Copyright ? 2009 by the Genetics Society of America
Transcriptional Silencing and Reactivation in Transgenic Zebrafish
Mary G. Goll,* Ryan Anderson,†Didier Y. R. Stainier,†Allan C. Spradling*
and Marnie E. Halpern*,1
*Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland 21218,†Department of Biochemistry and Biophysics,
Programs in Developmental Biology, Genetics and Human Genetics, University of California, San Francisco, California 94158
Manuscript received February 21, 2009
Accepted for publication April 30, 2009
Epigenetic regulation of transcriptional silencing is essential for normal development. Despite its
importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in
developing vertebrates. We describe a transgenic approach that allows monitoring of an epigenetically
regulated fluorescent reporter in developing zebrafish and their progeny. Using a self-reporting Gal4-VP16
gene/enhancer trap vector, we isolated tissue-specific drivers that regulate expression of the green
fluorescent protein (GFP) gene through a multicopy, upstream activator sequence (UAS). Transgenic
larvae initially exhibit robust fluorescence (GFPhigh); however, in subsequent generations, gfp expression is
mosaic (GFPlow) or entirely absent (GFPoff), despite continued Gal4-VP16 activity. We find that
transcriptional repression is heritable and correlated with methylation of the multicopy UAS. Silenced
transgenes can be reactivated by increasing Gal4-VP16 levels or in DNA methyltransferase-1 (dnmt1) mutants.
Strikingly, in dnmt1 homozygous mutants, reactivation of gfp expression occurs in a reproducible subset of
cells, raising the possibility of different sensitivities or alternative silencing mechanisms in discrete cell
populations. The results demonstrate the power of the zebrafish system for in vivo monitoring of epigenetic
processes using a genetic approach.
tance, several conserved mechanisms maintain repres-
gene promoters (Hsieh and Fire 2000; Bernstein et al.
2007; Girard and Hannon 2008). Histone modifica-
tions, small RNAs, and chromosome organization within
the nucleus contribute to transcriptional silencing
(Strahl and Allis 2000; Heard and Bickmore 2007;
of the silencing machinery are conserved, evolution of
transposons has driven organisms to evolve specialized
silencing in plants, vertebrates, and some fungi, but is
absent from other species, including many invertebrates
(Goll and Bestor 2005).
normal development. Mouse embryos lacking DNA
methyltransferase or histone-modifying enzymes are
abnormal(Lietal. 1992;Peters et al.2001; Tachibana
et al. 2002). Loss of methylation leads to H2AX
to genome maintenance. Underscoring its impor-
phosphorylation, ATM activation, and initiation of
the G2/M checkpoint in cell culture (Chen et al.
2007). Similarly, p53-dependent apoptosis is observed
in embryos lacking DNA methyltransferase-1 (Dnmt1)
(Jackson-Grusby et al. 2001; Stancheva et al. 2001).
Although these observations implicate programmed cell
death as an underlying cause of lethality in dnmt1
mutants, some evidence suggests that not all tissues are
similarly affected by the loss of DNA methylation. For
example, in zebrafish, methylation of DNA and histone
H3K9 appear to be required for terminal differentiation
of the intestine, exocrine pancreas, and retina, but not of
the endocrine pancreas (Rai et al. 2006). Similarly,
hypomethylation-induced cell death was observed only
in some Xenopus tissues at early stages of development
(Stancheva et al. 2001). Tissue-specific differences in
expression of chromatin-associated factors have been
reported in mice and zebrafish, suggesting that gene
requirements for silencing may not be equivalent in all
cells (Jarvis et al. 1996; Bourc’his et al. 2001; Rai et al.
2006; Sun et al. 2008).
Transcriptional silencing and methylation of trans-
genes have been studied in many organisms (for exam-
ple: Sapienza et al. 1989; Stuart et al. 1990; Weber et al.
in Dorer 1997). Transgenes containing the LacZ re-
porter allow transcriptional silencing to be visualized in
dissected tissues and fixed embryos (Allen et al. 1990;
for Science, 3520 San Martin Dr., Baltimore, MD 21218.
Genetics 182: 747–755 (July 2009)
for new genes involved in epigenetic regulation relied
on fluorescence-activated cell sorting of blood cells ex-
pressing a green fluorescent protein (GFP) transgene
(Blewitt et al. 2005; Ashe et al. 2008). While such ap-
proaches have yielded useful information, transgenic
tools to monitor silencing in live developing embryos
have been lacking.
The zebrafish is highly amenable to such an in vivo
strategy. The transparency of the embryo permits visual-
ization of fluorescent reporters at the level of individual
cells throughout development and over multiple gener-
ations. In addition, a short generation time and large
clutches of embryos facilitate unbiased genetic screens.
A gene/enhancer trap system was previously developed
that integrates a self-reporting Gal4-VP16; 143 upstream
activator sequence (UAS):GFP vector into the zebrafish
genomeby Tol2 transposition (Kawakami 2004; Davison
et al. 2007). Although recovered transgenic lines initially
exhibit robust tissue-specific patterns of GFP labeling,
mosaic expression is observed in subsequent generations.
Inthisreport, wesystematically characterize theonset
and progression of GFP silencing using a newly identi-
fied gene trap insertion that shows brain-specific ex-
pression. Through the generation of independent
epiallelic lines with high (GFPhigh), reduced (GFPlow),
or no GFP (GFPoff) labeling, we demonstrate that the
143 UAS is subject to DNA methylation, which corre-
lates with the heritable loss of gfp expression. We
examine the impact of Gal4-VP16 activity on methyla-
tion and demonstrate that mutation of the dnmt1 gene
overrides transcriptional repression. Reactivation of gfp
region of the brain, raising the possibility that discrete
cell populations possess different sensitivities to silenc-
ing mechanisms. The results highlight the value of an
in vivo system for monitoring epigenetic regulation of
transcription in developing tissues.
MATERIALS AND METHODS
Zebrafish strains and lines: Zebrafish were raised under
standard conditions at 27? (Westerfield 2000). The splice
acceptor-Gal4-VP16, UAS:GFP (SAGVG) construct (Davison
et al. 2007) and Tol2 transposase mRNA (Kawakami 2005)
were injected into one-cell embryos of the AB strain (Walker
1999), and injected G0larvae were raised to adulthood. Adults
were mated with AB fish, and the resultant F1progeny were
screened for restricted patterns of GFP labeling. One F1
founder male generated larvae with GFP labeling in the
forebrain, midbrain retina, and rhombomeres 5–6 (r5–6)
and was designated as carrying the c269 allele. Transgenic F2
adults carrying a single-gene trap insertion were used to
establish the c269 epiallelic lines. Additional Gt(Gal4-VP16;
UAS:EGFP) gene/enhancer trap lines and Tg(UAS-E1b:NfsB-
mCherry)c264/1were previously described (Davison et al. 2007)
(ENU)-induced mutation dnmt1s872(mixed AB, TU, and TL
background) was identified in a mutagenesis screen and is a
G-to-A transition at nucleotide 4376 (R. Anderson and D.
Stainier, unpublished results). To identify dnmt1s872homo-
zygotes, genomic DNA was isolated from individual larvae
and polymerase chain reaction (PCR) amplified using the
primers GCCTACATGCCATATGCTGA and TCTCCTGCT
Assessment of transgene copy number: Genomic DNA
(2.5 mg) was isolated from fin clips of c269 F2adults that had
been sorted for GFPhighor GFPlowlabeling at larval stages and
reared separately. DNA was digested with BamHI and sub-
jected to Southern blotting (Southern 1975). Membranes
were hybridized with radiolabled GFP coding sequence.
Transmission of epialleles: Progeny from the c269 F1
founder male were scored for high, low, or no GFP labeling
at 3 days post fertilization (dpf). Sorted groups of F2larvae
were raised separately to adulthood and mated to AB adults.
Resultant F3larval progeny were scored for GFP labeling.
GFPofflarvae that maintained the c269 transgene insertion
were identified by mating F2GFPlowadults with 143 UAS:
mCherry transgenic fish and raised to adulthood to establish
the stable, nonexpressing c269 GFPoffepiallelic line.
Methylation-sensitive Southern blots: GenomicDNA(5mg)
from pools of ?20 GFPhighor GFPofflarvae was digested with
SacIand then divided, with one-half further digested by HpaII.
Following digestion, samples were subjected to Southern
blotting (Southern 1975). Membranes were probed with
SAGVG (Davison et al. 2007) using the primers CGGCCAT
CAAGCTTAGGCTC and TCAAAGTGAGGCGAGACGC.
Bisulfite sequencing: Individual larval heads and bodies
were dissected by cutting anterior to the yolk at 3 dpf. DNA
isolation and bisulfite conversion were achieved using the EZ
DNA Methylation Direct kit (Zymo Research). Bisulfite con-
verted DNA was amplified using the primers TTTTATGTTT
TAGGTTTAGGG and CCCTTACTCACCATAATAAC followed
by TATGTTTTAGGTTTAGGGGGA and CCCTTACTCACCA
TAATAAC. The resultant fragment was purified and se-
quenced. Analysis and statistical comparison of bisulfite data
was performed using QUMA (Kumaki et al. 2008). P-values
were calculated using both Fisher’s exact test and the Mann–
Gal4-VP16 overexpression: Plasmid DNA (25 pg) encoding
Gal4-VP16 under the control of the EF1a promoter was
injected into one-cell embryos from GFPoffintercrosses. At
1 dpf, larvae were assessed for GFP-labeled cells under a
fluorescent stereomicroscope (Leica MZ16F). DNA was col-
reactivation or mock-injected controls and analyzed by bi-
Quantification of fluorescence intensity in c269 larvae:
Larvae showing the highest number of GFP-labeled cells in
Zeiss Axio Imager.D1 with the ApoTome system using
AxioVision digital imaging software. GFP- and mCherry-
integrated fluorescent intensities were independently as-
sessed in the mid/forebrain region and in r5–6 using
MetaMorph software (Version 7, Molecular Devices). The
ratio of fluorescence was calculated for each region by
dividing the integrated intensity of GFP by the integrated
intensity of mCherry.
gfp expression is epigenetically regulated in trans-
genic zebrafish lines: Larvae carrying Gal4-VP16; 143
748 M. G. Goll et al.
UAS:GFP gene/enhancer trap insertions exhibit ro-
bust tissue-specific patterns of GFP labeling in the F1
generation, but some showed variegated expression
(GFPlow) in subsequent generations (Figure 1A). To
assess the origins of the variegation systematically,
over multiple generations. F1larvae carrying the c269
insertion showed robust expression of gfp in the retina,
forebrain, and midbrain and in a broad stripe in the
pattern was evident at 1 dpf and persisted until at least
7 dpf (data not shown and Figure 1B). In the F2
generation, two classes of transgenic larvae were de-
tected: those with robust GFP labeling that recapitu-
lated the F1pattern (GFPhigh) and those showing only a
small subset of GFP-positive cells spatially restricted
within the F1pattern of labeling (GFPlow) (Figure 1C).
Using linker-mediated PCR, the c269 insertion was
mapped within the fourth intron of the zebrafish
homolog of the odd Oz/ten-m homolog 4 (odz4) gene (data
corresponded with the previously described expression
pattern for the odz4 gene (Mieda et al. 1999).
Differences in GFP labeling between c269 sibling
larvae could result from the segregation of multiple
independent insertions of the gene/enhancer trap
vector, mutation within the transgene, or a decrease
in the number of UAS sites due to DNA polymerase
slippage. Tol2-mediated trangenesis results in dispersed
single integration events rather than large concato-
meric arrays (Kawakami et al. 2000). Southern blots
revealedGFPhighand GFPlowindividuals that carried only
a single insertion of the gene/enhancer trap in the odz4
gene (supporting information, Figure S1). Therefore,
differences in transgene copy number cannot account
for the mosaicism observed in GFPlowindividuals. To
rule out mutation of the transgene, DNA fragments
encoding Gal4-VP16, the 143 UAS, or GFP were
amplified by PCR from GFPlowindividuals. Sequencing
of the amplified products failed to reveal any mutations
and confirmed the presence of 14 intact UAS copies
(data not shown). Therefore, GFPhighand GFPlowlarvae
have the identical transgene insertion yet are phenotyp-
ically distinct, suggesting that the transgene is regulated
Heritability of GFP epialleles: In mammals, genome-
wide erasure and reestablishment of epigenetic marks
early in development frequently leads to metastable
inheritance of silenced epialleles (Rakyan et al. 2002).
To test whether epialleles of the c269 transgene were
stably inherited, a single c269 F1adult was mated to wild
type. The resultant F2GFPhighand GFPlowsibling larvae
were separated and independently raised to adulthood.
All F2c269 GFPhighadults produced both GFPhighand
GFPlowF3progeny (Table 1). In contrast, F2c269 GFPlow
adults did not produce any GFPhighprogeny and GFPlow
F3 larvae were recovered at less than the expected
frequency of 50%. F2GFPlowadults also produced F3
larvae that retained the transgene but did not show any
GFP labeling (GFPoff). GFP-labeled cells were never
observed in progeny (n . 5000) from GFPoffadults in
the F3or F4generations (Table 1 and data not shown).
Similar inheritance of GFP epialleles was found upon
mating other transgenic lines and occurred irrespective
of the sex of the parent carrying the transgene (Table 1;
Table S1; Table S2). The results indicate that gfp ex-
pression is subject to silencing, which, once established,
is stably inherited.
Strain-specific modifiers that affect transgene expres-
sion have been previously reported (Sapienza et al.
lated GFP in zebrafish lar-
larva carrying unique inser-
tions of the gene/enhancer
trap vector designated by c
numbers. For a given inser-
tion, sibling F2 larvae ex-
pattern similar to the F1
panels) or in a smaller sub-
set of cells within this pat-
tern (GFPlow, right panels).
Larvae were imaged at 3
dpf. (B) Fluorescent label-
ing of the forebrain, mid-
(r5–6, arrowhead) of c269
GFPhighlarvae at 2 dpf.
(C) GFPlowc269 sibling larvae show significantly fewer GFP-labeled cells, which are restricted to within the c269 GFPhighpattern
of expression. (D) Corresponding images of GFP (top) and mCherry (bottom) fluorescence in c269 GFPhigh, GFPlow, and GFPoff
larvae carrying a newly integrated UAS:mCherry transgene at 2 dpf. Irrespective of the extent of GFP expression, mCherry labeling
recapitulates the complete c269 GFPhighexpression pattern.
Transgene Silencing in Zebrafish 749
1989; Allen et al. 1990; Martin and McGowan 1995).
Although the c269 transgene was introduced and
maintained in the AB background, this background is
unlikely to be homogeneous. To assess whether genetic
variation affected expression of the transgene, GFPoff
adults (AB) were mated to adults of the WIK, Tubingen
(TU), and mixed AB, TU, and Tupfel long fin (TL)
genetic backgrounds. Progeny expressing gfp werenever
observed (data not shown), indicating that genetic
background differences do not account for transcrip-
All transgenic lines express functional Gal4-VP16:
The bipartite nature of the gene trap construct meant
that loss of gfp expression could result from the failure
to produce functional Gal4-VP16 or of Gal4-VP16 to
activate UAS sites. To distinguish between the two
possibilities, c269 GFPhigh, GFPlow, and GFPoffadults were
mated to adults carrying a newly integrated 143 UAS-
regulated mCherry reporter. Expression of mCherry
depends on Gal4-VP16 from the trapped transgene.
Therefore, if Gal4-VP16 is absent in GFPoffcells,
mCherry will not be expressed. Alternatively, if activa-
tion of the 143 UAS:GFP is compromised, Gal4-VP16
should still transactivate UAS:mCherry in GFP-negative
cells. Labeling of mCherry was observed in GFPhigh,
GFPlow, and GFPoffc269 larvae in a pattern characteristic
of endogenous odz4 expression (Figure 1D). Robust
mCherry fluorescence was also observed in larvae from
other transgenic lines that exhibited variegated expres-
sion, irrespective of the degree of GFP labeling (Figure
S2). Thus, Gal4-VP16 is produced in GFPoffcells at levels
sufficient for activation of a second UAS-regulated
transgene. These results also suggest that epigenetic
modification of the multicopy UAS regulating GFP
prevents activation by Gal4-VP16. Further implicating
the 143 UAS in silencing, in later generations, some
larvae carrying the 143 UAS:mCherry transgene also
DNA methylation correlates with silencing: One
mechanism for transcriptional silencing is DNA meth-
ylation. In vitro methylation of UAS sites was previously
showntoprevent Gal4 activation ofaluciferase reporter
The 143 UAS (CGG-N11-CCG) may be particularly
susceptible to methylation in vivo because it is a CpG-
rich tandem repeat (Giniger et al. 1985). Methylation
occurs predominately at CpG dinucelotides in verte-
brates, and tandem repeats are frequent targets of DNA
methylation (Garrick et al. 1998).
To test for DNA methylation, genomic DNA was
collected from pools of c269 GFPhighor GFPoffheterozy-
gous larvae and digested with SacI to release an 840-bp
fragment corresponding to the 143 UAS upstream of
gfp. One-half of each sample was further digested with
HpaII, which cleaves only at unmethylated CCGG sites.
blots were performed on digested DNA and hybridized
using a radiolabeled 143 UAS DNA fragment as probe.
Full digestion at the six HpaII sites within the UAS
results in small fragments that are difficult to detect.
Therefore, decreased intensity of the 840-bp fragment
was used as a measure of the extent of HpaII digestion.
Following digestion with HpaII, the 840-bp fragment
from GFPhighlarvae was significantly decreased in in-
tensity when compared to the HpaII-digested sample
from GFPofflarvae, suggesting a correlation between
methylation of the 143 UAS and loss of gfp expression
To assess DNA methylation at higher resolution
and to determine if differences in methylation could
be detected between Gal4-VP16-expressing and -non-
expressing tissues, sodium bisulfite sequencing was
performed. Genomic DNA was separately isolated from
dissected heads and bodies of individual c269 GFPhighor
GFPoffheterozygous larvae. Following bisulfite treat-
ment, all unmethylated cytosine residues were con-
verted to thymidine while methylated cytosine residues
remained unconverted. Differences in methylation
were not observed when DNA from Gal4-VP16-express-
ingtissuesoftheheadwere compared tononexpressing
body tissues from the same individual (Figure 2B).
inFigure2A, astatisticallysignificantincrease(P , 0.001)
in the number of methylated CpG dinucleotides was
observed when GFPoffsequences were compared to
GFPhighsequences (Figure 2B). In addition, DNA from
GFPhighlarvae showed a higher number of clustered
unmethylated Gal4-binding site. Fully unmethylated
Gal4-binding sites were not observed in GFPofflarvae
(Figure 2B). Consistent with the heritability of the
GFPoffepiallele, DNA methylation patterns in sperm
from GFPoffadults were very similar to those in sperm
from GFPofflarvae (average of 88.5% of CpG’s methyl-
ated; Figure S3). Collectively, the bisulfite sequencing
data and Southern blot data indicate a correlation
between methylation and silencing of gfp expression
Inheritance of c269 epialleles
Class of progeny (%)
progenyc269/1 3 wild typeGFPhigh
750M. G. Goll et al.
and suggest that methylation directs the stable inheri-
tance of GFPoff.
Reactivation of gfp by exogenous Gal4-VP16: The
UAS remains methylated in GFPofflarvae despite the
presence of Gal4-VP16. In addition, differences in DNA
methylation were not observed between Gal4-VP16-
expressing and -nonexpressing tissues in GFPhighand
GFPlowindividuals. However, previous reports indicate
test whether higher levels of Gal4-VP16 could cause
UAS demethylation and reactivation of gfp expression,
GFPofflarvae were injected with plasmid DNA contain-
ing Gal4-VP16 under the control of a ubiquitous pro-
moter.Labeled cellswere not detected in mock-injected
controls or in controls injected with an unrelated
plasmid (Figure 3A). In contrast, GFP labeling was
detected throughout the entire larva in the presence
of excess Gal4-VP16 (Figure 3B). Reactivation of gfp in
Gal4-VP16-injected larvae correlated with a modest
decrease in DNA methylation as assessed by bisulfite
sequencing (Figure 3C). Therefore, increasing the
levels of Gal4-VP16 can reactivate gfp expression, likely
through partial demethylation of the UAS.
Mutation of dnmt1 reactivates selective gfp expres-
sion: If methylation is responsible for suppression of
UAS-regulated gene expression, then blocking the
methylation process should lead to reactivation of gfp
expression. Zebrafish homozygous for an ENU-induced
G1459D amino acid change in the AdoMet-binding
motif X of DNA methyltransferase-1 (dnmt1s872) show
morphological abnormalities and a reduction in global
methylation (Figure S4 A; R. Anderson and D. Stain-
ier, unpublished results). To test for reactivation of gfp,
the dnmt1s872mutation was introduced into the c269
GFPoff, UAS:mCherry transgenic background. Adults
heterozyogous for the mutation and the transgenes
gfp expression. At 1 dpf, GFP-labeled cells were not
detected despite transactivation of UAS:mCherry in the
complete c269 brain-specific pattern. The presence of
maternal Dnmt1 activity likely accounts for transgene
silencing at this stage. However, by 2 dpf, robust GFP
labeling appeared in the midbrain, forebrain, and
(Figure 4, D–F). Expression persisted until 7 dpf, when
dnmt1 homozygous mutants are no longer viable.
Genotyping confirmed that GFP-positive larvae were
homozygous for the dnmt1s872mutation (n ¼ 10), while
those without GFP-labeled cells had one or no mutant
alleles (n ¼ 6; Figure 4, A–C). Methylation-sensitive
Southern blots revealed significant demethylation of
the UAS in gfp-reactivated larvae compared to their
GFPoffsiblings (Figure S4). These results indicate that
Dnmt1-mediated methylation is important for silencing
of the UAS-regulated transgene.
the midbrain, forebrain, and retina, cells of the r5–6
hindbrain region were refractory to reactivation. Trans-
activation of UAS:mCherry confirmed that cells in r5–6
express Gal4-VP16 in homozygous mutant larvae. How-
ever, GFP-labeled cells were not detected in r5–6 in 65%
of larvae (n ¼ 135) that showed reactivation elsewhere
in the brain (Figure 4, D–F). In the remaining 35%, two
small groups of GFP-expressing cells were found at the
lateral edges of the r5–6 stripe (Figure 4, G–I, arrow-
head in I). The difference in reactivation between the
midbrain/forebrain and the hindbrain stripe was quan-
tified by determining the integrated fluorescent in-
tensity of GFP and mCherry for each brain region
using larvae that showed the greatest number of GFP-
labeled cells in r5–6. The average ratio of GFP to
of the multicopy UAS is
correlated with decreased
GFP labeling. (A) Genomic
DNA from pooled GFPhigh
or GFPofflarvae was di-
gested with SacI to produce
an 840-bp SacI fragment en-
compassing the 143 UAS
upstream of gfp. The 840-
bp SacI fragment has in-
creased sensitivity to HpaII
cleavage in GFPhighlarvae
compared to GFPofflarvae.
Transactivation of a 143
was used to identify GFPofflarvae that inherited the c269 transgene. The 143 UAS probe also recognizes the 143 UAS regulating
mCherry, although SacI cleavage releases a larger fragment (2.2 kb). This fragment, containing the 143 UAS upstream of
mCherry and the mCherry coding sequence, is largely digested, indicating that it is unmethylated in GFPhighand GFPoffsamples.
(B) DNA from heads and bodies of individual GFPhighor GFPofflarvae was subjected to bisulfite sequencing. The methylation status
of each CG site is reported as a percentage of total sites tested for 10 clones from the head and 10 clones from the body of each
individual. Methylated sites are solid circles; horizontal bars indicate pairs of CpG dinucleotides within individual Gal4-binding
sites. The final 8 CpG sites reside within the minimal promoter sequence.
Transgene Silencing in Zebrafish 751
mCherry fluorescence was 0.68 6 0.13 in the midbrain/
forebrain region and 0.05 6 0.07 in r5–6 (n ¼ 5),
indicating at least a 13-fold difference in labeling. Cells
in the hindbrain are therefore more refractory to
reactivation upon loss of Dnmt1 activity.
in the c269 transgenic background, even when the
(Figure 4, J–L). Among 112 GFPhighF2progeny with
robust gfp expression in the forebrain, midbrain, and
retina, only 16 had any labeled cells in r5–6. Cells in the
r5–6 stripe are therefore more susceptible to silencing
than those in the midbrain and forebrain regions.
In this study, we used a multicopy UAS and fluores-
in transgenic zebrafish. Independent GFPhigh, GFPlow,
and GFPoffepiallelic lines were established, which
allowed silencing of gfp expression to be followed in
developing tissues and over multiple generations. Com-
plete heritable silencing of single-copy GFPofftrans-
genes was observed and correlated with increased
methylation of the tandemly repeated UAS. Reactiva-
tion of GFPofftransgenes was demonstrated by increas-
ing Gal4-VP16 levels and by mutation of DNA
methyltransferase-1. In addition, we observed distinct
profiles of gfp silencing and reactivation in different
cell populations, underscoring the value of an in vivo
system for monitoring epigenetic regulation of tran-
ful system for studying transcriptional silencing during
vertebrate development using genetic approaches.
Although prior work in zebrafish provided evidence
for transgene silencing, integrated DNA existed in
concatemeric arrays and lacked trackable fluorescent
reporters (Gibbs et al. 1994; Martin and McGowan
Figure 4.—Distinct regions of the brain show differential
silencing and reactivation. Corresponding images of GFP
fluorescence (A, D, G, and J), mCherry fluorescence (B, E,
H, and K), and an overlay of mCherry, GFP, and differential
interference contrast (C, F, I, and L) from 3 dpf larvae. (A–C)
c269 GFPofflarvae that are wild type or heterozygous for the
dnmt1s872mutation do not show GFP labeling. (D–F) c269
GFPofflarvae that are homozygous dnmt1s872mutants show
GFP labeling in the midbrain and forebrain. Despite transac-
tivation of mCherry, GFP labeling is not observed in r5–6
(arrow in E and F). (G–I) The homozygous dnmt1s872mutant
with gfp reactivation in a small number of bilateral cells in
r5–6. The insert inI isan enlargement ofreactivated cells indi-
mated to wild type. Labeling of GFP is present in the forebrain
and midbrain, but not in r5–6 mCherry-positive cells.
Figure 3.—Reactivation of GFP expression by exogenous
Gal4-VP16. (A) Mock-injected larvae from c269 GFPoffinter-
crosses show no GFP labeling (n ¼ 100). (B) GFPoffsiblings
injected with ?25 pg of plasmid DNA encoding the EF1a
ubiquitous promoter driving Gal4-VP16 expression are exten-
sively labeled with GFP (98%; n ¼ 60). (C) DNA from injected
or mock-injected larvae was subjected to bisulfite sequencing,
and the methylation status of each CG site was reported as a
percentage of the total sites tested for 10 clones. Methylated
sites are solid circles. Data are representative of two biological
752 M. G. Goll et al.
1995). Silencing of a single-copy GFP transgene was
et al. 2006). Epiallelic transgenic lines were not previously
established, and inheritance of a fully silenced transgene
had not been followed over generations. In this study,
transgenesis was mediated by Tol2 transposition, resulting
in random, dispersed integrations rather than complex
concatemers (Kawakami 2005). Single insertions were
regulated transgenes are being used more widely in zebra-
fish research (Halpern et al. 2008); however, variegated
expression could be detrimental in experiments where
Gal4/UAS constructs may be necessary to increase their
utility, and decreasing UAS copy number could potentially
Silencing appears to be a stochastic event because only
some progeny from GFPhighadults showed variegated
expression, and only a subset of transgenic progeny from
GFPlowindividuals completely lacked GFP labeling. The
progression from GFPhighto GFPlowto GFPoffalso suggests
that more than one generation is required for extinction
of gfp expression. Widespread methylation of sperm from
GFPoffadults suggests that, once extensive methylation of
the transgene is achieved, it is propagated through the
gametes. The heritable complete silencing observed at
the UAS is in contrast to mouse transgenes, which
frequently show metastable inheritance of silencing
generation is a probabilistic event (Rakyan et al. 2002).
Heritable silencing in GFPofflarvae provides a tool to
explore the mechanisms of silencing. Previous studies
have shown that DNA-binding proteins can cause deme-
thylation of their recognition sites (Matsuo et al. 1998;
Hsieh 2000; Lin et al. 2000). It has been suggested that
protein binding during replication may protect sites
from methylation by Dnmt1, thereby demethylating
genes designated for activation (Matsuo et al. 1998;
Hsieh 2000). We similarly found that Gal4-VP16 could
cause demethylation of the UAS, leading to reactivation
of gfp. However, reactivation was achieved using very
high levels of Gal4-VP16. In contrast, methylation
and silencing persist in GFPofflines that express lower
levels of Gal4-VP16, and differences in methylation
were not observed between Gal4-VP16-expressing and
-nonexpressing tissues. These data imply that physio-
logical concentrations of DNA-binding proteins may
not generally be sufficient to drive demethylation as a
means of activating transcription.
Mutation of dnmt1 also resulted in reactivation of gfp
expression. Although transcriptional repression inde-
pendent of methyltransferaseactivity has been reported
for Dnmt1 (Milutinovic et al. 2004; Dunican et al.
2008), the dnmt1S872allele is a point mutation expected
to compromise catalytic function without disrupting
N-terminal regulatory domains. Therefore, it is most
likely that the methyltransferase activity of Dnmt1 is
directly required for silencing at the UAS.
Reactivation of gfp expression in dnmt1S872mutants
occurs in a cell-type-specific manner. Although strong
GFP labeling was observed in the forebrain, midbrain,
or no reactivation of gfp. There are several potential
explanations for this difference. It is possible that
partially methylated UAS sites may be more sensitive
to Gal4-VP16 levels. Transcription from the odz4 pro-
moter appears similar in the forebrain, midbrain, and
hindbrain as assessed by whole-mount RNA in situ
hybridization(Mieda etal.1999);however,subtle differ-
ences in expression between brain regions might not be
detected by this method. Slightly lower odz4 promoter
activity in the r5–6 stripe would selectively reduce Gal4-
VP16 activity in this region, which could account for the
differences in the onset of silencing and resistance to
reactivation at a partially methylated UAS.
Another explanation for differences in reactivation
between brain regions could be differences in the
timing of neurogenesis. Reactivation of gfp expression
in dnmt1 mutants was not observed at 1 dpf despite
transactivation of UAS:mCherry. The delay in reactiva-
tion likely reflects the presence of maternal Dnmt1 at
this time. If neurons in r5–6 differentiate prior to
depletion of maternal Dnmt1, they would retain meth-
ylation, and reactivation would not be expected. How-
ever, we do not favor this hypothesis. By 48 hours post
fertilization (hpf), reactivation is observed in the fore-
and midbrain, suggesting that maternal Dnmt1 activity
is no longer present. At this time point, cell prolifera-
(Mueller and Wullimann 2005). Moreover, another
1400 neurons are born in r4–5 after 48 hpf (Lyons et al.
2003). Finally, it is unclear how early maternal Dnmt1,
which is not spatially restricted, could specifically in-
fluence gfp expression in r5–6 of GFPhighlarvae.
A third hypothesisisthat different regionsofthebrain
have different gene requirements for silencing. In
addition to a single dnmt1 homolog, the zebrafish
genome also contains six DNA methyltransferase-3 (dnmt3)
roles for these de novo methyltransferases have not been
reported, increased activity of Dnmt3 proteins might
compensate for the loss of Dnmt1. Alternatively, over-
expression of the histone methyltransferase suv39h1 res-
cues phenotypes caused by depletion of dnmt1 in
zebrafish (Rai et al. 2006). Although suv39h1 expression
is higher in the forebrain compared to the hindbrain
(Rai et al. 2006), it is possible that other histone-
modifying enzymes or as-yet-unidentified molecular
pathways could influence silencing in r5–6.
principle that factors required for transcriptional si-
lencing may be uncovered genetically by screening for
Transgene Silencing in Zebrafish 753
reactivation of expression in GFPofflarvae. It is unlikely
in vertebrates has been identified, and cell-type-specific
roles for known factors may have been overlooked.
GFPofftransgenic lines provide an opportunity to probe
the mechanism of silencing through candidate testing,
mutagenesis, or drug screening approaches. Because
such experiments are performed in vivo, they have the
potential to further our understanding of developmen-
a developing embryo.
We thank Michelle Macurak and Brian Hollenback for technical
support and Steve Leach, Michael Parsons, and Courtney Akitake for
useful discussions and reagents. This project was supported in part by
the National Institute of Child Health and Human Development
(grant no. R01HD058530) to M.E.H. M.G.G. is a Damon Runyon
(DRG-#1945-07). A.C.S. is an investigator of the Howard Hughes
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In vitro DNA
Communicating editor: D. J. Grunwald
Transgene Silencing in Zebrafish755
Transcriptional Silencing and Reactivation in Transgenic Zebrafish
Mary G. Goll, Ryan Anderson, Didier Y. R. Stainier, Allan C. Spradling and
Marnie E. Halpern
Copyright © 2009 by the Genetics Society of America
M. Goll et al. 2 SI
FIGURE S1.—A single transgene insertion event generates both c269 GFPhigh and GFPlow larvae (A)
Schematic of the enhancer gene trap construct. (B) Southern blot of BamH1 digested genomic DNA from
individual GFPhigh (n=3) or GFPlow (n=3) adults sorted for GFP labeling at larval stages and probed with
radiolabeled gfp sequence. The probe recognizes restriction fragments which extend from the BamHI site
5’ to gfp (red) to the nearest BamH1 site in 3’ flanking genomic sequence (blue). Independent integrations
produce restriction fragments of distinct sizes because the nearest BamH1 in flanking DNA will vary in
distance from the transgene integration site. The lower band (arrow, 2.0 kb) corresponds to the predicted
molecular weight of the BamH1 fragment for the insertion mapping to the odz4 locus. Individuals from
both expression classes were identified that contain only this insertion (GFPhigh lanes 1 and 3, GFPlow
lanes 1 and 2). The arrowhead indicates a second insertion in two individuals that was unlinked to the
odz4 locus and was independent of the GFP labeling pattern.
M. Goll et al. 3 SI
FIGURE S2.—Transactivation of UAS:mCherry in GFPhigh and GFPlow transgenic lines. Corresponding
images of GFP (left) and mCherry (right) fluorescence in GFPhigh and GFPlow transgenic larvae from lines c251,
c218 and c223. Irrespective of the extent of gfp expression, mCherry labeling recapitulates the characteristic
GFPhigh pattern for each transgenic insertion.
M. Goll et al. 4 SI
FIGURE S3.—Widespread methylation of the UAS in sperm from GFPoff adults. DNA from sperm
isolated from GFPoff adults was subjected to bisulfite sequencing. The methylation status of each CG
site is reported as a percentage of total sites tested for 8 clones from one individual and ten clones
from a second GFPoff individual. Methylated sites are indicated black; bars indicate pairs of CpG
dinucleotides within individual Gal4 binding sites. The final 8 CpG sites reside within the minimal
M. Goll et al. 5 SI
FIGURE S4.—Methylation of the 14X UAS is decreased in larvae showing reactivation of gfp. (A-B) c269
GFPoff, UAS:mCherry adults heterozygous for the dnmt1S872 mutation were intercrossed and genomic DNA
was isolated from pooled progeny that showed either mCherry and GFP labeling in the brain (GFP+) or
only mCherry labeling (GFP-). DNA was digested with SacI and one half of each sample was further
digested with HpaII. (A) Ethidium bromide staining of GFP+ and GFP- DNA samples run on an agarose gel.
Increased smearing is observed in HpaII digested DNA from GFP+ larvae (lane 2) compared to GFP-
siblings (lane 4), indicating a genome wide decrease in methylation. (B) Southern blot of (A) using a DNA
fragment encompassing the 14X UAS as probe. SacI digestion isolates an 840 bp fragment corresponding to
the 14X UAS regulating gfp expression. Increased HpaII digestion in larvae showing reactivation of gfp
compared to their GFP- siblings (lane 4) indicates that a reduction in methylation at the 14X UAS
correlates with reactivation of gfp. The 14X UAS probe used fro hybridization also recognizes the 14X
UAS regulating mCherry, although SacI cleavage releases a significantly larger fragment (2.2 kb). This
fragment, containing both the 14X UAS upstream of mCherry and flanking mCherry coding sequence, is
largely digested, indicating that the mCherry fragment is unmethylated in both GFP+ and GFP- larvae.
M. Goll et al. 6 SI Download full-text
Inheritance of c218 epialleles
Class of Progeny
c218/+ X WT
GFPhigh GFPlow No GFP
F1 GFPhigh male 38% 18% 44% 83
F2 GFPhigh female 1 17% 37% 44% 124
F2 GFPhigh male 1 33% 5% 62% 200
F2 GFPhigh male 2 27% 6% 67% 54
F2 GFPlow female 1 0% 5% 95% 141
F2 GFPlow male 1 0% 15% 85% 200
F2 GFPlow male 2 0% 13% 87% 156
F3 GFPoff female 1 0% 0% 100% 69
F3 GFPoff male 1 0% 0% 100% 198