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First published online 9 July 2008
doi: 10.1242/dev.024539
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1 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA.
2 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104,
USA.
3 Department of Genetics and Penn Center for Bioinformatics, University of
Pennsylvania, Philadelphia, PA 19104, USA.
4 The Fels Institute for Cancer Research and Molecular Biology, and Department
of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140,
USA.
5 Laboratory of Immunopathology, NIAID, NIH, Rockville, MD 20852, USA.
Author for correspondence (e-mail:
bartolom{at}mail.med.upenn.edu)
Accepted 15 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: CTCF, Mouse, Oocyte, Preimplantation embryo, Meiosis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Ohlsson et al., 2001). It was
initially isolated as a factor that binds the Myc promoter and
represses transcription (Lobanenkov et
al., 1990). CTCF has since been shown to function as both a
classical repressor and activator of transcription. Moreover, it carries out a
multitude of other nuclear functions that depend on genomic and epigenetic
context. For example, CTCF was identified as an enhancer blocker at the
β-globin locus (Bell et al.,
1999), and is the only known protein required for insulator
activity in vertebrates. It can interact with both DNA and itself, providing a
means by which CTCF molecules bound at remote sites in the genome can be
brought together physically. According to a model for vertebrate insulator
activity, this would prevent enhancer-promoter interactions between elements
on different chromatin loops, while allowing or even facilitating those
interactions within the same loop (Engel
and Bartolomei, 2003; Wallace
and Felsenfeld, 2007). Additionally, CTCF can tether its binding
sites to the nucleolus, consistent with a model in which CTCF anchors
chromatin loops to nuclear substructures
(Yusufzai et al., 2004).
Subsequent to its discovery as an enhancer blocker at the β-globin
locus, CTCF was shown to bind four elements within the imprinting control
region (ICR) of the H19/Igf2 locus
(Bell and Felsenfeld, 2000;
Hark et al., 2000;
Kanduri et al., 2000;
Szabo et al., 2000). CTCF
binds to the maternal ICR, where it prevents the activation of Igf2
by downstream enhancers that are shared by H19 and Igf2.
This allows H19 exclusive access to enhancers on the maternal allele.
On the paternal ICR, DNA methylation prevents CTCF from binding, allowing
Igf2 access to the shared enhancers. Thus, CTCF serves as a
methylation-sensitive DNA-binding factor that mediates enhancer blocking at
the imprinted H19/Igf2 locus. Furthermore, CTCF binding is essential
for preventing ectopic methylation on the maternal ICR
(Engel et al., 2006;
Pant et al., 2003;
Szabo et al., 2004).
Additional CTCF-binding sites are described at other imprinted loci and on
the X-chromosome, where they are proposed to play a regulatory role
(Chao et al., 2002;
Fitzpatrick et al., 2007;
Hikichi et al., 2003;
Yoon et al., 2005). Other
CTCF-binding sites are located at boundaries between active and repressive
chromatin, pointing to a role for CTCF at barrier elements
(Barski et al., 2007;
Cho et al., 2005;
Filippova, 2008). Recently,
thousands of CTCF-binding sites have been identified throughout the genome,
consistent with a global role for CTCF in chromatin organization
(Barski et al., 2007;
Kim et al., 2007;
Xie et al., 2007). Moreover,
CTCF-binding sites overlap with cohesin-binding sites, and CTCF is essential
for localizing cohesins to defined sites in the genome
(Parelho et al., 2008;
Stedman et al., 2008;
Wendt et al., 2008). However,
despite significant advances made in the past year, there are still no
published data describing Ctcf deletions in mouse. Moreover, although
CTCF has been depleted in mammalian cell culture using RNAi, the in vivo
consequences of CTCF depletion are largely unknown. Thus, important
physiological aspects of CTCF function remain undescribed, particularly the
relevance of CTCF-binding sites throughout the genome, in different tissues of
an adult organism and during development.
To understand the role of CTCF at the H19/Igf2 locus, we
previously generated transgenic mice expressing Ctcf dsRNA that depletes CTCF
from growing oocytes (Fedoriw et al.,
2004). The resulting oocytes are hypermethylated at the
H19 ICR. Moreover, the incidence of development to the blastocyst
stage is markedly reduced. To gain insight into the molecular basis of these
observations, we now identify hundreds of genes that are misregulated in
CTCF-depleted oocytes using microarrays. More genes are downregulated than
upregulated; moreover, downregulated genes are preferentially closer to
CTCF-binding sites, especially in their upstream regions. These results are
consistent with a major role for CTCF in transcriptional activation and
derepression. In the oocyte, CTCF depletion delays the onset of meiosis and
reduces meiotic competence, while preventing chiasmata resolution in a small
proportion of eggs that have undergone polar body extrusion. After
fertilization, CTCF-depletion delays the second mitotic division, perturbs
zygotic genome activation, causes abnormal nuclear morphology and finally
leads to apoptotic death prior to the blastocyst stage. Maternal pronuclear
transfer and Ctcf mRNA microinjection experiments indicate that the
two-cell delay is a maternal effect that is not caused by persistent chromatin
changes arising in the egg, but is more likely to be caused by persistent
transcriptional defects. Thus, Ctcf is a mammalian maternal effect
gene that affects transcription during oocyte growth, and plays important
independent roles in meiotic maturation and early embryonic development.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Growing oocytes were collected from 10-day-old Tg and Ntg
littermates. Fully-grown germinal vesicle (GV) oocytes were collected from 8-
to 10-week-old littermates, and cultured in CZB medium at 37°C in an
atmosphere of 5% CO2 and air. Fertilized one-cell embryos were
collected from superovulated and naturally plugged 8- to 10-week-old mice, and
cultured in KSOM+AA medium (Ho et al.,
1995) at 37°C in an atmosphere of 5% CO2, 5%
O2 and 90% N2. All experiments involving mice were
approved by the University of Pennsylvania Institutional Animal Care and Use
Committee.
Immunofluorescence and TUNEL staining
Cells were fixed with 2% paraformaldehyde for 25 minutes at room
temperature. Embryos were TUNEL stained using In Situ Death Detection Kit
(Roche). Immunofluorescence staining was done as previously described
(Fedoriw et al., 2004). The
antibody working dilutions were 1:3 (anti-CTCF, BD Biosciences, 612149), 1:100
(anti-H2AZ, Abcam, ab4174), 1:500 (anti-SMC1, Bethyl Labs, A300-055A) and
1:1000 (anti-dimethylH3K4, Upstate, 07030; anti-HP1-β, Chemicon, MAB3448;
anti-
-Tubulin, Sigma, T6199). For SMC1 staining, embryos were pooled
and extracted with 0.1% Triton X-100 prior to fixation. Images were acquired
using confocal microscopy (Leica) and mean nuclear fluorescence was quantified
using ImageJ v1.36b software.
Karyotyping eggs
Chromosome spreads were prepared as previously described
(Tarkowski, 1966). Diakinesis
spreads were prepared from eggs having undergone germinal vesicle breakdown
(GVBD) by 2 hours post-culture. Metaphase II spreads were prepared from eggs
having undergone PBE by 16 hours post-culture. Chromosome spreads were
prepared from embryos arrested in prometaphase with 0.04 µg/µl colcemid
for 1-2 or 16 hours.
RNA isolation and microarray analysis
GV oocytes were collected from 8- to 10-week-old superovulated mice. Five
pairs of Ntg and Tg littermates from five different litters were used. Total
RNA was extracted from pools of 25 oocytes per mouse using PicoPure RNA
Isolation Kit (Acturus). cDNA was synthesized, amplified and biotin-labeled as
previously described (Pan et al.,
2005). One Affymetrix MOE430 2.0 GeneChip per mouse was probed
with 15 µg cDNA and processed according to Affymetrix instructions.
GeneChip tabular data are available at the Gene Expression Omnibus repository
(www.ncbi.nlm.nih.gov/geo;
accession #GSE11664). Raw microarray data were analyzed as previously
described using MAS5, GeneSpring v7, SAM and EASE software
(Pan et al., 2005). We used a
1.4-fold cutoff for EASE analysis because four biological replicates provided
sufficient statistical power and confidence to detect a 1.4-fold change in
transcript abundance (Zeng et al.,
2004).
Real-time PCR
Total RNA was extracted from GV oocytes using Absolutely RNA Microprep Kit
(Stratagene), and reverse transcribed using Superscript II reverse
transcriptase (Invitrogen) and random hexamer primers. cDNA was quantified by
real-time PCR using an ABI Prism 7000 thermocycler and Taqman probes (Applied
Biosystems; Ctcf, Mm00484027_m1; Pim1, Mm00435712_m1; Cbfa2t1h, Mm00486771_m1;
Gtl2, Mm00522599_m1; Grb10, Mm01180444_m1; Myc, Mm00487803_m1; Boris/Ctcfl,
Mm01242223_m1; Slc22a18, Mm00485426_m1; Phlda2, Mm00493899_g1; Fcgr1,
Mm00438874_m1; Tlr1, Mm00446095_m1; Ubft, Mm00456972_m1). Crossing points were
normalized to UBTF and converted to relative expression values representing
the average of three Tg samples over three Ntg samples. Each sample was
analyzed in duplicate wells.
TRC upregulation
Fertilized one-cell embryos were collected from 8- to 10-week-old
littermates 24 hours post-hCG, and cultured in CZB medium at 37°C in an
atmosphere of 5% CO2 and air. Embryos were examined for cleavage at
30-minute time intervals, and those that cleaved within a 2-hour time period
were cultured for an additional 6, 12 and 21 hours. Embryos were then
radiolabeled with 1 µCi/µl [35S]-methionine/CZB, as
previously described (Conover et al.,
1991). Samples were separated using 10% SDS-PAGE and exposed to a
phosphoimager. TRC levels were quantified using ImageQuant TL v2005
software.
Maternal pronuclear transfer experiments
One-cell embryos were collected from 8- to 10-week-old littermates 19 hours
post-hCG. Maternal pronuclear transfer was carried out as described
(Han et al., 2005). Embryos
were cultured until 72 hours post-hCG in KSOM+AA medium at 37°C in an
atmosphere of 5% CO2, 5% O2 and 90% N2.
Production of Ctcf mRNA and microinjection
An optimized Kozak sequence was added upstream of the start codon at
position 307 of a mouse CTCF cDNA sequence p5.1 (GB accession #U51037)
subcloned into a Bluescript plasmid
(Filippova et al., 1996). The
CTCF-coding sequence, 66 bp of the modified 5' UTR, and the first 267 bp
of the 3' UTR were then subcloned into an In-Vitro Transcription plasmid
(pIVT) containing a synthetic poly(A) tail. Capped and polyadenylated
Ctcf mRNA was transcribed using MEGAscript Kit (Ambion). RNA was
purified using MEGAclear Kit (Ambion), precipitated and resuspended at 2
µg/µl in water. One-cell embryos were collected from 8- to 10-week-old
littermates 19 hours post-hCG, and microinjected with 5-10 pl of Ctcf mRNA at
a concentration of 1 or 2 µg/µl. Control embryos were injected with an
equivalent amount of Gfp mRNA. Embryos were cultured until 72 hours
post-hCG in KSOM+AA medium at 37°C in an atmosphere of 5% CO2,
5% O2 and 90% N2.
Statistical analysis
Graphpad Prism 4 software was used to calculate statistical significance.
Two-tailed t-tests, one-way ANOVA or Fisher's exact tests were used
as appropriate.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
transgene depletes CTCF specifically in growing oocytes because the
Zp3 promoter is active only in these cells. Five independent Tg lines
have been previously described (Fedoriw et
al., 2004). Depending on the line, Ctcf transcripts were reduced
in fully-grown germinal vesicle (GV) oocytes by 50% to 99% relative to
controls. We selected the three most severely affected lines for further
study, all of which produced GV oocytes that were similarly CTCF-depleted
(> 99% reduction; Table 1).
Nevertheless, the three Tg lines exhibited phenotypic differences, described
below, that could have resulted from differences in the time course of CTCF
depletion during oocyte growth. We therefore examined CTCF protein levels in
growing oocytes, when the Zp3 promoter controlling transgene
expression is initially activated and maternal transcripts are beginning to
accumulate. Growing oocytes from Line 1 were the most severely CTCF-depleted
(86% reduction), followed by those from Line 21 (59% reduction) and Line 12
(33% reduction; Fig. 1A). CTCF
mRNA levels correlated with protein levels (data not shown). Therefore,
differences in CTCF depletion were apparent among the three most severely
affected lines.
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). Persistent RNAi should affect all embryos
derived from Tg females, regardless of whether the embryos themselves inherit
the transgene. We therefore determined the protein expression patterns of CTCF
in embryos derived from wild-type and Tg female littermates. In embryos
derived from Ntg females, CTCF was excluded from nucleoli, but was otherwise
diffusely localized in the nucleus throughout preimplantation development
(Fig. 1B). CTCF was upregulated
gradually from the morula to the late blastocyst stages of development (72 to
120 hours post-hCG). However, in embryos derived from Tg Line 1, CTCF was
depleted through the morula and into the early blastocyst stages of
development (72 and 96 hours post-hCG, respectively;
Fig. 1B). Because zygotic CTCF
is transcribed at the two-cell stage (48 hours post-hCG; data not shown),
depletion of CTCF from later stages of preimplantation embryo development was
most probably due to persistent RNAi (Fig.
1C).
Transcriptional misregulation in CTCF-depleted oocytes
Given the early and severe depletion of CTCF in Tg oocytes, and the
multitude of CTCF-binding sites throughout the genome, we expected that many
genes would be misregulated. We therefore compared the expression profiles of
Tg and Ntg GV oocytes using Affymetrix MOE430 2.0 GeneChips, which cover over
39,000 transcripts and variants. Oocytes from five pairs of Ntg and Tg
littermates were analyzed independently. To minimize false positives, we
restricted analysis to 20,396 transcripts that were `Present' according to
Microarray Analysis Suite 5 (MAS5) software, in four out of five Ntg or Tg
replicates. An unsupervised hierarchical cluster analysis revealed that all
replicates clustered according to their genotype (see Fig. S1 in the
supplementary material). Statistical Analysis of Microarrays (SAM) revealed
1590 significantly upregulated and 2282 significantly downregulated
transcripts (FDR<5%). Of these transcripts, 460 were upregulated and 934
were downregulated by more than 1.4-fold, while 115 were upregulated and 278
were downregulated by more than twofold (see Table S1 in the supplementary
material). Experimental Analysis Systematic Explorer (EASE) grouped 1.4-fold
misregulated transcripts into several over-represented functional categories.
(see Tables S2-S10 in the supplementary material). Interestingly, the most
significantly over-represented categories were related to embryogenesis,
suggesting that transcriptional defects in CTCF-depleted oocytes might affect
subsequent embryonic development.
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);
www.insulatordb.utmem.edu].
We obtained the genomic locations of all mouse genes from Ensemble Biomart
(www.ensembl.org).
For each mouse gene, we determined the distance to the closest CTCF-binding
site, using 0 as the distance between a gene and an overlapping site. We found
that upregulated and downregulated genes were both significantly closer to
CTCF-binding sites than background, and this trend was strong up to a distance
of 10 kb. In addition, the trend was much stronger for downregulated genes.
For example, 13% of all genes, 16% of upregulated genes (P<0.05)
and 20% of downregulated genes (P<10-8) were located
within 5 kb of a CTCF site (Fig.
2A). Next, we determined if CTCF binding sites were preferentially
upstream or downstream of misregulated genes. Among CTCF sites within 5 kb of
all genes, 19% were upstream and 18% were downstream of a gene. The remaining
sites overlapped with genes. Among CTCF sites within 5 kb of upregulated
genes, 15% were upstream and 18% were downstream. Most notably, among CTCF
sites within 5 kb of downregulated genes, 21% were upstream and only 12% were
downstream (P<0.05; Fig.
2B). Thus, among downregulated genes the closest CTCF-binding
sites were significantly biased toward upstream locations. These results
suggest that downregulated genes were CTCF targets rather than indirect
targets or off-targets of RNAi. Moreover, considering that the majority of
misregulated genes in CTCF-depleted oocytes was downregulated, these results
suggest that CTCF predominantly activates or derepresses transcription in
oocytes.
As expected, CTCF was the most highly downregulated gene
(Table 1). We validated eight
additional misregulated genes using real-time PCR
(Table 1). Because several
putative CTCF targets were not identified in our analysis, we examined two
putative targets (Myc and Ctcfl/Boris) using
real-time PCR. We found that although CTCF is a repressor of these genes in
cell culture (Filippova et al.,
1996; Qi et al.,
2003; Vatolin et al.,
2005), their expression was not upregulated in CTCF-depleted
oocytes (Table 1). This is
interesting in light of several studies showing that CTCF binding is largely
invariant across cell lines (Gombert et
al., 2003; Kim et al.,
2007). It is therefore likely that the transcriptional output of
CTCF binding varies in a cell-type specific manner, possibly as a consequence
of different binding co-factors and post-translational modifications.
Meiotic defects in CTCF-depleted oocytes
Given the high level of transcriptional misregulation in CTCF-depleted
oocytes, we examined oocyte nuclei using several anti-chromatin antibodies.
Nuclear protein levels of dimethylH3K4, H2Az and HP1-β were unchanged in
Tg oocytes derived from Line 1 (see Fig. S2A-F in the supplementary material).
We also scored the numbers of surrounded nucleolus (SN)-type and
non-surrounded nucleolus (NSN)-type GV oocytes, which can be distinguished
based on nuclear morphology. NSN-type nuclei contain several discrete foci of
heterochromatin on a background of decondensed euchromatin. Immediately prior
to ovulation, NSN-type nuclei may transition to SN-type nuclei that are
entirely heterochromatic and transcriptionally silent
(Zuccotti et al., 1995).
Although both subtypes can undergo meiotic maturation in vitro, SN-type
oocytes are more meiotically competent
(Liu and Aoki, 2002).
Interestingly, we found that the frequency of the SN subtype was slightly but
significantly decreased among Tg oocytes derived from all three lines
(Table 2).
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We observed no defects in spindle morphology using an antibody against
-tubulin (data not shown). However, we did find an increased proportion
of Tg eggs in anaphase or telophase of metaphase 1, which presumably resulted
from an overall delay in meiosis (Table
2). Eggs arrested at metaphase I can form 2n polar bodies upon
fertilization, leading to digynic triploid embryos that die at various stages
post-implantation (Kaufman and Speirs,
1987). Consistent with these observations, 8% of CTCF-depleted
eggs formed triploid embryos when fertilized in vivo, whereas no Ntg eggs
formed triploid embryos (see Fig. S3 in the supplementary material).
Early mitotic defects leading to apoptosis
We previously showed that although Tg eggs are fertilized in vitro at a
normal incidence, fewer develop to the blastocyst stage by 120 hours post-hCG
(Fedoriw et al., 2004). To
determine when the decrease in developmental competence was first apparent, we
cultured in vivo fertilized one-cell embryos and examined development at
various time points after hCG injection. CTCF-depleted one-cell embryos
cleaved at a normal rate, and by 48 hours post-hCG, almost all control and
CTCF-depleted one-cell embryos reached the two-cell stage (data not shown).
However, by 72 hours post-hCG, when control embryos were at the four- to
eight-cell stage, CTCF-depleted embryos were at the two- to four-cell stage
(Fig. 4A). Therefore, the
decrease in developmental competence was first apparent at the two- to
four-cell transition.
Because zygotic genome activation (ZGA) is essential for development beyond
the two-cell stage, we determined whether ZGA was defective in CTCF-depleted
embryos. An accepted marker for ZGA is the transcription requiring complex
(TRC), which is composed of three -amanitin-sensitive proteins 
70
kDa in weight (Conover et al.,
1991). In control two-cell embryos, TRC expression initiated by 6
hours after the first cleavage, peaked by 12 hours post-cleavage, and was
downregulated by 22 hours post-cleavage. In CTCF-depleted two-cell embryos,
TRC expression was reduced by 40% relative to control two-cell embryos at both
6 and 12 hours post-cleavage (Fig.
4B,C). It is therefore possible that a disruption in ZGA caused a
developmental delay at the two-cell stage.
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In mammalian cell culture, CTCF is essential for localizing cohesins to
specific sites throughout the genome; however, this localization is important
for gene regulation at interphase rather than for sister chromatid cohesion
during mitosis (Parelho et al.,
2008; Stedman et al.,
2008; Wendt et al.,
2008). Consistent with these findings, mitotic chromosomes from
CTCF-depleted two- to four-cell embryos exhibited no cohesion defects.
Moreover, no significant differences in the rates of sister chromatid
resolution or chromatid arm separation during mitotic arrest were observed,
indicating that cohesins dissociated from chromatin normally during prophase
and prometaphase (see Fig. S4A,B in the supplementary material). Finally,
antibody staining of CTCF-depleted and control two- to four-cell embryos
showed normal levels of chromatin-bound SMC1 during interphase (see Fig. S4C,D
in the supplementary material).
By 120 hours post-hCG, 94% of cultured control embryos were at the
blastocyst stage, whereas only 7% of cultured CTCF-depleted embryos were at
the blastocyst stage. The remaining embryos were at the morula stage or at
various stages prior to morula compaction. A limited amount of apoptosis is
normal in blastocysts, and may be required to eliminate suboptimal blastomeres
from the embryo prior to implantation. In agreement with previous reports
(Fabian et al., 2005), we
observed TUNEL-positive cells in the inner cell mass and trophectoderm of
control blastocysts, but prior to 120 hours post-hCG only the polar body was
TUNEL positive. Similarly, we did not observe apoptosis in CTCF-depleted
embryos at earlier timepoints; however, by 120 hours post-hCG many
CTCF-depleted embryos arrested at the morula stage exhibited abnormally high
levels of apoptosis (Fig. 4G;
data not shown). CTCF-depleted embryos having formed a blastoceol cavity
appeared small but otherwise normal (Fig.
4G, upper left `Tg' embryo). Thus, CTCF-depleted embryos failing
to reach the blastocyst stage at the time of implantation were probably
eliminated by apoptosis. Because CTCF levels were normal at this timepoint,
apoptosis was presumably a downstream effect of earlier defects.
Maternal pronuclear transfer and Ctcf mRNA-injection experiments
Given the importance of CTCF in chromatin organization, we initially
hypothesized that CTCF depletion caused a persistent nuclear change in the egg
that resulted in the early mitotic defect. Such persistent changes could have
included defects in meiosis or genome-wide epigenetic changes passed down from
the egg and inherited by the embryo. To test this hypothesis, we performed
maternal pronuclear transfer experiments in which maternal pronuclei of
CTCF-depleted one-cell embryos were exchanged with maternal pronuclei of
control one-cell embryos. By 48 hours after transfer, unmanipulated control
embryos were at the four- to eight-cell stage, whereas unmanipulated
CTCF-depleted embryos were at the two-cell stage. In control experiments, as
expected, exchanging maternal pronuclei among control embryos or among
CTCF-depleted embryos did not affect development. Surprisingly, introducing
CTCF-depleted maternal pronuclei into control embryos resulted in development
to the four- to eight-cell stage. These reconstructed embryos had normal
nuclear CTCF protein levels by 48 hours after transfer, presumably because
control embryos were replete with cytoplasmic stores of CTCF mRNA
(Fig. 5A; data not shown). This
result suggests that persistent nuclear changes arising in the egg did not
cause the early mitotic defect, or that the changes could be reversed at the
one-cell stage.
By contrast, introducing control maternal pronuclei into CTCF-depleted embryos resulted in development to the two-cell stage, suggesting that the early mitotic defect was caused by the depletion of maternal CTCF transcripts and possibly other maternal transcripts stored in the cytoplasm (Fig. 5A). However, persistent RNAi depleted CTCF until at least 48 hours after transfer, well after zygotic transcription of CTCF had begun (data not shown). Therefore, in order to separate maternal effects from the effects of persistent RNAi, we injected mouse Ctcf mRNA into one-cell embryos. By 48 hours after injection, control Gfp-injected embryos reached the four- to eight-cell stage, whereas CTCF-depleted Gfp-injected embryos reached the two- to four-cell stage. Injecting Ctcf mRNA into CTCF-depleted one-cell embryos restored nuclear protein levels by 6 and 48 hours after injection, but the resulting embryos were delayed at the two-cell stage (see Fig. S5 in the supplementary material; Fig. 5B,C). Therefore, maternal Ctcf transcripts were important for embryonic development, suggesting that CTCF is a maternal-effect gene. In addition, the data suggest that the two-cell delay was a consequence of transcriptional misregulation in the oocyte rather than a direct effect of CTCF depletion in the embryo.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Christians et al., 2000;
De Vries et al., 2004;
Gurtu et al., 2002;
Howell et al., 2001;
Leader et al., 2002;
Payer et al., 2003;
Roest et al., 2004;
Tong et al., 2000;
Wu et al., 2003). However,
useful this approach has been, it only uncovers maternal effect genes with
relatively minor functions in other essential processes. Because ZGA in
mammals occurs long before lineage specification and embryo patterning occur,
many mammalian maternal effect genes could have important general functions
that preclude the analysis of homozygous null females. More recent studies
have used promoter-driven transgenes to deplete maternal transcripts without
affecting maternal viability. For example, Ezh2 and Brg1
have been knocked out in growing oocytes using tissue-specific Cre/LoxP
(Bultman et al., 2006;
Erhardt et al., 2003), whereas
basonuclin transcripts have been depleted from growing oocytes using
transgenic RNAi (Ma et al.,
2006).
|
).
However, in the case of Formin2, the maternal effect is clearly
downstream of meiotic defects that lead to polyploidy and post-implantation
lethality in the embryo (Leader et al.,
2002). In many other cases, the molecular functions of
maternal-effect genes and the full extent of their effects on oocyte
development are simply unknown. Using transgenic RNAi, we have severely
depleted CTCF from growing oocytes and identify hundreds of misregulated
genes. However, despite such a high degree of gene misregulation in growing
oocytes, the importance of CTCF does not become grossly apparent until meiotic
maturation and the two-cell embryonic stage. In addition, we show that the
two-cell mitotic defect is not the result of earlier meiotic defects or other
persistent nuclear defects arising in the egg. Thus, Ctcf can be
added to a short list of mammalian maternal effect genes, a list that will
probably grow with the use of oocyte-specific gene-targeting methods.
Three recent studies have identified thousands of CTCF-binding sites
throughout the human and mouse genomes
(Barski et al., 2007;
Kim et al., 2007;
Xie et al., 2007). In the
first study, a ChIP-Chip approach was used to identify 13,804 CTCF-binding
sites, over 75% of which share a consensus motif
(Kim et al., 2007). In the
second study, a computational approach was used to identify 233 conserved
noncoding elements (CNEs) (Xie et al.,
2007). Three CNEs are bound by CTCF, and are found at 14,987 sites
in the genome, which constitutes 25% of the total number of sites
identified. In the third study, a ChIP-Seq approach was used to identify
20,262 CTCF-binding sites (Barski et al.,
2007). We have identified hundreds of misregulated genes in
CTCF-depleted oocytes. The number of downregulated genes is greater than the
number of upregulated genes, especially among highly misregulated genes.
Moreover, downregulated genes are enriched for nearby CTCF-binding sites,
especially in their upstream regions. Although it is likely that some
transcriptional changes are indirect effects of CTCF depletion, the results
suggest that many downregulated genes are direct CTCF targets. Moreover, the
results are consistent with recent findings that CTCF-binding sites are
enriched for active histone marks, and may activate genes by recruiting RNA
polymerase II (Chernukhin et al.,
2007) or by preventing the spread of heterochromatin. By contrast,
upregulated genes are not as strongly associated with CTCF-binding sites, and
nearby sites are not biased toward their upstream regions. These data could be
consistent with CTCF acting as an enhancer-blocker to repress gene
transcription from a distance, possibly by competing with promoters for nearby
enhancers (Yoon et al., 2007)
or by stalling the linear transfer of activating factors
(Zhao and Dean, 2004).
However, this mechanism of repression is less likely in oocytes and embryos
prior to two-cell ZGA because, lacking a required co-factor, they apparently
do not use enhancers (Majumder et al.,
1997).
Given the multitude of CTCF-binding sites throughout the genome, and its diverse roles in nuclear organization, it is notable that in the absence of appreciable levels of CTCF, oocyte growth is apparently not perturbed, and defects in meiotic maturation are relatively minor. For example, only 7% of Tg oocytes fail to segregate chromosomes at anaphase I, and all oocytes can be fertilized regardless of meiotic defects. By contrast, after fertilization most CTCF-depleted embryos are delayed at the two-cell stage, and only 7% of CTCF-depleted embryos are able to form a blastoceol cavity. Moreover, maternal pronuclear transfer and RNA microinjection experiments suggest that persistent transcriptional defects rather than persistent chromatin defects result in the early mitotic delay. Overall, these results point to an important role for CTCF in transcriptional regulation rather than in chromatin structure per se.
With respect to specific misregulated genes, several are imprinted. The
maternally expressed transcript Gtl2 is downregulated, consistent
with a role for CTCF in transcriptional activation at the Gtl2/Dlk1
locus (Paulsen et al., 2001).
In addition, the maternally expressed gene Grb10 is downregulated,
and although CTCF is hypothesized to function as an insulator on the paternal
allele (Hikichi et al., 2003),
our results suggest that CTCF may activate Grb10 on the maternal
allele. However, two adjacent maternally expressed genes, Slc22a18
and Phlda2, are upregulated. This is consistent with a role for CTCF
in transcriptional repression at the Kcnq1 locus
(Fitzpatrick et al., 2007).
Furthermore, the non-imprinted gene Cbfa2t1h is downregulated by
almost twofold. Cbfa2t1h is a putative maternal effect gene that was
identified as such because its transcripts are enriched in oocytes and
one-cell embryos (Mager et al.,
2006). However, Cbfa2t1h cannot easily account for the
phenotypes because its protein levels appear only slightly decreased (data not
shown). Nevertheless, it is possible that a combination of many
transcriptional defects contribute to the phenotypes in CTCF-depleted embryos,
or that a few highly misregulated genes have undiscovered maternal
effects.
The two-cell delay and disruption of ZGA are consistent with a maternal effect because maternally derived products are presumed to regulate these early processes. We have yet to determine whether TRC expression in CTCF-depleted embryos is reduced throughout the two-cell stage, or if TRC expression peaks at a timepoint between 12 and 22 hours post-cleavage. In addition, we have not yet determined if other markers of ZGA are disrupted. However, with respect to TRC expression, there is a high degree of heterogeneity among small groups of CTCF-depleted two-cell embryos, and this heterogeneity increases as the two-cell stage progresses. This suggests that transcriptional defects in the oocyte may be compounded to varying degrees in individual embryos. Near the end of preimplantation development, a small number of embryos survive and implant. This may be a consequence of transcriptional heterogeneity during ZGA, or it may reflect differences in the time course of CTCF depletion among individual growing oocytes. This also implies that CTCF can restore chromatin organization and transcription de novo, but we do not know whether more subtle consequences of early CTCF-depletion persist. It will be interesting to determine what long-term epigenetic effects persist in CTCF-depleted embryos subsequent to implantation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/16/2729/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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P<10-5.(B) Percentages of
CTCF binding sites within 5 kb of all, upregulated and downregulated genes.
Among sites within 5 kb of downregulated genes, the percentage of upstream
sites was significantly higher than the percentage of downstream sites,
compared with background. *P<0.05.
