Current Biology

Dispatches
12. Kullmann, D.M., Ruiz, A., Rusakov, D.M.,
Scott, R., Semyanov, A., and Walker, M.C.
(2005). Presynaptic, extrasynaptic and axonal
GABAA receptors in the CNS: where and why?
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Interlaminar connections in the neocortex.
Cerebr. Cort. 13, 5–14.
14. Gidon, A., and Segev, I. (2012). Principles
governing the operation of synaptic inhibition
in dendrites. Neuron 75, 330–341.
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J.M., and Somogyi, P. (1996). Differential
synaptic localization of two major

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16. Nicoll, R.A. (2004). My close encounter with
GABA(B) receptors. Biochem. Pharmacol. 68,
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Turecek, R., Shigemoto, R., Zhang, Y.P.,
Lujan, R., Jacobson, L.H., Biermann, B.,
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589–601.

18. Gentet, L.J., Kremer, Y., Taniguchi, H., Huang,
Z.J., Staiger, J.F., and Petersen, C.C. (2012).
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(2013). Distinct behavioural and network
correlates of two interneuron types
in prefrontal cortex. Nature 498, 363–366.

Small RNAs: Artificial piRNAs for Transcriptional
Silencing
Takamasa Hirano and Haruhiko Siomi*
Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan*Correspondence: awa403@z2.keio.jp
http://dx.doi.org/10.1016/j.cub.2015.02.009

Technologies have been developed in animal germ cells that produce artificial piRNAs from transgenes inpiRNA clusters to silence target genes by cleaving their transcripts. A new study provides a simple way togenerate artificial piRNAs to direct de novo DNA methylation in mice.

It is now known that although chromatin
modifications stabilize silencing oftransposable elements (TEs), a genetic
reinforcement loop based on germlinespecific small RNAs, PIWI-interacting
RNAs (piRNAs), is at the heart ofepigenetic regulation in animal gonads[1–3]. piRNAs are loaded onto PIWI
proteins, a germline-specific clade of the
Argonaute family, to form effector
complexes, termed piRNA-induced
silencing complexes (piRISCs). piRISCs
are guided to TEs by means of basepairing and direct posttranscriptionalsilencing by cleaving their transcripts inthe cytoplasm or by mediating thedeposition of repressive chromatin
modifications including methylation ofhistone H3 lysine-9 (H3K9me) and DNA
methylation at the target TE loci to induce
heterochromatization. The cleavage
process involves small RNA-directedendonuclease or Slicer activity of PIWI
proteins, whereas some PIWI proteins
form nuclear piRISCs with otherproteins to direct and maintain theepigenetic chromatin modification thatsuppresses transcription. These silencing
mechanisms act in trans as well as in cis.
Disruption of the piRNA pathway very
often disturbs germline development,
thereby leading to sterility. Thus TE
regulation by piRNAs has a profoundeffect on reproduction.
Single-stranded precursors,transcribed mostly from genomic locitermed piRNA clusters, are processed togenerate primary piRNAs by a Dicerindependent mechanism. In some cases,
they further initiate a Slicer-mediatedfeed-forward amplification loop, termed
the ping-pong cycle, to produce
secondary piRNAs. The piRNA clusters
mostly comprise various types of TEs andtheir remnants with sizes ranging from a
few kilobases (kb) to more than 200 kb. It
has been proposed that piRNA clusters
act as TE traps [4]; once a TE inserts into a
piRNA cluster by chance, it can become
fixed by evolutionary selection and can

R280 Current Biology 25, R269–R293, March 30, 2015 ª2015 Elsevier Ltd All rights reserved

start to produce corresponding piRNAs
that base-pair with other homologous
elements to regulate them in trans in germ
cells. This model implies that the greater
their movement activity, the higher the
chance that a TE will jump into a piRNA
cluster, thereby steering piRNA
production towards highly expressed andtranspositionally active TEs. piRNA
clusters also acquire processed
pseudogenes, which in turn become
piRNA sources and may adapt to regulate
cognate functional genes [5]. piRNA
clusters in Drosophila are mostly locatedin heterochromatin and proximal
heterochromatin–euchromatin boundary
zones [6]. Synteny of piRNA cluster
genomic locations is highly conserved
among mammals, although the primary
sequence of each piRNA shows noapparent similarity [5,7–9]. These findings
suggest that the relative chromosomal
position has specific features that allow
TE insertion and the production ofpiRNAs. However, a new study by Itou

Current Biology

Dispatches
B

GFP

A

GFP

piRNA cluster?

BAC clone
MILI–MIWI ping-pong (?)
MILI

?
MIWI

C
GFP

D

PiggyBac-based
random insertion

Pro
lacO

GFP

Rhi

lacO

GFP

Pro
lacO

GFP

GFP

LacI

lacO

asGFP

E
Random integration

Nuclear import

Dnmt3lPing-pong (?)
MILI

Dnmt3let al. [10] reported in this issue of Current
Biology now demonstrates that piRNAs
can be produced in mice to mediatetarget silencing through DNA methylation,
independently of piRNA clusters.
Recent studies have started toelucidate how a specific locus turns into a
piRNA-producing site. In addition to TEcontaining clusters, piRNAs are also
produced from the 3’UTRs of proteincoding genes, which are referred to as
genic piRNA clusters [11,12]. Only a few
3’UTRs give rise to piRNAs, suggestingthat specific primary sequences and/or
secondary structures may exist in genic
piRNA clusters to direct piRNA
production. BAC clones containing
piRNA clusters can be inserted intoeuchromatic sites in flies and mice andcorresponding piRNAs are produced,
showing that piRNA clusters can function
even when separated from their native
genomic locations [13]. In addition, transcription of a piRNA cluster named 42AB
in Drosophila germline cells is initiated by
Rhino (Rhi), an HP1a family protein [14].
Rhi forms a complex with Deadlock
and Cutoff, which is anchored to
H3K9me3-marked chromatin [15,16]. The
association of the complex appears toallow the cluster to be transcribed. It also
protects the transcripts from transcription
termination and suppresses splicing of
the cluster transcript. Thus thesefindings indicate that the cis-elementsthat funnel their RNA products into thepiRNA-generating pathway must
reside within the clusters themselves.
These cis-elements in piRNA cluster
chromatin and/or their RNA products
must be recognized by trans-factors,
which in turn distinguish piRNA clusters
and their transcripts from cellularcounterparts.
Indeed, targeted or random knockinof a GFP reporter into a piRNA cluster
gives rise to piRNAs corresponding to the
GFP reporter in flies, silkworm and mice
[13,17,18] (Figure 1A–C). The resulting
piRNAs, when they are derived from anantisense GFP sequence, can repress theexpression of GFP genes integrated
elsewhere in the genome by cleaving GFP
mRNA. These findings are also
consistent with the model in which piRNA
clusters act as TE traps; by being inserted
into piRNA clusters, new elements
become incorporated into the piRNA
repertoire to repress homologous ele-

MIWI2

DNMT3L

MILI

Dnmt3lDnmt3lEndogenous Dnmt3l

Dnmt3lMIWI2

Cytoplasm

Current Biology

Figure 1. Transgenic systems that produce artificial piRNAs in animal germ cells.
(A) Targeted knockin of a GFP reporter into a piRNA cluster. Muerdter et al. used BAC clones to generate
artificial piRNAs in flies and mice [13]. BAC clones containing piRNA clusters were recombineered to insert
a cassette comprising a GFP sequence and a bacterial neomycin resistance gene. Then the BAC clones
were inserted into genomes to produce transgenic animals. (B) Targeted knockin of a GFP reporter into a
piRNA cluster. Yamamoto et al. introduced a targeting vector containing a GFP reporter into ES cells,
which were then used to generate chimeric mice [18]. The GFP reporter was targeted into an adult/
pachytene and bidirectional piRNA cluster. piRNAs corresponding to the knockin sequences were
produced and they appear to be amplified in the ping-pong cycle. (C) Random knockin of a GFP reporter
into a piRNA cluster. Kawaoka et al. introduce a GFP-containing cassette into the genome of a silkworm
germ cell line, BmN4, using the piggyBac transposase. They then screened and selected clonal BmN4 celllines that produce GFP-derived piRNAs [17]. (D) Tethering a piRNA cluster factor to an ectopic locus.
Zhang et al. used a transgenic LacO–LacI system to tether Rhino (Rhi) to a locus containing a transgenewith the GFP sequence [16]. Expression of antisense GFP sequences driven by a second transgene was
required for piRNA production in fly ovarian germ cells. Binding of Rhi–LacI fusion to the LacO site maylead to Rhi spreading into the downstream transcription unit. (E) Expression of both sense and antisense
RNAs using promoters active in the piRNA biogenesis phase of spermatogenesis. Itou et al. introduce a
simple transgenic system to produce artificial piRNAs that leads to de novo DNA methylation in the
gonocytes of fetal testes [10].

ments in trans. This implies that thisknockin system can be used to express
artificial piRNAs to repress the expressionof a gene of interest in animal germ
cells. Artificial piRNAs can also be expressed in fly germ cells using a transgenic LacO–LacI system to tether a factor
required for piRNA production to an
ectopic locus [16] (Figure 1D). Tethering ofRhi, as a LacI fusion protein, to a transgene encoding a GFP fusion protein issufficient to trigger piRNA production

from a trans combination of transgenic
GFP reporters that express complementary transcripts. This suggests that,
although it is not known how Rhi is
specifically recruited to piRNA clusters,
Rhi binding has a function in differentiating clusters or cluster transcripts from
cellular genes and producing piRNA
precursors.
Itou et al. introduce a simple transgenic
system in the embryonic mouse testis to
produce artificial piRNAs that mediate

Current Biology 25, R269–R293, March 30, 2015 ª2015 Elsevier Ltd All rights reserved R281

Current Biology

Dispatches
target silencing independently of piRNA
clusters [10] (Figure 1E). In mouse testes,
three PIWIs (MIWI, MIWI2, MILI) are
expressed at different stages during
spermatogenesis [2]. In embryonic malegerm cells, MILI associates with primarypiRNAs and hands secondary piRNAs
to MIWI2, which in turn is imported into
the nucleus to direct specific DNA
methylation of TE loci [2]. Itou et al.
produce transgenic mice that express
both sense and antisense GFP transcripts
driven by Oct4 and Miwi2 promoters,
both of which are active in the piRNAbiogenesis phase of spermatogenesis.
These transgenes are not integrated into
any known piRNA clusters, though it has
not escaped our notice that thesetransgenes are inserted in the middle of aTE [10]. It is known that unlike MILI- andMIWI-associated piRNAs, MIWI2associated piRNAs often arise fromdispersed euchromatic TE copies [19].
Itou et al. find that high levels ofantisense GFP transcripts correlate with
DNA methylation of Oct4 and Miwi2
promoters [10]. This DNA methylation
state also correlates with the productionof sense and antisense GFP piRNAs.
Thus, this finding suggests that apiRNA response for a given gene, whose
locus is outside of the known piRNA
clusters in male germ cells, can beinitiated by simple production of sense
and antisense transcripts for the gene,leading to DNA methylation and silencing.
This work also adds to the current
hypothesis that piRNAs guide specificde novo DNA methylation to silence
their targets in mammals. Because
endogenous Oct4 and Miwi2 promoterswere not methylated in the transgenic
mice analyzed, signals of piRNA-mediated DNA methylation may be spreadfrom the GFP gene body to the promoter
region.
In proof-of-principle experiments, Itouet al. demonstrate that this system canbe applied to silence an endogenous
gene [10]. Transgenic mice expressing
antisense transcripts of Dnmt3l driven
by the Miwi2 promoter exhibit DNA
methylation of the endogenous Dnmt3l
gene and produce piRNAs corresponding
to the gene. These findings suggestthat as long as a sense transcript ormRNA is expressed in the piRNAbiogenesis phase of spermatogenesis,
only the production of a corresponding

antisense transcript from a transgene issufficient for piRNA production andsubsequent DNA methylation of theassociated locus. As mentioned above,
several transgenic systems have beendeveloped to express artificial
piRNAs. However, these piRNAs are notthought to be involved in de novo DNA
methylation. The work by Itou et al.
opens the door to studies aimed atunderstanding how piRNAs mediate
DNA methylation. The work, however,does not address the fundamental
mechanism by which expression ofsense and antisense transcripts elicits apiRNA response. Itou et al. find that
production of an antisense transcript
leads to activation of the ping-pongcycle with corresponding sense transcripts. In the mouse ping-pong cycle,
MILI-associated primary piRNAs arenormally sense-oriented, which initiates
the production of MIWI2-associatedantisense secondary piRNAs [2]. How
then can the production of antisensetranscripts activate the ping-pongcycle? Could pre-existing sense
piRNA-like molecules function as aseed to initiate the ping-pong cycle?
There is precedence from a fission
yeast system for a decisive influence ofDicer-independent transcriptome
degradation products, referred to as
primal RNAs, to act as the initial trigger ofsmall RNA production [20]. It will beinteresting to examine if primal RNA-likemolecules exist to initiate the ping-pongcycle in mice and see how these stories
unfold.
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Bioluminescence: A Fungal Nightlight with an Internal
Timer
Etelvino J.H. Bechara
Departamento de Quı́mica Fundamental, Instituto de Quı́mica, Universidade de São Paulo, Brazil*Correspondence: ejhbechara@gmail.com
http://dx.doi.org/10.1016/j.cub.2015.01.004

A recent study shows that green light emission by Neonothopanus gardneri mushrooms, endemic to coconut
forests of Northern Brazil, is controlled by a circadian clock. Furthermore, insects are attracted by the light,
raising the possibility that bioluminescence functions in spore dispersal and fungal dissemination.

Enchantment and curiosity are
immediately evoked when, entering
humid woods under a new moon, one
sees green, shining mushrooms popping
up on the surface of rotten logs. The
photo of a colony of Mycena lucentipis
mushrooms (Agaricomycetes) should
help the reader appreciate these rare
and splendid creatures [1] (Figure 1).
Seventy-one out of thousands offungus species occurring mainly intropical and temperate zones of the
globe are documented to be bioluminescent, of which twenty-six species
belong to the genus Mycena [1,2]. As
reported in this issue of Current Biology
by Oliveira et al. [3], light emission
by Neonothopanus gardneri mushrooms
found in Brazilian coconut forests
is controlled by a circadian clock andserves to attract insects for spore
dispersal.
Although observed all over the world
and documented by Aristotle and Pliny
the Elder, natural philosophers of theAncient World [4], fundamental questions
about bioluminescent fungi — how, when,
and why they emit light — have not beenanswered. Their biochemical andbiological features are still murky, partly
because it is difficult to spot them indense forests, even with dark-adapted

eyes, and to collect, transport andcultivate their mycelia and mushroomsin the laboratory. The chemical mechanism and function of light production byfungi is still controversial [5,6]. Up until
recently, a key question has concerned
whether molecular oxygen or hydrogen
peroxide oxidizes a luciferin substrate in
the presence of the enzyme luciferase.
Alternatively, it was possible that lightemission is a byproduct of somemetabolic process such as lignin
degradation? Almost all bioluminescentorganisms known use oxygen to produce
the electronically excited product(oxyluciferin), which decays to theground state by photon emission (see reaction scheme below). Thus, the visibleand ‘cold’ light emission results fromefficient conversion of energy fromchemical bonds to light without heatdissipation.
L + NADðPÞH + H + !LH2+ NADðPÞ +soluble reductase

insoluble luciferase

LH2 + O2 !LO + H2 O + light
L; luciferin; LH2 ; reduced luciferin;
LO; oxidized luciferin

In contrast to fungi, the luciferin/
luciferase systems of dozens ofluminescent organisms — from bacteria
to fishes and insects — have long been
identified. Furthermore, the bioluminescence produced by such systems has
been implicated in courtship andmating, prey attraction or visual
localization, predator warning(aposematism), camouflage, and species
recognition/grouping [6]. Various
chemically and phylogeneticallyunrelated luciferins have been isolated,
identified and synthesized since the1950s, among them firefly luciferin(a benzothiazole), sea-pansy
coelenterazine and jellyfish aequorin(imidazopyrazinones), dinoflagellate
luciferin (an open chain tetrapyrrole),
bacterial luciferin (flavins), annelid
luciferin (an oligoamide) and thelimpet luciferin (a formylated aldo-enol)[5,6]. In a number of bioluminescent
reactions (e.g., fireflies, crustaceans
and coelenterates), chemical electronic
excitation of the light emitter involves
an intermediate consisting of a highly
unstable four-membered ringperoxide named dioxetanone(or a-peroxylactone), whose thermalcleavage yields CO2 and the excitedproduct [7].

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