolutionary
knocked out Bari2 elements was also difﬁcult to understand. While the comparison of
the ORF (and its protein product) of Bari1 and the reconstructed consensus sequence of
Bari2 clearly suggested their evolutionary relationship, their TIRs are very dissimilar both
in sequence and structure. Bari2 is indeed featured by long TIRs, identical to each other.
Only at the DRs level the two families share evident homology, a sign that both derived
from an ancestral Bari transposon [
]. Why the TIRs of Bari2 have been preserved in
extant Drosophila species is not clear. An intriguing hypothesis, that parallelizes the one
proposed for the human Hsmar1 element, is that Bari2 TIRs have been co-opted to titrate
some endogenous nuclear protein [
] involved in the maintenance of the chromatin
status or in chromatin remodeling or to titrate the transposase of active Bari elements.
The missing evolutionary link between Bari1, possessing short TIRs and intact ORF,
and Bari2, possessing long TIRs and disrupted ORF, was subsequently identiﬁed with
the discovery of the third Bari family in D. mojavensis [
]. Bari3 was next identiﬁed in
species of the obscura and the willistoni groups [
]. Many intact Bari3 insertions and
the polymorphism observed in geographically distinct populations suggested that it is an
active transposon [
]. Bari3 transposases share 80% similarity with Bari1, and it is featured
by long TIRs with an IR/DR structure (Figure 1A) [24,42].
2.2. New Cognate Elements in New Species: The Crew Grows up in the Post-Genomic Era
The advent of the post-genomic era has offered the opportunity to perform compara-
tive studies that were very difﬁcult to perform without the availability of genome sequence
assembly. Evidence of the presence of Bari elements in distant Drosophila species were
mounting in a former work which identiﬁed homologous sequences in the Sophophora and
Drosophila genera [
]. In a genomic survey study conducted in 23 species of Drosophila,
several other elements related to the three known Bari sub-families were identiﬁed in newly
sequenced Drosophila genomes (Figure 1B) [
]. In this study, Bari-like elements were
identiﬁed and annotated in all but the D. grimshawi species. The extended annotation of
Bari-like transposons suggests that, despite the diversity observed in the TIR structure, the
DRs are well-conserved across Bari sub-families (Figure 1A) [
]. It is worth noting that
an interesting Bari1-type element with long TIRs was identiﬁed in D. rhopaloa (Figure 1B),
which further entangles the evolutionary dynamics involving the terminal ends of the
The evolutionary scenario observed in the extant Drosophila species is complicated by
the presence of an additional group of non-autonomous sequences called MITEs (
lements) are frequently found in eu-
karyotic genomes [
] and they are considered as evolutionary byproducts, originating
from a rearranged (i.e., internally deleted) ancestral form of TIR elements. Their sub-
sequent ampliﬁcation in the genome occurred through trans-complementation with the
functional transposase expressed by active TEs. Bari-derived MITEs have been identiﬁed in
D. sechellia [
] and in other Drosophila species [
]. Bari-derived MITEs can be categorized
either as short- or long-MITEs, depending on their sequence length. While both forms
share the same terminal sequences, the internal sequence can be highly variable in length,
with the long form exhibiting sequences unrelated to Bari transposons [
]. It has been
proposed that the TIRs of both functional and defective copies (including MITEs) that
retain the transposase binding activity can act as buffer to titrate endogenous levels of the
transposase, . Therefore, it is possible that also Bari-derived MITEs are maintained for
this regulatory purpose.
Cells 2022,11, 583 6 of 17
3. Horizontal Transfer Events Involving Bari Elements
Horizontal transfer (HT) is one of the most obscure yet fascinating aspects underly-
ing the evolutionary dynamics of the genomes [
]. TEs are among the most prone
DNA sequenced to take part in HT events [
]. While during the pre-genomic era
horizontal transposon transfer (HTT) events could be only detected using molecular as-
says, currently we have potent bioinformatic tools that allow HTT inference supported
by statistical methods [
]. It is now more evident from genome sequence comparison
that HTT commonly occurs during evolution but it is hard to detect only for two basic
limitations in our approaches. The ﬁrst is intrinsic to the HTT process, in that we could
detect it if occurs in germline cells. In this case, the horizontally transferred DNA can be
transmitted in the population and we can detect it as an “alien” piece of DNA. Somatic
HTT events could conversely result in a small number of mosaic organisms and would not
be detected because of the low (or very low) representation of the transferred sequence
in whole genome extracts. The second limitation in detecting HTT is due to the restricted
number of sequenced genomes, compared to the number of extant species. Furthermore,
even if there are more than 20,000 genome projects in NCBI (different advancement status—
last access early December 2021) they refer to small individual samples, representative of
entire populations or species. This gives us a poor vision of the sequence variability caused
by HTT, with a consistent loss of event detection at the population level.
Several studies suggest that Bari1 moved horizontally several times during the evolu-
tion among Drosophilidae species. Several works provide evidence that Bari1 HT occurred
between D. melanogaster and D. simulans [
], between D. melanogaster and D. yakuba [
and between D. melanogaster and D. sechellia [
]. Moreover, an additional study in 23 se-
quenced species of Drosophila showed that horizontal transfer involving Bari1 elements
also occurred between D. biarmipes and D bipectinata [
]. Bari1 elements in the two species
are nearly identical in sequence, despite that the host species divergence dates back to
27 million years ago .
4. The Missing Jump to the Next Level
4.1. Why We Cannot Use Bari Transposons as Tools for Chromosomal Integration
An initial effort to develop a new chromosomal integration tool based on the Bari1
transposon was made in Drosophila [
]. A binary transposition system was constructed
along the same lines of the P-element system: a helper plasmid (the transposase source)
and a donor plasmid (the transposon source marked with a white reporter gene), were
injected into genotypically suitable ﬂy embryos with the goal of genetically transforming
the recipient strain. After the initial excitement due to the identiﬁcation of few transpo-
sition events (i.e., red-eyed individuals), their molecular characterization turned out to
be puzzling and frustrating, since the observed phenotype was due to transposition of
the NOF-FB transposon rather than the transposition of Bari1 from the donor plasmid. To
make a long story short, the expression of the Bari1 transposase from the helper plasmid
has possibly deregulated the NOF-FB transposon [
], whose transposition is not currently
known and may depend on the activity of unrelated active transposases. Taken together,
these fortuitous observations remind us that unpredictable and complex interactions occur
when we attempt to manipulate the genome. Following a stress condition, the genome
reacts, and the fastest response is often given by the de-repression of transposable elements
in several species [
]. We are currently investigating the possible cross-interaction
between the overexpression of the Bari1 transposase and the transposition of NOF-FB
Similar attempts to observe the transposition of Bari elements through a transposition
assay were made using cultured cells as experimental systems. Setting up a binary system,
in which the donor transposon was marked with a reporter cassette and the transposase
was expressed by a helper plasmid, did not result in a signiﬁcant integration over the
background in Drosophila and human cultured cells [
]. Finally, even correcting the
diverging aminoacidic residues in critical transposase subdomains, with the guide of a
Cells 2022,11, 583 7 of 17
multiple transposase alignment containing other active Tc1-like transposons, does not
signiﬁcantly improve the transposition efﬁciency of Bari1 in heterologous transposition
assays (RMM unpublished observations).
4.2. Active or Non-Active? That Is the Question
The reiterated and unsuccessful attempts to develop an efficient transposition system
from Bari elements led to question whether Bari transposons are transposition-competent. Ex-
cluding the Bari2 sub-family, which is entirely composed of non-autonomous elements [
members of the Bari1 and Bari3 sub-families have what it takes to be regarded as functional.
Standing to the general architecture of the IS630/Tc1/mariner transposase, the funda-
mental domains of this enzyme are the DNA binding domain, the GRPR-like domain
(which mediates protein–protein interactions), the nuclear localization signal, the homeo-
like domain, and the catalytic domain [
]. All these domains can be predicted in the
transposase of both Bari1 and Bari3. Furthermore, the functionality of the DNA binding
domain has been tested
for both transposons [
]. In addition, there is indi-
rect evidence of the transposition ability of Bari transposons coming from population
A series of population genetics analyses strongly suggest that Bari1 is an active trans-
poson in natural populations of D. melanogaster. Early studies were performed in 46 pop-
ulations of D. melanogaster, which suggested both Bari1 inter- and intra-stock polymor-
]. Junakovic and collaborators performed additional studies on a Charolles
laboratory population using Southern blot hybridization of single-ﬂy genomic digestions.
A strong difference in the insertion pattern can be highlighted in unstable (Charolles)
versus stable laboratory strains, suggesting that host factors control the transposition fre-
] and the insertion preference [
] of Bari1. The strongest evidence that Bari1 is
a functional transposon comes from the observation of an excision event in a population
established from ﬁeld-collected ﬂies. The excision event, involving one of the two adaptive
Bari1 insertions in the genome of D. melanogaster, was characterized at the molecular level,
demonstrating that it was due to genuine transposition (i.e., presence of the transposition
In conclusion, combining the indirect genetic evidence of the mobility of Bari1 transpo-
son with the outcome of the experimental transposition assays, we argue that Bari1 could
be tightly regulated, or alternatively we speculate that Bari1 could be subjected to some
unknown types of activation to become transposition-competent. Further and extensive
-directed molecular evolution studies would clarify whether hyperactive variants
can be obtained to develop Bari-based transposition systems.
5. What Do We Know about the Regulation Bari Transposons?
The transposition activity of TEs is tightly regulated at various levels. The copy number
per haploid genome is a critical factor for the resulting ﬁtness of the whole organism since an
excessive TE load can be deleterious for various reasons, ranging from the gene inactivation
to the disturbance of the physiological expression networks .
Standing to the current knowledge on Tc1/mariner elements, two possible types of
regulation can be predicted. One is the transposon self-regulation while the other one is
the epigenetic regulation.
One mode of self-regulation is exerted through the dissemination throughout the
genome of inactive TE copies that still retain the transposase binding ability. Defective
copies can act as buffer to titrate endogenous levels of the transposase [
]. Since de-
fective copies of Bari elements are abundant, it is conceivable to hypothesize this mode
There is some experimental evidence in favor of the self-regulation of Bari elements
through post-transcriptional processing of the transposase mRNA. Few reported data come
from overexpression of the transposase in S2R
cultured cells and in D. melanogaster em-
] (Figure 2A). Under these experimental conditions, Bari1 and Bari3 transcripts
Cells 2022,11, 583 8 of 17
undergo alternative splicing that is potentially translated into a truncated transposase pro-
tein. It can be predicted that the transcriptional de-repression of Bari elements can inhibit the
transposition through the expression of a dominant-negative transposase form. The pres-
ence of alternatively spliced transcripts of Bari1 has also been reported in a D. melanogaster
HSP83 mutant that deregulates many transposons, including Bari1 [
]. Possibly, the
construction of synthetic Bari1 and Bari3 transposase genes in which all the conventional,
unconventional and cryptic splicing sites have been eliminated, could hopefully enhance
the transposition efﬁciency of both systems.
Bari Transposons Regulation Relies on the piRNA Pathways
The second mode of regulation of TEs is at the chromatin level. The transcriptional
control of TEs is intimately connected to chromatin control at the TE insertion site, and this
is possible through the recruitment of speciﬁc chromatin remodeling complexes that either
open or close the chromatin, thus inﬂuencing the transcription.
Genomic structural and transcriptional changes are at the basis of stress responses. In
this view, TEs are an amazing source of variability in the short temporal timeframe that
allows organisms to promptly react to virtually any kind of stress .
The regulation of Bari transposons. (
) Spliced transposase mRNA of Bari1 (green)
identiﬁed in [
] and Bari3 (red) identiﬁed in [
]. Both spliced transcripts encode a transposase
with non-functional catalytic domain. (
) Distribution of piRNA mapping to the Bari1 transposon.
The amount of piRNA is purely representative of the relative fraction matching the ORF (green)
or the heterochromatic inter-monomer junctions (blue) of Bari1 (see text for further details). A
Piwi-interacting RNAs (piRNAs) are the most abundant non-coding RNAs in the
germline, aiming at the genome safeguard against TE movement [
]. Their action is
mainly exerted through the transcriptional silencing. piRNAs usually originate from the
so-called piRNA clusters, genomic loci riddled of TE relics that form long non-coding
RNAs that are further processed into piRNAs that selectively degrade TE transcripts [
Subsequently, a heterochromatic repressive state can be induced at the TE insertion site,
reinforcing the transposition control [70–72].
In several published reports, piRNA sequences matching Bari1 and Bari2 are de-
], confirming that Bari transposons are among the plethora of Drosophila
TEs that are regulated by the piRNA pathway in the germline (Figure 2B). In addition, im-
pairment of the piRNA pathway leads to the transcriptional activation of Bari1, such as in the
HSP83 mutant [
], suggesting that transcriptional activation occurs upon piRNA depletion.
Despite the lack of experimental evidence, both Bari clusters of D. melanogaster might
support the transcriptional repression of the Bari elements. The clusters might indeed act as
Cells 2022,11, 583 9 of 17
piRNA clusters, through the expression of small RNA molecules (i.e., piRNA and siRNA)
that in turn regulate the transposition frequency.
Several databases offer the opportunity to search in silico annotated Drosophila piR-
NAs, using sequence similarity criteria. In the piRBase database [
], more than 5800 piR-
NAs can be retrieved using Bari1 as a query. Roughly 3800 of them recognize the Bari1
transposase gene. Notably, a small proportion of piRNAs matching the sequence across
the monomer-to-monomer junctions—typical of the Bari1 heterochromatic clusters—can
also be found (Table 1and Figure 2B). Why a piRNA should be directed against a piece
of untranscribed DNA is, however, unclear. Are they byproducts of the piRNA loci tran-
script maturation or could they have some regulatory or structural role? Time (and further
investigations) will tell.
However, direct genetics evidence is currently lacking in support of the piRNA cluster
role of the Bari1 arrays. The currently available chromosomal heterochromatic deletion
that removes the Bari1 cluster in the h39 region also deletes the adjacent Responder locus
] and references therein; [
]), while deletion only involving one of the main satellites
would be more useful. In addition, the lack of precise mapping of the small X-linked
cluster currently makes it impossible to perform detailed genetic studies in a Bari-cluster
null genetic background. Hopefully, such kind of chromosomal aberrations will be soon
available due to the application of the most modern genome editing techniques.
Cells 2022,11, 583 10 of 17
piRNAs targeting the Bari1 inter-monomer junction. Data extracted from the piRBase database [
] (last accessed March 2021). The sequence used to
query the database encompasses an inter-monomer junction of the heterochromatic Bari1 cluster (TTTGACCACCTCTGGTCATGGTCAAAATTAT). Sequence
matching either the left or the right monomers are marked with different colors (blue and red, respectively). O = ovaries; F = follicle cells; E = eggs; W = wild type;
T = transgenic; M = mutant.
Name Tissue Genetic
Background Methods Reads Sequence Length Reference
piR-dme-2858217 O; E W; T; M
oxidized small RNA
1–3 TTTGACCACCTCTGGTCATGGTCAAAA 27 [77–79]
piR-dme-3826713 O W; T; M
oxidized small RNA
1–6 TCTGGTCATGGTCAAAATTATTTT 24 [77,79–81]
piR-dme-8496440 O T small RNA 1–3 TTTGACCACCTCTGGTCATGGTCAA 25 
piR-dme-13381112 O T small RNA 1 CCACCTCTGGTCATGGTCAAAATTAT 26 
piR-dme-21388569 F; O W; T small RNA 1–9 TTTGACCACCTCTGGTCATGGTCAAAAT 28 [79,81,82]
piR-dme-21631816 O W small RNA 1 CACCTCTGGTCATGGTCAAAATTAT 25 
piR-dme-26496558 O T small RNA 1 ACCACCTCTGGTCATGGTCAAAATTA 26 
piR-dme-26779428 O T small RNA 1 CCACCTCTGGTCATGGTCAAAAT 23 
piR-dme-27814712 O T small RNA 1 TCTGGTCATGGTCAAAATTATTT 23 
piR-dme-29438648 O T small RNA 1 TGACCACCTCTGGTCATGGTCAAA 24 
piR-dme-29670403 O T small RNA 1 TTGACCACCTCTGGTCATGGTCAAAA 26 
piR-dme-30537191 O T small RNA 2 TTTGACCACCTCTGGTCATGGTCAAA 26 
piR-dme-31705044 O T small RNA 1 CTCTGGTCATGGTCAAAATTATTT 24 
piR-dme-32470189 O W small RNA 1 TTTGACCACCTCTGGTCATGGTCA 24 
piR-dme-33774286 O W small RNA 1 TGACCACCTCTGGTCATGGTCAAAAT 26 
piR-dme-38817646 O T small RNA 1 CTCTGGTCATGGTCAAAATTATTTT 25 
Cells 2022,11, 583 11 of 17
6. Do Bari Transposons Have a Role in The Genome?
6.1. Contribution in Creating Somatic Mosaicism and Adaptive Insertions
It has been reported that TEs are unleashed in some circumstances, to create somatic
variability that could have a physiological relevance in certain tissue types [
somatic instability of several TEs has been described in two recent papers. The ﬁrst paper
described the genetic mosaicism of the neurons in the mushroom bodies [
], although this
phenomenon has been resized after further studies [
]. The second study revealed the
somatic transposition in the intestinal stem cells [
]. In both cases, Bari1 was found
among the mobilized TEs responsible for the somatic mosaicism in the two tissue types.
The early distribution studies [
] suggested a patchy distribution of Bari1 euchromatic
insertions among 46 different populations of D. melanogaster collected worldwide. By
contrast, at least two insertions were invariably detected in all the populations analyzed.
These insertions occurred at the 55F and 91F bands of the polytene chromosomes. After
the completion of the Drosophila genome-sequencing project [
], it appeared clear that
both insertions were intimately connected to host genes. Indeed, the insertion in the
55F region falls near the Juvenile hormone epoxy hydrolase (Jheh) gene cluster (Bari-Jheh
insertion), while the 91F insertions are associated to the cyp12a4 gene (Bari1-Cyp12a4
insertion). Further studies in which ChIP-seq data were integrated with gene expression
analyses and phenotypic assays demonstrated that the Bari-Jheh insertion introduced extra
antioxidant response elements upstream of Jheh1 and Jheh2 genes [
]. Furthermore, Bari-
Jheh is differentially associated to H3K27me3 in stress vs. non-stress oxidative conditions,
suggesting the addition of histone marks to the intergenic region between Jheh2 and Jheh3
genes, and its association with histone marks enrichment in the promoter of Jheh1 gene [
The Bari1-Cyp12a4 insertion overlaps with the 3
end of the Cyp12a4 gene and it
contributes to the stability of the mature transcript. It has been demonstrated that Bari1-
Cyp12a4 supplies the polyadenylation sequence to the Cyp12a4 transcript .
In addition, the Bari1-Cyp12a4 showed an enrichment of H3K27me3 under oxidative
stress conditions [
], suggesting an active role of the insertion in altering the epigenetic
context in response to stress.
6.2. Possible Role in Chromatin Assembly
TEs can also act as potent epigenetic modiﬁers that could change both gene expression
and chromatin structure [
] with important evolutionary implications [
]. This is possible
thanks to the ability to recruit chromatin proteins .
Large-scale mapping of
binding sites of chromatin proteins using tethered dam
] showed that the clustered Bari1 elements in the h39 region of the
2nd chromosome are targets of HP1, a chromatin protein mainly involved in heterochro-
matin formation [
]. It has been reported that Bari1 also contains Polycomb responsive
elements that could collaborate in recruiting additional heterochromatin components [
This could be of particular relevance when clustered copies of Bari1 are exploited in the
genome. It is therefore possible that both Bari1 clusters could have acquired a structural
role in the heterochromatin of D. melanogaster, such as to ensure proper chromosome
conformation and stability over the cell cycle.
7. The Blurry Promoter: Who Wants to Live Forever?
Along with the characterization of the Bari1 transposition system, the strength of the
Bari1 promoter was tested. Luciferase promoter assays [
] conducted in ﬂy cell cultures
) revealed that Bari1 has a weak promoter if compared to the strong constitutive copia
]. Weak promoters are typical of the Tc1/mariner transposons, which suffer
from overproduction inhibition and therefore tend to limit the amount of transposase [
Unexpectedly, the Bari1 promoter drives the reporter transcription also in human, bacteria,
and yeast cells [
]. This is quite counterintuitive, since evolutionarily distant genomes
(e.g., animal and bacteria) have profoundly different modes of transcription and promoter
]. Surprisingly, the promoter of Bari3 also showed a similar promiscuous
Cells 2022,11, 583 12 of 17
activity (Figure 3). Due to this feature, the promoters were named “blurry”, to highlight
that they do not “sharply” activate the transcription in a single or few closely related
species. These ﬁndings led to the hypothesis that the members of the Bari family can take
advantage of this feature to engage successful HTT events. The ability of TEs promoter
to sustain the transcription of associated genes upon HTT is recognized as one of the
bottlenecks that restricts the success of HTT [
]. The presence of blurry promoters was
also assessed in other Tc1- and mariner-like TEs. This observation is in agreement with the
known predisposition of Tc1/mariner elements to be horizontally transferred [
]. It also
suggests a common origin of this promoter type, and a possible common survival strategy
for the elements belonging to the Tc1/mariner superfamily [
]. The discovery of the blurry
promoters in the Bari family has also opened the possibility to implement them, together
with many other TE-derived transcriptional control sequences, into expression modules
in order to expand the repertoire of the existing expression vectors with a considerable
improvement of their performances .
Activity of the native promoter of Bari1 and Bari3 in heterologous cellular systems. The
transposon fragment tested in promoter-luciferase assays is depicted in the upper part of the ﬁgure.
The % activity compared to strong species-speciﬁc constitutive promoters (SV40, copia, URA3, and
CAT for human, ﬂy, yeast, and bacteria cells, respectively), arbitrarily assumed as 100%, is reported
on Y axis of the chart.
8. Concluding Remarks
What We Have Learned and What We Still Have to Learn from Bari Transposons
In this review, we summarized the current scientiﬁc literature concerning the trans-
posable elements belonging to the Bari family. Although the scientiﬁc interest around this
transposon was mainly focused on its evolutionary history, several important experimental
outcomes are worth noting, which have been (and hopefully will be in the future) important
to deepen our understanding of TE biology.
Many aspects of the Bari transposons’ biology are still not completely known and
should be clariﬁed, such as the low transposition rate
. Studies on wild-collected
ﬂy populations will likely be the key to obtaining insights into the regulation of the Bari
elements, as suggested by studies of Junakovic et al. .
It is possible that, as suggested by different studies, the Bari transposase could be
intrinsically error-prone, and only occasionally it gives rise to productive transposition
Cells 2022,11, 583 13 of 17
events. Fixing the transposase could possibly allow to obtain a robust transposition system.
We still have to understand how the blurry promoter has evolved, and how it can be used
to develop new tools for the genetic manipulation.
Finally, the determination of the structural and functional role of the heterochromatic
clusters is a task that deserves further investigations, and will be accomplished with the
aid of the genome editing technologies.
We think that more secrets associated to TEs remain to be discovered, and the continu-
ous effort in dissecting the most diverse TEs in nature will contribute to clarifying the role
of these genetic elements in evolution, hopefully attracting more interest in the ﬁeld of TE
biology, especially from emerging scientists.
Writing—original draft preparation, A.P., R.C., R.M., and R.M.M.; writing—
review and editing, A.P., R.C., R.M., and R.M.M. All authors have read and agreed to the published
version of the manuscript.
A.P. is supported by a grant from Regione Puglia “Research for Innovation (REFIN)”-POR
PUGLIA FESR-FSE 2014/2020. Codice Pratica: B39303C8.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
We are grateful to Z. Ivics for constructive discussion and for supporting
site-directed mutagenesis experiments in his lab. We are also grateful to all past members of the lab
and all the graduating students for stimulating discussions.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
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