gradient, suggesting that, in the case of Thus, by the most general conceptual

Bcd, the target expression domains do criteria, Bcd should still be considered
Figure 1. Activator and Repressor Gradi-
ents Cooperate for Differential Gene
Expression
Anterior target genes (cyan, yellow, and purple
bars) integrate activating inputs from Bicoid (Bcd,
blue) and repressive inputs from Runt (orange) and
Capicua (not shown) in order to establish different
posterior borders of expression in fruit fly embryos.
Chen et al. (2012) shows that many anterior genes
share a common activation threshold (AT) but
respond to different repressive thresholds (RTN).
Their work also indicates that the Runt gradient is
generated through the action of one or more Bcd
target genes (purple line).
The results of these experiments are
quite striking. Even flat Bcd gradients
may result in sharply defined head gap
gene domains that are correctly ordered
along the anterior posterior axis. These
domains are established at Bcd concen-
trations that are lower than the corre-
sponding concentrations of the wild-type
not depend on absolute morphogen
levels. This conundrum can be partially
explained by the observation that even
flat Bcd gradients result in sharp bound-
aries of runt expression and thus in
opposing Runt repressor gradients.
The work of Chen et al. shows that
neither the wild-type gradient nor the
specific levels of Bcd protein are either
necessary or sufficient for establishing
precise borders of target gene expression
at the anterior of the embryo. Given this, is
it possible to continue to classify Bcd as
a ‘‘morphogen’’? Based on the criteria
of the French flag model, clearly not.
However, although the cellular concentra-
tion of Bcd protein does not set all thresh-
olds of gene expression in the fly embryo,
multiple read-outs of the gradient are
detectable. In addition, in the case of
runt, it appears that the levels and activity
of this antagonistic gradient are a function
of those of Bcd, indicating that the Bcd
gradient is indeed generating most of
the positional information in the anterior
half of the embryo. Finally, it has been
demonstrated in other systems that
morphogen interpretation is largely an
emergent property of the target gene
network (Balaskas et al., 2012).
a morphogen, just one that is getting
even more interesting than we might
have imagined.
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A piRNA to Remember
Danesh Moazed1,*
1Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

*Correspondence: danesh@hms.harvard.edu
DOI 10.1016/j.cell.2012.04.008

In this issue of Cell, Rajasethupathy et al. report a surprising role for piRNAs, previously thought to
act mainly in the animal germline to silence transposons, in transcriptional regulation of plasticity-
related genes in the central nervous system of the sea slug Aplysia californica. The findings expand
the functions of small RNAs and have important implications for our understanding of how transient
signals can give rise to long-term memories.

During the past decade, small noncoding from fungi to mammals. Based on their miRNAs and siRNAs, is produced from
RNAs have emerged as widely recog- mechanism of biogenesis, small RNAs cleavage of double-stranded RNA precur-
nized regulators of gene expression and can be divided into at least two major sors by the Dicer ribonuclease. The
genome stability in eukaryotes ranging classes. The first class, which includes second class, the Piwi-associated small

512 Cell 149, April 27, 2012 ª2012 Elsevier Inc.

RNAs (piRNAs), is produced function of Piwi and piRNAs
independently of Dicer by in modulation of synaptic
a mechanism that involves activity. This system allows
the endonuclease slicer the study of long-term facilita-
activity of the Piwi clade of tion (LTF) based on the
Argonaute proteins. piRNAs measurement of serotonin
are larger than miRNAs and (5HT)-induced electrical acti-
siRNAs (
27 versus
22 nt) vity across synaptic connec-
and specifically associate tions that can be formed
with Piwi proteins, and their between one or more sensory
accumulation requires ampli- neurons and a single motor
fication by the so-called neuron. LTF requires change
ping-pong mechanism in- in synaptic strength and
volving at least two distinct shares requirements with
Piwi proteins (Aravin et al., memory formation in the
2006; Brennecke et al., 2007; animal. The authors’ first key
Gunawardane et al., 2007; observation using this system
Houwing et al., 2007). Unlike is that exposure of cultured
the other small RNAs, which neurons to 5HT results in
are abundantly present in a marked increase, up to 5-
somatic cells of many organ- fold, in the level of a subset
isms, piRNAs are thought to of piRNAs, suggesting that
be restricted to germ cells piRNA synthesis in neurons
and germline tissues in which may be regulated by neuro-
they function in silencing Figure 1. Genome and Gene Regulation by piRNAs transmitter-induced activity.
transposons by RNA degra- (A) In many multicellular organisms, piRNA expression from a few clusters is Depletion of Piwi impaired
developmentally regulated in germ cells and silences transposon expression.
dation at the posttranscrip- piRNAs associate with Piwi proteins and mediate silencing at the post-
LTF after exposure of syn-
tional level and by DNA transcriptional level and are also thought to promote DNA methylation at apsed neurons to 5HT,
methylation leading to tran- targeted transposons. revealing a role for Piwi in
(B) In Aplysia, in addition to germline cells, piRNAs are expressed in the CNS. In
scriptional gene silencing synaptic plasticity. Remark-
cultured neurons, specific piRNAs are induced by the serotonin neurotrans-
(Figure 1A). In a series of mitter and mediate the methylation of a promoter-proximal CpG island at the ably, a search for plasticity-
beautifully executed and CREB2 locus in a Piwi- and DNMT-dependent manner. Disruption of this related genes whose ex-
compelling experiments, methylation event impairs long-term facilitation. pression changes after Piwi
Kandel, Tuschl, and co- knockdown revealed a 2-fold
workers now provide evidence that at their 30 ends, suggesting that they are upregulation in the levels of CREB2
piRNAs are expressed in the central 20 -O methylated, which is also the case protein, a transcriptional repressor and
nervous system (CNS) and other somatic with piRNAs in other organisms. In all, major inhibitory constraint on LTF.
tissues in Aplysia and mediate CpG the authors define 372 distinct piRNA CREB2 mRNA levels were increased to
methylation and transcriptional silencing clusters in Aplysia. Consistent with the even higher levels than CREB2 protein,
of a key plasticity-related gene, CREB2 presence of piRNAs, the authors cloned suggesting that this regulation involves
(Rajasethupathy et al., 2012). a cDNA from the CNS that encodes the transcriptional control. Interestingly, and
Several previous studies have provided 96.4 kD Piwi protein and, using both in line with previous findings pointing to
evidence that the miRNA pathway plays antibody staining and expression of a role for piRNAs in directing DNA methyl-
a critical role in posttranscriptional regula- GFP-Piwi, showed that the protein is ation in the germline, the 5HT-induced
tion of specific mRNAs in response to predominantly nuclear. Thus, a convincing reduction in CREB2 protein and RNA
neuronal stimulation (for example, see set of results establishes the presence of was abolished by treatment with RG108,
Schratt et al. [2006]). In the course of CNS piRNAs and Piwi in Aplysia. An a DNA methyltransferase inhibitor.
using high-throughput sequencing to intriguing aspect of Aplysia piRNAs, noted These findings raise the tantalizing
screen for miRNAs in the Aplysia CNS by the authors, is that, unlike in other possibility that piRNAs may be directing
that might regulate long-term memory, systems, one or a few individual piRNAs long-lasting changes at the CREB2 locus
the authors noticed the presence of are cloned hundreds of times more by inducing DNA methylation. In fact,
a second class of small RNAs 27–30 nt frequently than surrounding piRNAs in examination of the CREB2 locus reveals
in length, the characteristic size of the same cluster. The mechanism for that methylation of a promoter-proximal
piRNAs. This class comprised 15% of this uneven distribution is unclear but CpG island is increased by treatment of
the total small RNA reads in the CNS suggests that amplification may be regu- neurons with 5HT in a Piwi-dependent
and could also be detected in other lated in interesting ways. manner. The authors go on to find several
somatic cells, and further examination of The authors next use the elegant relatively abundant piRNAs complemen-
a subset showed that they were blocked Aplysia coculture system to explore the tary to the CREB2 locus and reveal a key

Cell 149, April 27, 2012 ª2012 Elsevier Inc. 513

role for one of these, piR-F, in CREB2
silencing in response to 5HT. As would
be expected if the 5HT-mediated silencing
of CREB2 were mediated by piR-F, its
knockdown results in increased CREB2
RNA and protein levels, and piR-F levels
increase about 2-fold with a time course
that is consistent with the corresponding
reduction in CREB2 mRNA levels.
The findings in this study raise many
questions about the scope and extent of
piRNA/Piwi-mediated regulation of gene
expression in somatic cells. Although
piRNAs are thought to be mainly
restricted to the germline tissue, there is
some previous evidence suggesting that
they also function in heterochromatic
gene silencing outside of the germline in
Drosophila and can be detected in mouse
hippocampal neurons (Brower-Toland
et al., 2007; Lee et al., 2011). The findings
of Rajasethupathy et al. raise the exciting
possibility that piRNAs play a conserved
role in regulation of plasticity-induced
genes in parallel with miRNAs, which
play major roles in regulation of gene
expression in neurons and other cells. In
contrast to miRNAs, which act in post-
transcriptional gene silencing, piRNAs
have been suggested to promote the
methylation of specific DNA sequences.
This clearly seems to be the case with
the Aplysia CNS piRNAs, which mediate
CpG methylation at the CREB2 locus. By
analogy to mechanisms in fission yeast,
plants, and C. elegans (reviewed in
Moazed, 2009), the authors suggest that
514 Cell 149, April 27, 2012 ª2012 Elsevier Inc.
piRNA-programmed Piwi associates with
nascent CREB2 transcripts and recruits
DNMT to promote CpG methylation
(Figure 1B). Although this is a plausible
mechanism, it remains to be demon-
strated that Piwi associates with the
CREB2 locus and that the observed
CpG methylation reduces CREB2 tran-
scription. Future studies are also likely to
provide insight into how serotonin regu-
lates the accumulation of specific piRNAs
and the nature of repressive histone
modifications that are likely to be coupled
to the induced DNA methylation.
The observation that serotonin induces
CpG methylation at the CREB2 locus is
fascinating. This finding provides support
for the concept that, in addition to well-
established changes at synapses, the
formation of long-term memories involves
stable, long-lasting molecular changes in
the nucleus and, together with other
observations, reveals a mechanism for
how these changes can be directed by
activity-induced small RNAs. CpG meth-
ylation is perhaps the most stable epige-
netic mark and is maintained by the
activity of maintenance DNA methyltrans-
ferases over numerous rounds of DNA
replication. The requirement for DNMT in
repression of CREB2 in cultured Aplysia
neurons suggests that CpG methylation
in these nondividing neurons is dynamic
and requires continued DNMT activity,
which presumably maintains the methyla-
tion marks as it does in dividing cells.
Previous studies have suggested a role
for DNA methylation in memory formation
in mammals (Miller and Sweatt, 2007). It
will be exciting to see whether piRNAs
mediate specific DNA methylation events
in the mammalian brain.
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