Retrotransposon activation contributes to fragile X premutation rCGG-mediated neurodegeneration.
ABSTRACT Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with fragile X premutation carriers. Previous studies have shown that fragile X rCGG repeats are sufficient to cause neurodegeneration and that the rCGG-repeat-binding proteins Pur α and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 could modulate rCGG-mediated neuronal toxicity. Mobile genetic elements or their remnants populate the genomes, and the activities of these elements are tightly controlled for the fitness of host genomes in different organisms. Here we provide both biochemical and genetic evidence to show that the activation of a specific retrotransposon, gypsy, can modulate rCGG-mediated neurodegeneration in an FXTAS Drosophila model. We find that one of the rCGG-repeat-binding proteins, hnRNP A2/B1, is involved in this process via interaction with heterochromatin protein 1. Knockdown of gypsy RNA by RNAi could suppress the neuronal toxicity caused by rCGG repeats. These data together point to a surprisingly active role for retrotransposition in neurodegeneration.
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ABSTRACT: L1 retrotransposons are an abundant class of transposable elements that pose a threat to genome stability and may have a role in age-related pathologies such as cancer. Recent evidence indicates that L1s become more active in somatic tissues during the course of ageing; however the mechanisms underlying this phenomenon remain unknown. Here we report that the longevity regulating protein, SIRT6, is a powerful repressor of L1 activity. Specifically, SIRT6 binds to the 5'-UTR of L1 loci, where it mono-ADP ribosylates the nuclear corepressor protein, KAP1, and facilitates KAP1 interaction with the heterochromatin factor, HP1α, thereby contributing to the packaging of L1 elements into transcriptionally repressive heterochromatin. During the course of ageing, and also in response to DNA damage, however, we find that SIRT6 is depleted from L1 loci, allowing the activation of these previously silenced retroelements.Nature Communications 09/2014; 5:5011. · 10.74 Impact Factor
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ABSTRACT: Fragile X syndrome (FXS) is the most common single gene cause of intellectual disability and it is characterized by a CGG expansion of more than 200 repeats in the FMR1 gene, leading to methylation of the promoter and gene silencing. The fragile X premutation, characterized by a 55 to 200 CGG repeat expansion, causes health problems and developmental difficulties in some, but not all, carriers. The premutation causes primary ovarian insufficiency in approximately 20% of females, psychiatric problems (including depression and/or anxiety) in approximately 50% of carriers and a neurodegenerative disorder, the fragile X-associated tremor ataxia syndrome (FXTAS), in approximately 40% of males and 16% of females later in life. Recent clinical studies in premutation carriers have expanded the health problems that may be seen. Advances in the molecular pathogenesis of the premutation have shown significant mitochondrial dysfunction and oxidative stress in neurons which may be amenable to treatment. Here we review the clinical problems of carriers and treatment recommendations.Brain disorders & therapy. 01/2014; 3.
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ABSTRACT: Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a highly conserved family of RNA-binding proteins able to associate with nascent RNAs in order to support their localization, maturation and translation. Research over this last decade has remarked the importance of gene regulatory processes at post-transcriptional level, highlighting the emerging roles of hnRNPs in several essential biological events. Indeed, hnRNPs are key factors in regulating gene expression, thus, having a number of roles in many biological pathways. Moreover, failure of the activities catalysed by hnRNPs affects various biological processes and may underlie several human diseases including cancer, diabetes and neurodegenerative syndromes. In this review, we summarize some of hnRNPs' roles in the model organism Drosophila melanogaster, particularly focusing on their participation in all aspects of post-transcriptional regulation as well as their conserved role and involvement in the aetiology of human pathologies.Chromosoma 06/2014; · 3.26 Impact Factor
Retrotransposon activation contributes to fragile X
premutation rCGG-mediated neurodegeneration
Huiping Tan1,2, Abrar Qurashi2, Mickael Poidevin2, David L. Nelson3, He Li1,∗and Peng Jin2,∗
1Division of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, People’s Republic of China2Department of Human Genetics, Emory University School of Medicine, Atlanta,
GA 30322, USA and3Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
Received July 7, 2011; Revised August 29, 2011; Accepted September 19, 2011
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with fragile
X premutation carriers. Previous studies have shown that fragile X rCGG repeats are sufficient to cause neu-
rodegeneration and that the rCGG-repeat-binding proteins Pur a and heterogeneous nuclear ribonucleopro-
tein (hnRNP) A2/B1 could modulate rCGG-mediated neuronal toxicity. Mobile genetic elements or their
remnants populate the genomes, and the activities of these elements are tightly controlled for the fitness
of host genomes in different organisms. Here we provide both biochemical and genetic evidence to show
that the activation of a specific retrotransposon, gypsy, can modulate rCGG-mediated neurodegeneration
in an FXTAS Drosophila model. We find that one of the rCGG-repeat-binding proteins, hnRNP A2/B1, is
involved in this process via interaction with heterochromatin protein 1. Knockdown of gypsy RNA by RNAi
could suppress the neuronal toxicity caused by rCGG repeats. These data together point to a surprisingly
active role for retrotransposition in neurodegeneration.
Neurodegenerative diseases are a heterogeneous group of dis-
orders characterized by the progressive loss of structure and/or
function of neurons (1). Many neurodegenerative disorders are
caused by genetic mutations within the coding regions, such as
CAG repeat expansions that can directly alter the function of
specific proteins; however, recent studies also suggest that
toxic RNAs can directly cause several neurodegenerative dis-
orders, among them fragile X-associated tremor/ataxia syn-
drome (FXTAS), which is associated with fragile X
premutation carriers (2).
Fragile X syndrome (FXS) is usually caused by expansion
of the CGG trinucleotide repeat in the 5′untranslated region
of the fragile X mental retardation 1 (FMR1) gene (3).
Whereas normal individuals generally possess between 5 and
54 repeats, fully affected individuals have .200 CGG
repeats on what are referred to as full mutation alleles (4). Pre-
mutation alleles (55–200 CGG repeats) of the FMR1 gene are
known to contribute to the fragile X phenotype through
genetic instability, and they can expand into the full mutation
during germline transmission (5). Within the last decade,
FXTAS, a late-onset neurodegenerative disorder, has been
recognized among many male premutation carriers in or
beyond their fifth decade of life (6), and FXTAS is distinct
from the neurodevelopmental disorder, FXS. The most
common clinical feature of FXTAS is a progressive action
tremor with ataxia. Nearly, all autopsy studies on the brains
of symptomatic premutation carriers show degeneration in
the cerebellum, which includes Purkinje neuronal cell loss,
Bergman gliosis, spongiosis of the deep cerebellar white
matter and swollen axons (7,8). The major neuropathological
hallmark and postmortem criterion for definitive FXTAS is
broadly distributed throughout the brain in neurons, astrocytes
and in the spinal column (7).
One unique molecular signature of the fragile X premuta-
tion allele is that the level of FMR1 mRNA is significantly ele-
vated, while the FMR1 protein (FMRP) remains relatively
unchanged in cells from premutation carriers (9,10), so the
neurodegenerative phenotypes associated with FXTAS are
suspected of being caused by a gain of function in fragile X
premutation rCGG-repeat RNAs (5,11). It has been hypothe-
sized that overproduced rCGG repeats in FXTAS sequester
∗To whom correspondence should be addressed. Email: email@example.com (H.L.); firstname.lastname@example.org (P.J.)
# The Author 2011. Published by Oxford University Press. All rights reserved.
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Human Molecular Genetics, 2012, Vol. 21, No. 1
Advance Access published on September 22, 2011
specific RNA-binding proteins and reduce their ability to
perform their normal cellular functions, thereby contributing
significantly to the pathology of this disorder. The presence
of FMR1 mRNA in inclusions found in the brains of
FXTAS patients, as well as the formation of similar inclusions
upon ectopic expression of rCGG repeats in model systems,
have provided strong support for this hypothesis (11–14).
Two RNA-binding proteins, Pur a and heterogeneous
nuclear ribonucleoprotein (hnRNP) A2/B1, could bind rCGG
repeats specifically in both mammalian and Drosophila
brains (15,16). Both Pur a and hnRNP A2/B1 are found to
be present in the inclusions of FXTAS brain tissues, and
could modulate rCGG-mediated neuronal toxicity.
Mobile genetic elements or their remnants populate the
genomes of nearly every living organism (17). Transposable
elements (TEs) include members of both DNA and RNA fam-
ilies of transposons (retrotransposons). Retrotransposons can
be further subdivided into long-terminal repeat (LTR),
non-LTR (nLTR) groups, inverted repeat (IR) elements and
repeat-containing elements. So far there is no evidence that
the DNA elements are currently active, whereas retrotranspo-
sons are considered active (18). The potential negative effects
of mobile elements on the fitness of their hosts have led to the
development of strategies for transposon control in different
organisms. Active retrotranspositions are reported to cause
human diseases, including several types of cancer, through
insertional mutagenesis of genes critical for preventing or
driving malignant transformation, and active retrotransposi-
tions contribute to inter-individual genetic variation (17,18).
New retrotransposition is found to generate genomic plasticity
in neurons by causing variation in genomic DNA sequences
and by altering the transcriptome of individual cells (19).
More recently, aberrant overexpression of satellite repeats
was seen in pancreatic and other epithelial cancers (20). Fur-
thermore, Alu RNAs are also found to directly cause
age-related macular degeneration (AMD) (21). These findings
point to the direct involvement of retrotransposons in the
pathogenesis of human diseases.
Here we show that fragile X rCGG repeats can induce the
activation of specific retrotransposons, including gypsy, in an
FXTAS Drosophila model. One of the rCGG-repeat-binding
proteins, hnRNP A2/B1, could modulate the activation of
gypsy via interaction with heterochromatin protein 1 (HP1).
Furthermore, knockdown of gypsy RNA by RNAi could sup-
press the neurodegeneration caused by fragile X rCGG
repeats. Our results reveal an unexpectedly active role for ret-
rotransposition in neurodegeneration.
Fragile X rCGG repeats cause the activation of specific
retrotransposons in the brain
expression profiling using rCGG-repeat transgenic flies that
we generated previously (11). In these flies, the severity of
their phenotype depends on both dosage and length of the
rCGG repeat. To analyze the effect of rCGG repeats in adult
brains, we used RNAs isolated from the age- and sex-matched
brains of wild-type (WT) flies and flies expressing rCGG60
repeats in neurons for gene expression profiling experiments.
Surprisingly, we observed a consistent upregulation of
several retrotransposons. Based on this finding, we systematic-
ally examined the expression of retrotransposons in our
FXTAS Drosophila model. We determined the expression
levels of different retrotransposons, LTR elements (mdg1,
roo, gypsy, copia and flea), nLTR elements (I-element,
HeT-A, R1Dm, R2Dm and Juan), IR elements (pogo, hobo
and NOF) and a repetitive locus (Mst40) in the brains of
both rCGG-repeat flies (elav-GAL4; UAS-CGG60-EGFP) and
control flies (elav-GAL4) by quantitative reverse transcriptase
polymerase chain reaction (RT-PCR). Compared with the
control flies, we saw a significant increase in three retrotran-
sposons (I-element, gypsy and copia) and one repetitive se-
quence (Mst40) in rCGG-repeat transgenic flies (Fig. 1A).
We further examined the expression of these transposon ele-
ments in transgenic flies expressing reduced rCGG repeats
reduced increase in gypsy and copia expression, but detected
no elevation in the expression of I-element and Mst40 transcripts
in these flies (Fig. 1B). These results together suggest that the
Figure 1. Activation of selective retrotransposons in rCGG-repeat transgenic flies. (A) The relative steady-state levels of retrotransposon transcripts in the brains
of rCGG-repeat flies (elav-GAL4; UAS-CGG60-EGFP) versus control flies (elav-GAL4) were determined by quantitative reverse transcriptase polymerase chain
reaction (RT-PCR). (B) Relative quantity of retrotransposon transcripts in rCGG-repeat heterozygous flies (elav-GAL4/+; UAS-CGG60-EGFP/+) compared
with control flies (elav-GAL4/+). For all experiments, n ≥ 3; error bars indicate mean+SEM.
58Human Molecular Genetics, 2012, Vol. 21, No. 1
rCGG-repeat transgenic flies is rCGG-repeat dosage-dependent.
flamenco mutants with increased levels of gypsy RNA could
modulate rCGG-mediated neurodegeneration
To determine the role of retrotransposon activation in
rCGG-mediated neurodegeneration, we further examined the
rCGG-mediated neuronal toxicity based on the fragile X pre-
phenotype we observed previously (11). Prior genetic
mapping of gypsy resistance determinants led to a discrete
locus in the pericentric b-heterochromatin of the X chromo-
some that was named flamenco (22). Drosophila Piwi-family
proteins, including Piwi, Aubergine (Aub) and Argonaute 3
(Ago3), have been implicated in transposon control in repro-
ductive systems (23,24). Piwi-interacting RNAs (piRNAs)
generated from discrete loci were found to be the master reg-
ulators of transposon activity in Drosophila (23). Recent
studies have shown that flamenco is a piRNA cluster that
can regulate specific retroelements, including gypsy, Idefix
and ZAM (25). Indeed, the presence of one flamenco allele
could further elevate the gypsy mRNA level caused by
fragile X rCGG60repeats (data not shown). We saw no signifi-
cantly increased expression of either Idefix or ZAM in
rCGG-repeat transgenic flies (Fig. 2A). To determine the
role of gypsy in rCGG-mediated neurodegeneration, we
crossed gmr-GAL4, UAS-(CGG)90-EGFP transgenic flies
with two flamenco mutant fly lines that are permissive for
gypsy expression (26). As shown in Figure 2B–F, derepres-
sing gypsy expression could enhance the neurodegenerative
eye phenotype of rCGG-repeat transgenic flies, suggesting
HnRNP A2/B1 and its Drosophila orthologs are
associated with HP1
Since Piwi clade is known to be capable of controlling trans-
poson activity in reproductive systems, we further tested the
genetic interaction between Piwi proteins and rCGG-mediated
neurodegeneration and found that the loss of Piwi proteins had
no effect on rCGG-repeat-induced neuronal cell death (data
not shown). This is consistent with the observation that Piwi
proteins only function in the germline. Besides Piwi clade,
transposon control also requires the presence of heterochroma-
tin (27). Indeed, HP1, a conserved critical component of het-
erochromatin, is known to be required for retrotransposon
silencing (28). Intriguingly, HP1 is found to interact with mul-
tiple hnRNPs to modulate heterochromatin formation (29).
Given that one of the rCGG-repeat-binding proteins that we
identified previously is hnRNP A2/B1, we examined the
potential association between HP1 and hnRNP A2/B1 pro-
teins. We performed immunoprecipitation using FLAG anti-
body from S2 cells expressing FLAG-tagged hnRNP A2/B1
and found that HP1 could be co-immunoprecipitated with
hnRNP A2/B1 (Fig. 3A). Similarly, HP1 could also be
co-immunoprecipitated with two Drosophila orthologs of
hnRNP A2/B1, Hrb87F and Hrb98DE (Fig. 3A). These
results were further confirmed by immunoprecipitating HP1
from the lysate of S2 cells expressing FLAG-tagged hnRNP
A2/B1, HRB87F and HRB98DE (Fig. 3B). Our data suggest
that hnRNP A2/B1 is associated with HP1, and potentially
HnRNP A2/B1 binds to selective retrotransposon DNAs
and modulates their expression
HP1 is required for retrotransposon silencing (28), and previ-
ous chromatin immunoprecipitation (ChIP) assays indicated
that HP1 could associate with the majority of the flamenco
locus to control retroelement activity (27). Since hnRNP
A2/B1 and its Drosophila orthologs are associated with
Figure 2. Activation of gypsy is involved in rCGG-mediated neurodegenera-
tion. (A) Expression of specific retrotransposons that are regulated by the fla-
menco locus in rCGG-repeat transgenic flies. flamenco has been shown to
increasedin rCGG-repeat transgenic flies.For all experiments, n ≥ 3; error bars
indicate mean+SEM. (B–F) Derepressing gypsy expression enhances the
rCGG-mediated neurodegenerative eye phenotype. SEM eye images from
(B). This CGG90eye phenotype could be enhanced by upregulating gypsy
expression in flies carrying a heterozygous mutation in flamenco (C and D).
flamenco itself along with gmr-GAL4 does not cause any abnormality in
the eye (E and F). Genotypes are B-gmr-GAL4, UAS-CGG90-EGFP/
+; C-flamBG02658/+; gmr-GAL4, UAS-CGG90-EGFP/+; D-flamKG00476/+;
Human Molecular Genetics, 2012, Vol. 21, No. 159
HP1, we examined whether hnRNP A2/B1 could directly bind
to genomic DNA. Given that fragile X rCGG repeats could
cause the activation of selective retrotransposons and hnRNP
A2/B1 is one of the rCGG-binding proteins, we determined
the interaction between hnRNP A2/B1 and retrotransposons
induced by fragile X rCGG repeats using a ChIP assay. As
shown in Figure 3C, hnRNP A2/B1 is significantly associated
with the genomic regions containing retrotransposons with
increased expression in the presence of fragile X rCGG
repeats, including gypsy, copia and Mst40. The retrotranspo-
sons without altered expression in the presence of fragile X
rCGG repeats are not associated with hnRNP A2/B1 (Fig. 3C).
To further examine the role of hnRNP A2/B1 in regulating
retrotransposons, we chose to test the possible interaction
Figure 3. HnRNP A2/B1, an rCGG-repeat-binding protein, could modulate retrotransposon activation via interaction with HP1. (A) HP1 could be
co-immunoprecipitated with hnRNP A2/B1 and its Drosophila orthologs. Immunoprecipitation (IP) was performed in S2 cell lysates transfected with
Flag-hnRNPA2/B1, Flag-Hrb87F and Flag-Hrb98DE, respectively, using anti-Flag M2 antibody. The immunoprecipitates were analyzed by western blot
with anti-HP1 antibody. S2 cells transfected with pUAST vector were used as a negative control. (B) HnRNP A2/B1 and its Drosophila orthologs could be
co-immunoprecipitated with HP1. Immunoprecipitation (IP) was performed in S2 cell lysates transfected with Flag-hnRNPA2/B1, Flag-Hrb87F and
Flag-Hrb98DE, respectively, using anti-HP1 antibody. The immunoprecipitates were analyzed by western blot with anti-FLAG M2 antibody. S2 cells transfected
with the pUAST vector were used as a negative control. (C) HnRNP A2/B1 associates with selective retrotransposons in vivo. HnRNP A2/B1 ChIP assay indi-
cates that hnRNP A2/B1 could bind to the genomic regions containing selective retrotransposons, such as gypsy, copia, Mst40 and I-element, but not Het_A,
Idefix or ZAM. Relative enrichment is calculated relative to IgG-only non-specific control and normalized to the empty vector (n ¼ 3, error bars indicate
mean+SEM). (D) Overexpression of hnRNPA2/B1 only (elav-GAL4/+; UAS-hnRNPA2/B1/+) shows no effect on gypsy transcript expression compared
with control (elav-Gal4/+). However, overexpression of hnRNPA2/B1 (elav-GAL4/+; UAS-CGG60-EGFP/+; UAS-hnRNPA2/B1/+) could suppress the upre-
gulated gypsy transcript expression caused by rCGG repeats (elav-GAL4/+; UAS-CGG60-EGFP/+) (n ¼ 3 for all experiments,∗P , 0.05; ns, P . 0.05). (E)
Overexpression of hnRNPA2/B1 in normal fly brain (elav-GAL4/+; UAS-hnRNPA2/B1/+) has no effect on copia activity, whereas overexpression of
hnRNPA2/B1 in rCGG-repeat transgenic flies (elav-GAL4/+; UAS-CGG60-EGFP/+; UAS-hnRNPA2/B1/+) could rescue the elevated expression of copia
(n ¼ 3 for all experiments,∗P , 0.05; ns, P . 0.05).
60 Human Molecular Genetics, 2012, Vol. 21, No. 1
between hnRNP A2/B1 and selective retrotransposons. As
shown in Figure 3D, overexpression of hnRNPA2/B1 in
control fly brain has no effect on gypsy expression, whereas
overexpression of hnRNPA2/B1 in rCGG-repeat transgenic
flies could suppress upregulated gypsy expression; similar sup-
pression was also seen with copia expression (Fig. 3E). Our
assays and genetic studies suggest that hnRNP A2/B1 could
bind to selective retrotransposons and recruit HP1 for trans-
poson silencing. In the presence of fragile X rCGG repeats,
hnRNP A2/B1 will be sequestered by excess rCGG repeats.
The depletion of hnRNP A2/B1 potentially leads to less HP1
being recruited to the genomic regions containing those retro-
transposons, and subsequent activation of retrotransposons.
Reduction of gypsy expression by RNAi suppresses
To further investigate the physiological relevance of retro-
transposon activation in fragile X premutation rCGG-mediated
neurodegeneration, we generated fly UAS lines that could
express dsRNAs against either gypsy or copia in the presence
of a GAL4 driver (Fig. 4A). Once synthesized, those dsRNAs
could produce siRNAs against corresponding retrotransposons
and knockdown their expressions (Fig. 4B and C). We then
crossed these transgenic lines with the rCGG-repeat transgenic
lines that exhibit eye neurodegeneration. To our surprise, we
found that expression of dsRNAs against gypsy, but not
copia, could suppress rCGG-mediated neurodegeneration
(Fig. 4D–F). Expression of these dsRNAs has no effect on
control fly eyes (Fig. 4G and H). This observation strongly
indicates that the dysregulation of retrotransposon activation
plays a significant role in the molecular pathogenesis of
rCGG-mediated neuronal toxicity.
FXTAS is a neurodegenerative disorder associated with fragile
X premutation carriers. Previous studies have shown that
fragile X rCGG repeats are sufficient to cause neurodegenera-
tion and that the rCGG-repeat-binding proteins Pur a and
hnRNP A2/B1 could modulate rCGG-mediated neuronal tox-
icity. Mobile genetic elements or their remnants populate the
genomes, and the activities of these elements are tightly con-
trolled for the fitness of host genomes in different organisms.
Here we demonstrate that activation of specific retrotranspo-
sons could contribute to fragile X rCGG-repeat-mediated neu-
rodegeneration using a FXTAS fly model. We show that
fragile X premutation rCGG repeats could induce the activa-
tion of specific retrotransposons. HnRNP A2/B1, one of the
rCGG-binding proteins that we identified previously, could
regulate the activity of these retrotransposons by interacting
Figure 4. Knockdown of gypsy expression by RNAi suppresses rCGG-mediated neurodegeneration. (A) Shown is the diagram depicting the strategy for gen-
eration of transgenic dsRNA lines. A DNA fragment corresponding to the gene of interest is inserted twice into the pWIZ vector, with inserts in opposite orienta-
tions on each side of the white intron. IRs that are head–head or tail–tail are placed downstream of the UAS promoter. When these UAS lines are crossed to
GAL4 driver lines, the F1 progeny generate tissue- and cell-specific expression of loopless hairpin RNA to induce RNAi in Drosophila. (B) Knockdown effi-
ciency of gypsy and copia expression in transgenic dsRNA fly lines. Real-time PCR data indicate that both gypsy and copia could be efficiently downregulated in
transgenic dsRNA fly lines. Data are plotted as mean+SEM. (D–H) Reduction in gypsy RNA suppresses rCGG-mediated neuronal toxicity. SEM (D–H)
eye images from 14-day-old flies. Flies expressing CGG90show disorganized, fused ommatidia (D). Knockdown of gypsy could rescue the neurodegenerative
eye phenotype (E). However, knockdown of copia has no such effect on the eye phenotype (F). Knockdown of gypsy or copia alone does not cause an abnormal
eye phenotype (G–H). Genotypes are D-gmr-GAL4, UAS-CGG90-EGFP/+; E-gmr-GAL4, UAS-CGG90-EGFP/UAS-Gypsy-dsRNA; F-gmr-GAL4, UAS-CGG90-
EGFP/UAS-Copia-dsRNA; G-gmr-GAL4/UAS-Gypsy-dsRNA; H-gmr-GAL4 /UAS-Copia-dsRNA.
Human Molecular Genetics, 2012, Vol. 21, No. 161
with HP1. More importantly, reduction in a specific retrotrans-
poson, gypsy, could suppress rCGG-repeat-mediated neuronal
toxicity. Our biochemical and genetic studies demonstrate a
surprisingly active role for retrotransposition in fragile X pre-
mutation rCGG-repeat-mediated neurodegeneration.
TEs make up a significant portion of most eukaryotic
genomes: for example, 80% of the maize (30), 45% of the
human (31) and 5.3% of the fruit fly (32,33) genomes are
by contributing recombination substrates, both during and long
after their integration (17). Barbara McClintock has proposed
that, in addition to causing chromosome breakage and acting
as insertional mutagens, these transposons might also act as
‘controlling elements’ to play an important role in gene regula-
sion of host genes (35). They may function as part of
genome-wide regulatory networks (36). LINE-1 (L1) elements,
the major group of nLTR retrotransposons, are known to play
important roles in mammalian genome evolution (18,37,38).
Early studies ofL1 expression put a strong emphasis on primar-
ily germline expression of these elements (39,40). Recent evi-
dence suggests a somatic function for L1 transcripts,
including cell proliferation (41), differentiation (42) and early
embryo development (42). Moreover, somatic retrotransposi-
tion events have been found in the human brain, and the retro-
transposition may have the potential to contribute to
neurogenesis and/or affect neuronalfunction(43).Morerecent-
ly, aberrant overexpression of satellite repeats has been seen in
pancreatic and other epithelial cancers (20). Furthermore, Alu
RNAs are also known to directly cause AMD, and knockdown
of Alu could mitigate AMD (21). These findings point to the
direct involvement of retrotransposons in the pathogenesis of
human diseases. In this study, for the first time, we show an
active role for retrotransposition in neurodegeneration, as
well. We saw a significant increase in several retrotransposon
transcripts in fly brains from a FXTAS Drosophila model.
More importantly, we show that the activation of these retro-
transposons is directly related to neuronal cell death, since the
reduction in a specific retrotransposon RNA, gypsy RNA,
could suppress rCGG-mediated neurodegeneration.
(44,45). In Drosophila, gypsy is the most abundant among the
four main groups of LTR retroelements (gypsy, copia, DEL
and DIRS) (32,46). In our study, we observed much higher
fly brain than in normal fly brain. Moreover, the increased ex-
pression of these retrotransposons is rCGG-repeat dosage-
dependent. Intriguingly, the knockdown of gypsy RNA only,
but not copia RNA, could suppress rCGG-mediated neuronal
toxicity. This may imply that different active retrotransposons
could influence the fate of neuronal cells differently, which
requires further investigation.
One unique molecular signature of the fragile X premuta-
tion allele is that the level of FMR1 mRNA is significantly ele-
vated, while the FMRP remains relatively unchanged in cells
from premutation carriers (9,10). Given that the neurodegen-
erative phenotype of FXTAS is associated specifically with
premutation carriers, but not with the full mutation, FMRP
deficiency per se is not likely to be the culprit behind
FXTAS (6). Instead, the neurodegenerative phenotypes asso-
ciated with FXTAS are suspected of being caused by a gain
of function in fragile X premutation rCGG-repeat RNAs
(5,11). It has been hypothesized that overproduced rCGG
repeats in FXTAS sequester specific RNA-binding proteins
and reduce their ability to perform their normal cellular func-
tions, thereby contributing significantly to the pathology of
this disorder. The presence of FMR1 mRNA in inclusions
found in the brains of FXTAS patients, as well as the forma-
tion of similar inclusions upon ectopic expression of rCGG
repeats in model systems, provide strong support for this hy-
pothesis (11–14). Two RNA-binding proteins, Pur a and
hnRNP A2/B1, have been shown to bind rCGG repeats specif-
ically in both mammalian and Drosophila brains (15,16). Both
Pur a and hnRNP A2/B1 are present in the inclusions of
FXTAS brain tissues. Furthermore, overexpression of either
Pur a or hnRNP A2/B1 can alleviate neurodegeneration in
the fly model of FXTAS (15,16). In this study, we show that
hnRNP A2/B1 could also directly bind to the genomic
regions containing specific retrotransposons and interact with
HP1, a conserved critical component of heterochromatin that
is known to be required for retrotransposon silencing (28).
Moreover, our study found that overexpression of hnRNP
A2/B1 could suppress upregulated gypsy and copia expression
in rCGG-repeat flies. These data together suggest that hnRNP
A2/B1 is also required in retrotransposon modulation. In
FXTAS, our results imply that the sequestration of the
rCGG-repeat-binding proteins, particularly hnRNP A2/B1,
could limit the amount of hnRNP A2/B1 protein that can
bind to retrotransposons and recruit HP1 for efficient retro-
In summary, we provide both biochemical and genetic data
to support an active role for retrotransposon activation in
rCGG-mediated neurodegeneration. The consequences of
such retrotransposon activation in post-mitotic neurons could
be genomic instability and neuronal apoptosis. It would be
interesting to explore further whether the activation of TEs
could be a common mechanism underlying neurodegeneration
MATERIALS AND METHODS
All flies were maintained under standard culture conditions.
The rCGG-repeat transgenic flies (UAS-CGG60-EGFP and
UAS-CGG90-EGFP) and UAS-hnRNPA2/B1 were generated
in the lab as described previously. The flamenco mutant fly
lines (flamBG02658and flamKG00476) were obtained from
Bloomington Stock Center. The UAS-Gypsy-dsRNA transgenic
flies were generated as described previously (47). In brief, a
595 bp DNA fragment corresponding to gypsy was amplified
by PCR (forwardprimer:
CGGTGCAGTCC; reverse primer: GCTCTAGAGCAGTGA
ATAGCGTTCACGA). The PCR products were inserted
twice by two ligation steps into the pWIZ vector. After the
second ligation step, inserts were in an opposite orientation.
IRs that are head–head or tail–tail repeats were confirmed
62Human Molecular Genetics, 2012, Vol. 21, No. 1
by DNA sequencing. The constructs were then injected in the
w1118strain by standard methods.
The fly heads from genotypes indicated were collected. Trizol
(Invitrogen) was used to isolate total RNA from each geno-
type. RNA samples were reverse-transcribed into cDNA
with oligo(dT)20and SuperScript III (Invitrogen). Real-time
PCR was performed with gene-specific primers and Power
SYBR Green PCR Master Mix (Applied Biosystems) using
the 7500 Standard Real-Time PCR System (Applied Biosys-
tems). RpL32 (Qiagen) was used as an endogenous control
for all samples. Primers for retrotransposon transcripts were
designed using Primer Express 3.0 software (Applied Biosys-
tems) and were as follows:
Sequence (forward primer, reverse primer)
Mst40 CCTAAGTCCCTCGCAATCAAGT, ACGCTTA
R1Dm TGCGCCTACGGTGAGGTT, CCGACAGCTAC
Hobo CAAGTGCGACCGTCGACAT, AAGTGATGCC
NOF TGCATCGAAGCTGTTTGCA, GAGTTTTAAC
All real-time PCR reactions were performed in triplicate,
and RQs were calculated using the DDCt method, with calibra-
tion to control samples.
Scanning electron microscopy
For scanning electron microscopy (SEM) images, whole flies
were dehydrated in gradient concentration ethanol (25, 50,
75 and 100%), dried with hexamethyldisilazane (Sigma), and
analyzed with an ISI DS-130 LaB6 SEM/STEM microscope.
S2 cells were co-transfected with pMT-GAL4 and pUAST
plasmid coding for Hrb87F, Hrb98DE and hnRNPA2/B1
reagent (Qiagen) following the manufacturer’s procedure.
Cells were induced 24 h with 500 mM copper sulfate (Sigma)
beginning 12 h after transfection. Cells were then harvested
by centrifugation and incubated in lysis buffer [10 mM Tris
(pH 7.4), 150 mM NaCl, 30 mM EDTA, 0.5% Triton X-100]
with 2× complete protease inhibitors on ice for 30 min, fol-
lowed by centrifugation at 15 000g for 20 min. The nuclear
lysate was precleared with 100 ml recombinant protein A
agarose (Invitrogen) for 1 h. Ten-microgram precleared
nuclear lysate was used as an input in western blot analysis.
The remaining nuclear lysate was immunoprecipitated with
anti-Flag M2 beads (Sigma) or anti-HP1 antibody (Covance)
with protein A agarose (Invitrogen) at 48C overnight. The pre-
cipitated complexes were used for western blot. Anti-HP1
antibody (Covance) at a dilution of 1:500 or anti-Flag M2 anti-
body at a dilution of 1:1000 was used for western blot. Detec-
tion of horseradish peroxidase was performed using ECL
Western Blotting Detection reagents (GE Healthcare).
Chromatin immunoprecipitation (ChIP)
ChIP was performed using a ChIP Assay Kit (Millipore). S2
cells were crosslinked with 1% formaldehyde (Sigma-Aldrich)
for 10 min at room temperature. Chromatin was fragmented to
an average size of 500 bp by sonication (Sonicator 3000;
Misonix) and immunoprecipitated with an anti-Flag M2 anti-
body (sigma). Immunoprecipitated and purified DNA frag-
ments were diluted to 1 ng/ml in nuclease-free water. We
used 8 ng of DNA in 20 ml SYBR Green real-time PCR reac-
tions consisting of 1× Power SYBR Green Master Mix and
0.5 mM forward and reverse primers. Reactions were run on
an SDS 7500 Fast Instrument (Applied Biosystems). Primers
were designed using Primer Express 3.0 software (Applied
Biosystems) and were as above. DNA relative enrichment
was determined by taking the absolute quantity ratios of spe-
cific IPs to non-specific IPs (normal mouse IgG only), IP/
IgG and normalizing to control (pUAST only). Independent
chromatins were prepared for all ChIP experiments, and real-
time PCR reactions were performed in triplicate for each
sample on each amplicon.
The authors would like to thank J. Taylor of The Integrated
Microscopy and Microanalytical Facility for her help with
SEM, the members of the Jin lab for their assistance and
C. Strauss for critical reading of the manuscript.
Conflict of Interest statement. None declared.
H.T. is supported by the China Scholarship Council. A.Q. was
supported by and is a recipient of National Ataxia Foundation
Postdoctoral Award. H.L. is supported by the National Natural
Human Molecular Genetics, 2012, Vol. 21, No. 1 63
Science Foundation of China 30430260 and 30225024. P.J. is
NS067461). P.J. is a recipient of the Beckman Young Investi-
gator Award and the Basil O’Connor Scholar Research Award,
as well as an Alfred P Sloan Research Fellow in Neuroscience.
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