"Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1".

Pengpeng Ma ³, Shu Lin ⁴, Marisa S. Bartolomei ⁴, and Richard M. Schultz ^{2, 3}

³ Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania,
⁴ Department of Cell, Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

¹ Supported National Institutes of Health grants HD042026 to M.S.B. and R.M.S., and HD022681 to R.M.S.
² Correspondence: Richard M. Schultz, Department of Biology, University of Pennsylvania, 433 South University Ave., Philadelphia, PA 19104-6018.   FAX: 215 898 8780;  e-mail:  rschultz@sas.upenn.edu

Received June 1, 2010.   First decision July 7, 2010.   Accepted August 11, 2010.

The epigenetic mechanisms involved in establishing and maintaining genomic imprinting are steadily being unmasked. The nucleosome remodeling and histone deacetylation (NuRD) complex is implicated in regulating DNA methylation and expression of the maternally expressed H19 gene in preimplantation mouse embryos. To dissect further the function of the NuRD complex in genomic imprinting, we employed an RNA interference (RNAi) strategy to deplete the NuRD complex component Metastasis Tumor Antigen 2 (MTA2). We found that Mta2 is the only zygotically expressed Mta gene prior to the blastocyst stage, and that RNAi-mediated knockdown of Mta2 transcript leads to biallelic H19 expression and loss of DNA methylation in the differentially methylated region in blastocysts. In addition, biallelic expression of the paternally expressed Peg3 gene, but not Snrpn, is also observed in blastocysts following Mta2 knockdown. Loss of MTA2 protein does not result in a decrease in abundance of other NuRD components, including methyl-binding-CpG-binding domain protein 3 (MBD3), histone deacetylases 1 and 2 (HDACs 1 and 2), and chromodomain helicase DNA-binding protein 4 (CHD4). Taken together, our results support a role for MTA2 within the NuRD complex in genomic imprinting.

Keywords:
DNA methylation   embryo   genomic imprinting  H19   MTA   NuRD complex  Peg3   RNAi

Dynamic alterations in chromatin structure due to ATPdependent
remodeling and covalent histone modifications play
important roles in regulating gene expression [1]. The
nucleosome remodeling and histone deacetylation (NuRD)
complex is ideally situated to modulate gene expression
because it possesses both nucleosome remodeling and histone
deacetylase activities [2–7]. The NuRD complex is approximately
2 MDa in size and in mammalian cells is composed of
at least seven polypeptides [7–10]. Four of the components,
histone deacetylases 1 (HDAC1) and HDAC2 and two histone-binding
proteins (RBBP4/RbAp46 and RBBP7/RbAp48), form
a deacetylase complex that is also found in the SIN3A histone
deacetylase complex [11]. The other three components,
chromodomain helicase DNA-binding protein 4 (CHD4 [Mi-2b]),
methyl-binding-CpG-binding domain protein 3 (MBD3),
and Metastasis Tumor Antigen (MTA), however, appear to be
unique to the NuRD complex [12].

CHD4 is an ATP-dependent nucleosome-remodeling enzyme
[7, 13]. Although mammalian MBD3 does not bind to
methylated DNA [14], when NuRD binds MBD2, the resulting
NuRD complex becomes an integral component of the MeCP1
complex [13, 15] that can bind to methylated DNA, thereby
leading to histone deacetylation of DNA-methylated nucleosomes
[13]. MTA proteins, which are the last characterized
proteins of the NuRD complex [16], are encoded by three
genes [17], and it appears that only one family member is
found within a given NuRD complex [18].

MTA1, the founding member of the MTA family, is
associated with metastatic growth of cell lines in vitro and with
invasive growth of tumors [19]. In addition to MTA1, MTA3
represses WNT4 function in mammary epithelial cells and plays
important roles in invasive growth of breast cancer cells by
repressing transcription of Snail [18, 20]. In contrast to the
restricted expression patterns of MTA1 and MTA3, MTA2 is
ubiquitously expressed. Targeted mutation of the Mta2 gene
results in multiple phenotypes that include partial embryonic and
perinatal lethality, female infertility, abnormal T-cell activation,
and lupuslike autoimmune disease in mice [21]. Although it is
likely that the three MTA family members form different NuRD
complexes with distinct functions [16, 22–25], the role of MTA
proteins in the function of NuRD complexes remains elusive.

Genomic imprinting is an epigenetic process that results in
parent-of-origin-specific gene expression of a small number of
genes [26, 27]. In mammals, many imprinted genes are located in
approximately 1-Mb clusters, with regulation of the linked genes
controlled by a differentially methylated region (DMR), which
exhibits allele-specific epigenetic modifications, such as DNA
methylation or posttranslational histone modifications. Deletion
of DMRs results in the loss-of-imprinting of multiple genes in
cis. Allele-specific epigenetic modifications are established in
the germline or zygote and must be subsequently maintained for
development to proceed normally. Maintaining these marks is
especially critical during preimplantation development because
the paternal genome is actively demethylated shortly after
fertilization, and then both genomes are passively demethylated
during preimplantation development [28]. Moreover, a dramatic
reprogramming of gene expression occurs during preimplantation
development, first being observed during the two-cell stage,
with changes in histone acetylation serving a regulatory function
[29]. Remarkably, despite these dramatic changes in DNA
methylation and gene expression, the imprinting marks that are
established during gametogenesis are maintained by mechanisms
that remain to be elucidated fully.

Although chromatin remodeling and histone modifications
play crucial roles in genomic imprinting [30–32], a direct link
between the NuRD complex and genomic imprinting is
lacking. Results of our previous study implicate NuRD in
regulating imprinted gene expression, because knockdown of
MBD3 in preimplantation mouse embryos results in both
biallelic expression of H19 and loss of DNA methylation
within the imprinting control region (ICR) at the blastocyst
stage [33]. We also noted in that study that MBD3 depletion is
accompanied by a reduction in the amount of nuclear-localized
MTA2, suggesting a role for MTA2 in genomic imprinting.

To define further the function of the NuRD complex in
imprinting, and in particular MTA2, we used an RNA
interference (RNAi) strategy to deplete MTA2 selectively.
We found that Mta2 is the only zygotically expressed Mta
gene, and that depleting Mta2 transcripts leads to biallelic
expression of H19 and loss of DNA methylation on the ICR in
blastocysts. In addition, biallelic expression of the paternally
expressed Peg3, but not Snrpn, is also observed in blastocysts
following Mta2 knockdown.

MATERIALS AND METHODS:

Collection and Culture of Oocytes and Preimplantation Embryos

For double-stranded RNA (dsRNA) injections, oocytes and embryos were
obtained as previously described from either CF1 (Harlan, http://www.harlan.com/)
females (6–8 wk of age) mated to B6D2F1/J males (Jackson Laboratory)
for embryo assays [34], or from C57BL/6J (Jackson Laboratory, http://www.jax.org/)
females mated to B6(P12X) males for allelic assays [33, 35]. This latter
strain has Mus musculus castaneus chromosomes 7, 12, and X in a C57BL/6
background. All experiments were conducted with the approval of the
Institutional Animal Care and Use Committee at the University of Pennsylvania.

Cumulus cell-free, germinal vesicle (GV)-intact oocytes were obtained
from equine chorionic gonadotrophin (eCG)-primed females as previously
described [36]. The collection medium for oocytes was bicarbonate-free
minimal essential medium (Earle salts) containing 25 mM Hepes, pH 7.3; 3 mg/
ml polyvinylpyrrolidone; and 2.5 lM milrinone to prevent GV breakdown.
Oocytes were matured in vitro in Whitten medium [37] containing 0.01%
polyvinyl alcohol (PVA) (Whittens/PVA).

Meiotically incompetent oocytes (i.e., oocytes that do not spontaneously
resume meiosis when placed in a suitable culture medium) were obtained from
12-day-old female mice. Ovaries were placed in Ca²⁺- and Mg²⁺-free CZB
medium [38] containing collagenase I (Worthington, Lakewood, NJ) and
DNase I (Sigma, St. Louis, MO); prior to use, the medium was filtered using a
0.2-lm filter. The ovaries were minced into small pieces and then incubated for
5–10 min in an atmosphere of 5% CO₂ in air at 378C. Oocytes were freed from
follicles by repeated pipetting with a mouth-operated pipette, and liberated
oocytes were removed and transferred to CZB medium. The remaining ovarian
tissue was returned to the incubator, and after another 15–30 min the procedure
was repeated.

Embryos were cultured in KSOM containing amino acids [39] for up to 4
days in 5% CO₂ in air at 37^oC. One-cell, two-cell, four-cell, and eight-cell
embryos and blastocysts that developed in vivo were flushed from either
oviducts or uteri at 20–21, 41–44, 60–61, 68, and 92–96 h after eCG,
respectively [29].

Microinjection of one-cell embryos was performed essentially as previously
described [29]. Prior to pronucleus formation, the embryos were injected with
10 pl of dsRNA using a Picoliter Injector Microinjection System (Harvard
Apparatus, Holliston, MA); the culture medium was bicarbonate-free Whitten
medium containing 0.01% PVA and 25 mM Hepes, pH 7.3. Following
microinjection, the embryos were cultured in KSOM containing amino acids as
described above.

dsRNA Preparation

Double-stranded RNA was prepared by annealing two complementary
RNAs transcribed by T7 or SP6 polymerase in vitro. The cDNA fragments
were initially subcloned into the PCRII vector. Mta2 dsRNA was a 529-bp
fragment prepared with 5'-CACCAATCAACAGAAACCAGC-3' and 5'-
ACACACACACACCAGGGTCAA-3'.

After in vitro transcription using T7 and SP6 RNA polymerase (Ambion),
DNA templates were removed by DNase I treatment. The RNA products were
extracted with phenol:chloroform and then precipitated with ethanol. To anneal
sense and antisense RNAs, equimolar quantities of sense and antisense RNA
were mixed in annealing buffer (10 mM Tris, pH 7.4, and 0.1 mM
ethylenediaminetetraacetic acid) to a final concentration of 2 mM each, heated
for 2 min at 94^oC and then incubated at room temperature for at least 16 h. To
remove unhybridized RNA, the mixture was treated with 2 mg/ml RNaseT1
(Calbiochem, San Diego, CA) and 1 mg/ml RNaseA (Sigma) for 30 min at
37^oC. The dsRNA products were extracted with phenol:chloroform and ethanol
precipitated, then dissolved in water. The quality of dsRNA was confirmed by
electrophoresis in an agarose gel. Gfp dsRNA was prepared as previously
described [40]. The dsRNA samples were diluted to a final concentration of
1–2 mg/ml and stored at -80^oC until used.

RNA Extraction and Real-Time RT-PCR

Total RNA from 5 to 50 embryos was extracted using the Absolutely RNA
Microprep Kit (Stratagene, La Jolla, CA). The RT reaction, primed with
random hexamers, was performed using Superscript II reverse transcriptase
(Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Total
RNA isolated was reverse transcribed in a 20-ll reaction volume. The resulting
cDNA was quantified by real-time PCR (qRT-PCR). The qRT-PCR analysis
was performed with the ABI Taqman Assay-on-demand probe/primer sets for
Mta1(Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3
(Mm00475365_m1). One embryo equivalent of cDNA was used for each
real-time PCR reaction with a minimum of three replicates as well as a minus
RT and minus template controls for each gene. Unless otherwise stated,
quantification was normalized to Ubtf (Mm00456972_m1) and histone H2A
mRNA (Mm00501974_s1).

Allele-Specific Expression Analysis of Blastocyst-Stage Embryos

H19 and Snrpn expression assays were conducted on cDNA using the
LightCycler Real Time PCR System (Roche Molecular Biochemicals) as
described previously [33]. Peg3 RT-PCR expression assays were conducted on
cDNA from single blastocysts by allele-specific restriction digests as previously
described [41]. The digested PCR products were resolved by PAGE. The
contribution of each parental allele to the total expression was determined using
the Quantity One software (Bio-Rad).

Antibodies

The following antibodies were used in immunofluorescence (IF) and
Western blotting (WB): anti-MTA1 goat polyclonal (sc-9445; Santa Cruz
Biotechnology; WB, 1:1000; IF, 1:100), anti-MTA2 mouse monoclonal
(ab50209; Abcam; WB, 1:10 000; IF, 1:200), anti-MTA3 rabbit polyclonal
(sc-48799; Santa Cruz Biotechnology; WB, 1:1000; IF, 1:100), anti-CHD4/
Mi2b rabbit polyclonal (A301-082A; Bethyl Laboratories; WB, 1:1000; IF,
1:150), anti-RBBP4 rabbit monoclonal (2566–1; Epitomics; WB, 1:10 000),
anti-HDAC1-rabbit polyclonal (06–720; Millipore; WB, 1:5000), anti-HDAC2-
mouse monoclonal (05–814; Millipore; WB, 1:10 000), anti-MBD3 rabbit
polyclonal (3896; Cell Signaling Technology; WB, 1:1000), anti-POU5F1/
OCT4 mouse monoclonal (sc-5279; Santa Cruz Biotechnology; IF, 1:100),
anti-NANOG rabbit polyclonal (ab80892; Abcam; IF, 1:200), anti-hyperacetylate
histone H4 rabbit polyclonal (06–946; Millipore; IF, 1:500), anti-YY1
rabbit polyclonal (ab12132; Abcam; WB, 1:1000; IF, 1:100), anti-b-tubulin
(T8328; Sigma; WB, 1:2000).

Immunostaining of Oocytes/Eggs/Embryos and Quantification of Fluorescence Intensity

Oocytes or embryos were fixed in 2% paraformaldehyde in PBS for 20 min
at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for
10 min. Immunocytochemical staining was performed by incubating the fixed
samples with primary antibodies for 60 min, followed by secondary antibodies
conjugated with Cy5 or fluorescein isothiocyanate for 60 min; no signal was
observed when the primary antibody was omitted. Polyclonal antibodies against
MTA1, MTA2, and MTA3, hyperacetylated histone H4, and monoclonal
antibodies against POU5F1 and SOX2 (Upstate Biotechnology) were diluted as
listed above. The DNA was stained with 1mlM SYTOX Green (Molecular
Probes, Eugene, OR). The cells were then washed and mounted under a
coverslip with gentle compression in VectaShield antibleaching solution
(Vector Laboratories). Fluorescence was detected on a Leica TCS SP laserscanning
confocal microscope.

For each MTA immunostaining, all samples—i.e., oocytes, eggs, and
embryos—were processed simultaneously. For each MTA, the laser power was
adjusted so that the signal intensity was below saturation for the developmental
stage that displayed the highest intensity, and all images were then scanned at
that laser power. The intensity of fluorescence was quantified using National
Institutes of Health Image J software.

Immunoblot Analysis

Protein samples from embryos were solubilized in Laemmli sample buffer
[42], resolved by SDS-PAGE (10% gel, except when MBD3 or CHD4 were
analyzed, in which case the gel concentration was 15% or 8%, respectively),
and transferred to a nitrocellulose membrane. The membrane was blocked by
soaking in Blotto (Tris-buffered saline with 0.1% Tween-20 and 5% nonfat
dried milk) for 1.5 h and incubated overnight with the primary antibody in
blocking solution. The membrane was then washed three times with Trisbuffered
saline with 0.1% Tween-20 (TBST), incubated with a secondary
antibody conjugated with horseradish peroxidase for 45 min, and washed five
times with TBST. The signal was detected with the ECL Advance Western
blotting detection reagents (Amersham, Piscataway, NJ) following the
manufacturer’s recommendations. The primary antibodies were diluted as
described above, and secondary antibodies (Amersham ECL-HRP Linked
Secondary Antibody; GE Healthcare) were diluted 1:20 000.

Bisulfite Mutagenesis

The allele-specific DNA methylation patterns were examined for DMRs of
H19 [43], Peg3 [44], and Snrpn [41]. DNA was isolated from 2–12 blastocysts
using The QIAamp DNA Micro Kit (Qiagen, Valencia, CA). The DNA was
then denatured and treated with bisulfite using the Imprint DNA Modification
Kit according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO).
Partial mutagenized DNA was amplified by the Epitech Whole Bisulfitome Kit
(Qiagen) according to the manufacturer’s instructions. Half of a blastocyst
equivalent of the mutagenized DNA or 2 ml of the amplified DNA was used for
each PCR reaction, and the products were cloned and sequenced as described
previously [33, 43]. Six or more clones were sequenced for each PCR reaction.
The sequencing data was analyzed using the online tool BDPC
(http://biochem.jacobs-university.de/BDPC/index.php) [45]. Strands from a PCR that contained
an identical pattern of methylated cytosines and that could not be distinguished
from other strands by polymorphisms were only counted once. DNA strands
were considered hypomethylated when at least 50% of the CpGs assayed were
not methylated.

Statistical Analysis

Student t-test and chi-square test were used, and differences of P < 0.05 were considered significant.

RESULTS:

Temporal and Spatial Pattern of MTA Expression During Oocyte Maturation and Preimplantation Development

Transcripts for Mta1, Mta2, and Mta3 were assayed by qRT-PCR in oocytes and throughout preimplantation development (Fig. 1A).

FIG. 1. Mta RNA and protein expression patterns in oocyte and preimplantation embryos.

FIG. 1. Mta RNA and protein expression patterns in oocyte and preimplantation embryos.

A) Temporal pattern of expression of Mta1, Mta2, and Mta3. The experiment was conducted twice, and the data are expressed as mean 6 range and are expressed relative to the value obtained for full-grown oocytes. The amount of Mta RNA was normalized to Gfp mRNA that was added as an external control prior to RNA isolation.

B) Immunoblot analysis of MTA1, MTA2, and MTA3 expression. One hundred oocytes/embryos were loaded per lane, and beta-tubulin (TUBB) was used as a loading control. The experiment was conducted three times, and similar results were obtained in each case; a representative experiment is shown.

C) Immunocytochemical analysis of MTA1, MTA2, and MTA3 expression. All samples for a given MTA were processed for immunocytochemistry together, and all images were taken at the same laser power, thereby enabling direct comparison of signal intensities. The experiment was conducted three times, and at
least 25 oocytes/embryos were analyzed for each sample. Shown are representative examples. INC, incompetent oocyte; GV, fully grown oocyte; MII, metaphase II; 1C, one-cell embryo; 2C, two-cell embryo; 4C, four-cell embryo; 8C, eight-cell embryo; BL, blastocyst.

Original magnification x80.

In contrast to Mta1 and Mta3 mRNA, which
are degraded during maturation and preimplantation develop-
ment and then zygotically expressed between the eight-cell and
blastocyst stages, Mta2 expression is dramatically increased in
the two-cell embryo, which corresponds to the time of the
maternal-to-zygotic transition [46]. Consistent with these
changes in transcript abundance, immunoblotting experiments
demonstrated that the amounts of MTA1 and MTA3 were
relatively constant during preimplantation development,
whereas there was a dramatic increase in the amount of
MTA2 protein starting in the two-cell embryo (Fig. 1B). This
increase in Mta2 expression and protein accumulation was also
observed by immunocytochemistry, in which staining was
predominantly observed in the nucleus (Fig. 1C). Nuclear
staining for MTA1 and MTA3 was also relatively constant up
to the eight-cell stage. The amount of MTA1 assayed by
immunoblotting remained essentially constant during preimplantation
development, suggesting that the MTA1 decrease in
nuclear signal intensity was due to the increased number of
nuclei. In contrast, although the amount of MTA3 also
remained essentially constant, little nuclear signal was detected
in blastocysts, suggesting that the decrease is due to
translocation from the nucleus to the cytoplasm.

RNAi-Mediated Ablation of MTA2 Reduces the Amount of MTA2 but Not Other NuRD Complex Components

We previously demonstrated that RNAi-mediated targeting
of Mbd3 resulted in reduced amounts of nuclear MTA2 protein
in blastocysts and biallelic H19 expression [33]. The
observation that Mta2 is upregulated during the two-cell stage
made Mta2 an ideal candidate to be studied in genomic
imprinting by an RNAi approach. Accordingly, one-cell
embryos were injected with Mta2 dsRNA to ablate both
maternal and zygotic Mta2 transcripts. Control embryos were
injected with dsRNA for Gfp. The embryos were then cultured
to the blastocyst stage in vitro. The RNAi strategy specifically
targeted Mta2, and not Mta1 and Mta3 transcripts (Fig. 2A),
although there was a transient increase in the relative
abundance of Mta1 transcripts after injection.

FIG. 2. Effects of RNAi-mediated knockdown of Mta2 on Mta1, Mta2, and Mta3 expression, and localization and amount of MTA1, MTA2, and MTA3.

FIG. 2. Effects of RNAi-mediated knockdown of Mta2 on Mta1, Mta2, and Mta3 expression, and localization and amount of MTA1, MTA2, and MTA3.

A) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured for 24, 48, or 72 h, at which time the relative abundance of Mta1, Mta2, and Mta3 transcripts was assayed by qRT-PCR and expressed relative to controls. The experiment was performed three times, and the data are expressed as mean +/- SEM.

B) The experiment was performed as described in A, and the samples were processed for immunocytochemical detection of MTA 1, 2, or 3 at the indicated times. The experiment was conducted three times, and at least 25 oocytes/embryos were analyzed for each sample. Original magnification x80.

C) The experiment was performed as described in A, and the relative amount of MTA1 and MTA2 was determined by immunoblot analysis after 72 h of culture. The experiment was performed twice, and
similar results were obtained. One hundred embryos were used for immunoblot analysis.

The amount of MTA2 protein was reduced within 48 h
following injection as determined by immunocytochemistry
(Fig. 2B) and remained low 72 h following injection as
determined by immunocytochemistry and immunoblotting
(Fig. 2, B and C). Although the amount of MTA1 protein
was unchanged following depletion of Mta2 transcripts (Fig.
2C), the amount of nuclear-associated MTA1 protein was
significantly reduced (Fig. 2B), suggesting that MTA1 (and
any MTA1-associated proteins) was translocated to the
cytoplasm, where dilution would account for the decreased
immunocytochemical signal. Targeting Mta2 transcripts had
little, if any, effect on MTA3 (Fig. 2, B and C).

To determine whether expression of other components of
the NuRD complex was perturbed in MTA2-depleted embryos,
we examined expression of CHD4 (Mi-2b), RBBP4 (RbAp48),
MBD3, HDAC1, and HDAC2, as well as MTA1 and MTA2,
by immunoblot analysis and found no differences in these other
components in blastocysts (Fig. 3A);

FIG. 3. Effect of Mta2 knockdown on expression of other components of the NuRD complex.

FIG. 3. Effect of Mta2 knockdown on expression of other components of the NuRD complex.

A) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured 96 h in
vitro. Equal numbers of dsGfp- and dsMta2-injected embryos were collected for immunoblot analysis; 100 embryos were loaded per lane, and TUBB was used as a loading control. The experiment was performed
twice, and similar results were obtained.

B) dsGfp- or dsMta2-injected embryos were processed for immunocytochemical detection of histone H4 acetylation state. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images.

C) dsGfp- or dsMta2-injected embryos were processed for immunocytochemical detection of histone POU5F1 and NANOG. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images.

Original magnification x80.

CHD3 (Mi-2a) is not expressed in oocytes based on microarray studies [47] and
therefore was not assayed. Immunocytochemical detection of
these NuRD components was consistent with the immunoblotting
data and revealed no change in the intensity of the nuclear
signal (Supplemental Fig. S1, all supplemental data are
available online at: http://www.biolreprod.org/content/83/6/1027/suppl/DC1

HDAC1 appears to be the HDAC largely responsible for the
global state of histone acetylation in preimplantation mouse
embryos [29]. Consistent with the observation that HDAC1
expression appeared unperturbed in MTA2-depleted blastocysts,
there was no obvious effect on the global state of histone
acetylation in these blastocysts (Fig. 3B). Last, the NODE
complex (for NANOG- and POU5F1-associated deacetylase)
identified in embryonic stem (ES) cells is composed of MTA1/
MTA2, HDAC1/2, NANOG, and POU5F1 [48]. Expression of
NANOG and POU5F1 in dsMta2-injected embryos was not
different compared with control embryos (Fig. 3C). Results of
these experiments suggest that Mta2 dsRNA effectively
reduces MTA2 protein but does not affect other components
of the NURD complex or another complex that contains MTA2.

Biallelic H19 Expression and Loss of DNA Methylation on the Paternal DMR in MTA2-Depleted Blastocysts

We previously reported that depletion of MBD3 is
accompanied by biallelic H19 expression and reduced amount
of MTA2 in blastocysts [33]. We therefore examined the effect
of directly depleting MTA2 on imprinted H19 expression.
Using an allele-specific real-time RT-PCR assay for H19
expression in blastocysts, we found that H19 expression was
biallelic in 32% of the embryos (16/50), whereas only 10% (4/
39) of the dsGfp-injected embryos exhibited biallelic H19
expression (P < 0.05, chi-square). The loss of DNA
methylation in the DMR, which is located 2 kb upstream from
the transcription start site, in the MTA2-depleted blastocysts
provides a likely explanation for the observed biallelic H19
expression in these embryos (Fig. 4A).

FIG. 4. Loss of H19 paternal DMR methylation in MTA2-depleted blastocysts.

FIG. 4. Loss of H19 paternal DMR methylation in MTA2-depleted blastocysts.

Onecell embryos were injected with either Gfp dsRNA or Mta2 dsRNA, cultured to the blastocyst stage, and collected in pools of 2–12 blastocysts that were then subjected to bisulfite mutagenesis.

A) H19, paternal DNA stands are shown.

B) Snrpn, maternal strands are shown.

C) Peg3, maternal strands are shown. The H19 maternal and the Snprn and Peg3 paternal strands were
unmethylated in all samples. Each line of circles represents a single DNA strand, with the number to the left of the line corresponding to the number of times this pattern was seen.

Each circle represents a single CpG. If the CpG was methylated, the circle is filled. Those strands with less than half of the CpGs methylated are considered hypomethylated.

We observed a significant increase in the number of DNA hypomethylated
paternal strands in MTA2-depleted embryos (42.5%) compared
with controls (11.7%). These results strongly suggest that
MTA2 is necessary for both the proper imprinted gene
expression of H19 and maintenance of H19 paternal methylation
in mouse early embryos.

Snrpn and Peg3 Imprinted Gene Expression in MTA2-Depleted Embryos

Our previous study showed that reduced MBD3 had an
impact on proper imprinted expression of H19 but not
expression of other imprinted genes, including Snrpn and
Peg3 [33]. To ascertain whether a similar situation occurs for
MTA2, we examined the expression status of Snrpn and Peg3,
which are paternally expressed and imprinted in blastocysts, in
MTA2-depleted blastocysts. Snrpn expression remained
monoallelic from the paternal allele, and there was no change
in DNA methylation of the maternal allele of the Snprn DMR
compared with dsGfp-injected controls (Fig. 4B). In contrast,
32% (12/38) of the MTA2-depleted embryos showed biallelic
Peg3 expression compared with control embryos (2 [7.1%] of
28; P < 0.05, chi-square). Curiously, DNA methylation of the
maternal allele of a potential Peg3 DMR was not significantly
affected in these embryos (Fig. 4C), suggesting that this
region is not responsible for controlling paternal Peg3
expression.

YY1 is implicated in proper monoallelic Peg3 expression
[49–52], and YY1 can interact with MTA2 in vitro [25].
Therefore, it was formally possible that biallelic Peg3
expression was due to perturbed Yy1 expression in Mta2-
depleted embryos. Such is not the case, however, because the
amount of YY1 protein was unchanged in Mta2-depleted
embryos (Supplemental Fig. S2). Consistent with this finding
was that there was no decrease, as determined by qRT-PCR, in
the relative amount of Yy1 mRNA in Mta2-depleted embryos
compared with controls (data not shown).

Increased Number of Metaphase Chromosome Pairs in MTA2-Depleted Blastocysts

MTA2-depleted embryos developed to the blastocyst stage
at an incidence similar to controls and contained a comparable
number of cells (Table 1). Nevertheless, we noted a significant
increase of the number of metaphase chromosome pairs in
MTA2-depleted embryos compared with dsGfp-injected embryos
(Table 1), suggesting a role for MTA2 in cell division.

TABLE 1. Cell numbers and metaphase chromosome pairs of blastocysts after dsRNA injection.

DISCUSSION:

Genomic imprinting is established in the germline and early
embryos. How imprinted marks are maintained during early
development, and how they survive the genome-wide reprogramming
after fertilization, is essentially unknown. Our
previous finding [33] show that depletion of MBD3, which
led to a decrease in both the amount of nuclear MTA2 and
biallelic H19 expression, as well as a loss of DNA methylation
in the DMR, implicated a NuRD complex in maintaining
imprinted marks in preimplantation embryos. Results of
experiments reported here in which the amount of MTA2 is
specifically reduced further implicate a role for the NuRD
complex in maintaining imprinted marks in preimplantation
embryos, as well as provide new insights into NuRD function.

The composition of the NuRD complex in preimplantation
embryos is not known, and the limited amounts of biological
material effectively preclude undertaking such analyses. In
systems in which the NuRD complex has been isolated and
characterized, it appears that different MTAs are associated
with different NuRD complexes [18]. Our finding of the
different changes in nuclear concentration of the three MTAs
between the eight-cell and blastocyst stages is consistent with
each MTA residing in a distinct NuRD complex. For example,
although the amount of MTA3 remains relatively constant
between these two stages, there is a dramatic decrease in the
nuclear MTA3 signal in blastocysts, suggesting that NuRD
complexes containing MTA3 translocate to the cytoplasm. In
contrast, MTA1, whose amount also remains essentially
unchanged between the eight-cell and blastocyst stages,
displays only a modest decrease in the nuclear signal, much
of which is accounted for by the increase in the number of
nuclei.

The failure of MBD3 to interact with CHD4 and the
inability of antibodies against MBD3 to immunoprecipitate
MBD3-containing NuRD complexes—the antibody could
immunoprecipitate recombinant MBD3—suggest that MBD3
is embedded within the NuRD complex [53]. These findings,
coupled with the ability of MBD3 to interact directly with
MTA2 [53], provide an explanation that targeting Mbd3
mRNA results in a decrease in nuclear MTA2 [33], whereas
targeting Mta2 mRNA has no apparent effect on the amount of
MBD3. In this scenario, loss of MBD3 would lead to a reduced
association of MTA2 with the complex and a concomitant loss
from the nucleus, and possibly degradation. In contrast, a
decrease in the amount of MTA2 would be predicted to have
little, if any, effect on the stability of MBD3, as was observed.
These observations would also account for the observation that
a similar increase in the number of blastomeres arrested in
metaphase is observed following depletion of either MBD3
[33] or MTA2, and that the effect is mediated by MTA2.

HDACs 1 and 2, and RBBPs 4 and 7 can form a core
complex whose activity is stimulated by association with
MTA2 to a level similar to that of intact NuRD; neither CHD4
nor MBD3 stimulates this core complex [53]. HDAC1 appears
to be the HDAC responsible in determining the extent of global
histone acetylation in preimplantation mouse embryos [29].
The finding that there is no apparent change in global histone
acetylation in MTA2-depleted embryos—the amount of
HDAC1 is unchanged in these embryos—suggests that either
MTA2 does not stimulate HDAC activity in NuRD complexes
present in preimplantation embryos or, more likely, that
another HDAC1-containing complex(es), e.g., SIN3 [8, 54],
is responsible.

A seminal finding reported here is that reducing the amount
of MTA2 in preimplantation embryos results in biallelic
expression of both H19 and Peg3, which are maternally and
paternally expressed, respectively. Biallelic Peg3 expression
contrasts with our previous finding that paternal monoallelic
Peg3 expression is observed in MBD3-depleted preimplantation
embryos [33]. A possible explanation for this difference is
that MTA1- and MTA2-containing NuRD complexes control
expression of Peg3 and H19, respectively. Loss of nuclear
MTA1 in MTA2-depleted embryos would account for biallelic
expression of both Peg3 and H19; the molecular basis for the
nuclear exit of MTA1-containing NuRD complexes that occurs
following MTA2 depletion is enigmatic, especially in light of
finding that the amount of MTA1 protein determined by
immunoblotting appears unchanged. The greater loss of
MTA2, when compared with MTA1 in nuclear extracts from
Mbd3-/- ES cells [55], provides an explanation for our prior
finding that H19, but not Peg3, expression is biallelic
following RNAi-mediated reduction of MBD3. Should a
similar difference in the amounts of MTA1 and MTA2 exist
in these embryos, sufficient quantities of MTA1-containing
NuRD complexes may remain, thereby maintaining monoallelic
paternal Peg3 expression.

In contrast to the loss of DNA methylation observed in the
H19 DMR, MTA2 depletion does not affect DNA methylation
in the DMR adjacent to the Peg3 promoter. This finding is not
surprising, because the Peg3 DMR has not been demonstrated
to function as an ICR. Szeto et al. [56] analyzed three different
lines of transgenic mice that carry a bacterial artificial
chromosome covering 120 kb around the Peg3 locus. Only
one of three lines exhibited partial imprinted expression of
Peg3 and differential methylation in the Peg3 DMR. These
results suggest that the Peg3 DMR alone is not sufficient to
control the imprinting in this locus, and our data indicate that
MTA2 functions through an unknown element to repress the
maternal Peg3.

At a molecular level, how the loss of MTA2 is
mechanistically linked to biallelic expression of both H19
and Peg3, and DNA methylation of the DMR of H19 (and,
presumably, a differentially methylated DNA sequence responsible
for paternal Peg3 expression) remains unresolved.
Attempts to conduct chromatin immunoprecipitation experiments
to localize the NuRD complex to H19 and Peg3 genes
were unsuccessful, largely in part because it is difficult to
conduct such experiments given the small amounts of
biological material that can be readily isolated. Similar
experiments were also unsuccessful when model mouse cell
lines were used, e.g., mouse embryonic fibroblast cells, ES
cells, and trophoblast stem cells (data not shown). Nevertheless,
the results presented here provide further evidence for a
critical role of NuRD complexes in directing appropriate
expression of imprinted genes.

ACKNOWLEDGMENTS:

The authors thank Richard A. Jimenez for his assistance with the allelespecific
expression assays, Mellissa R.W. Mann and Rocio M. Rivera for
their useful suggestions for DNA methylation analysis, and Gerd Blobel for
his generous gifts of MTA1 and CHD4 antibodies.

REFERENCES:

1. Wang GG, Allis CD, Chi P. Chromatin remodeling and cancer, part II:
ATP-dependent chromatin remodeling. Trends Mol Med 2007; 13:373–380.

2. Denslow SA, Wade PA. The human Mi-2/NuRD complex and gene
regulation. Oncogene 2007; 26:5433–5438.

3. Humphrey GW, Wang Y, Russanova VR, Hirai T, Qin J, Nakatani Y,
Howard BH. Stable histone deacetylase complexes distinguished by the
presence of SANT domain proteins CoREST/kiaa0071 and Mta-L1. J Biol Chem 2001; 276:6817–6824.

4. Tong JK, Hassig CA, Schnitzler GR, Kingston RE, Schreiber SL.
Chromatin deacetylation by an ATP-dependent nucleosome remodelling
complex. Nature 1998; 395:917–921.

5. Wade PA, Gegonne A, Jones PL, Ballestar E, Aubry F, Wolffe AP. Mi-2
complex couples DNA methylation to chromatin remodelling and histone
deacetylation. Nat Genet 1999; 23:62–66.

6. Xue Y, Wong J, Moreno GT, Young MK, Cote J, Wang W. NURD, a
novel complex with both ATP-dependent chromatin-remodeling and
histone deacetylase activities. Mol Cell 1998; 2:851–861.

7. Zhang Y, LeRoy G, Seelig HP, Lane WS, Reinberg D. The
dermatomyositis-specific autoantigen Mi2 is a component of a complex
containing histone deacetylase and nucleosome remodeling activities. Cell 1998; 95:279–289.

8. Ahringer J. NuRD and SIN3 histone deacetylase complexes in
development. Trends Genet 2000; 16:351–356.

9. McDonel P, Costello I, Hendrich B. Keeping things quiet: roles of NuRD
and Sin3 co-repressor complexes during mammalian development. Int J
Biochem Cell Biol 2009; 41:108–116.

10. Zhang Y, Iratni R, Erdjument-Bromage H, Tempst P, Reinberg D. Histone
deacetylases and SAP18, a novel polypeptide, are components of a human
Sin3 complex. Cell 1997; 89:357–364.

11. Knoepfler PS, Eisenman RN. Sin meets NuRD and other tails of
repression. Cell 1999; 99:447–450.

12. Feng Q, Zhang Y. The NuRD complex: linking histone modification to
nucleosome remodeling. Curr Top Microbiol Immunol 2003; 274:269–290.

13. Feng Q, Zhang Y. The MeCP1 complex represses transcription through
preferential binding, remodeling, and deacetylating methylated nucleosomes.
Genes Dev 2001; 15:827–832.

14. Hendrich B, Bird A. Identification and characterization of a family of
mammalian methyl-CpG binding proteins. Mol Cell Biol 1998; 18:6538–6547.

15. Le Guezennec X, Vermeulen M, Brinkman AB, Hoeijmakers WA, Cohen
A, Lasonder E, Stunnenberg HG. MBD2/NuRD and MBD3/NuRD, two
distinct complexes with different biochemical and functional properties.
Mol Cell Biol 2006; 26:843–851.

16. Manavathi B, Kumar R. Metastasis tumor antigens, an emerging family of
multifaceted master coregulators. J Biol Chem 2007; 282:1529–1533.

17. Kumar R, Wang RA, Bagheri-Yarmand R. Emerging roles of MTA family
members in human cancers. Semin Oncol 2003; 30:30–37.

18. Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS, Wade PA.
MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth
pathway in breast cancer. Cell 2003; 113:207–219.

19. Toh Y, Oki E, Oda S, Tokunaga E, Ohno S, Maehara Y, Nicolson GL,
Sugimachi K. Overexpression of the MTA1 gene in gastrointestinal
carcinomas: correlation with invasion and metastasis. Int J Cancer 1997;74: 459–463.

20. Zhang H, Singh RR, Talukder AH, Kumar R. Metastatic tumor antigen 3
is a direct corepressor of the Wnt4 pathway. Genes Dev 2006; 20:2943–2948.

21. Lu X, Kovalev GI, Chang H, Kallin E, Knudsen G, Xia L, Mishra N, Ruiz
P, Li E, Su L, Zhang Y. Inactivation of NuRD component Mta2 causes
abnormal T cell activation and lupus-like autoimmune disease in mice.
J Biol Chem 2008; 283:13825–13833.

22. Bowen NJ, Fujita N, Kajita M, Wade PA. Mi-2/NuRD: multiple
complexes for many purposes. Biochim Biophys Acta 2004; 1677:52–57.

23. Fujita N, Kajita M, Taysavang P, Wade PA. Hormonal regulation of
metastasis-associated protein 3 transcription in breast cancer cells. Mol
Endocrinol 2004; 18:2937–2949.

24. Wang Y, Zhang H, Chen Y, Sun Y, Yang F, Yu W, Liang J, Sun L, Yang
X, Shi L, Li R, Li Y, et al. LSD1 is a subunit of the NuRD complex and
targets the metastasis programs in breast cancer. Cell 2009; 138:660–672.

25. Yao YL, Yang WM. The metastasis-associated proteins 1 and 2 form
distinct protein complexes with histone deacetylase activity. J Biol Chem
2003; 278:42560–42568.

26. Ideraabdullah FY, Vigneau S, Bartolomei MS. Genomic imprinting
mechanisms in mammals. Mutat Res 2008; 647:77–85.

27. Verona RI, Mann MR, Bartolomei MS. Genomic imprinting: intricacies of
epigenetic regulation in clusters. Annu Rev Cell Dev Biol 2003; 19:237–
259.

28. Bartolomei MS. Genomic imprinting: employing and avoiding epigenetic
processes. Genes Dev 2009; 23:2124–2133.

29. Ma P, Schultz RM. Histone deacetylase 1 (HDAC1) regulates histone
acetylation, development, and gene expression in preimplantation mouse
embryos. Dev Biol 2008; 319:110–120.

30. Greciano PG, Goday C. Methylation of histone H3 at Lys4 differs between
paternal and maternal chromosomes in Sciara ocellaris germline
development. J Cell Sci 2006; 119:4667–4677.

31. Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic
imprinting. Nature 1993; 366:362–365.

32. Yamasaki-Ishizaki Y, Kayashima T, Mapendano CK, Soejima H, Ohta T,
Masuzaki H, Kinoshita A, Urano T, Yoshiura K, Matsumoto N, Ishimaru
T, Mukai T, et al. Role of DNA methylation and histone H3 lysine 27
methylation in tissue-specific imprinting of mouse Grb10. Mol Cell Biol 2007; 27:732–742.

33. Reese KJ, Lin S, Verona RI, Schultz RM, Bartolomei MS. Maintenance of
paternal methylation and repression of the imprinted H19 gene requires
MBD3. PLoS Genet 2007; 3:e137.

34. Temeles GL, Ram PT, Rothstein JL, Schultz RM. Expression patterns of
novel genes during mouse preimplantation embryogenesis. Mol Reprod
Dev 1994; 37:121–129.

35. Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, Bartolomei
MS. Disruption of imprinted gene methylation and expression in cloned
preimplantation stage mouse embryos. Biol Reprod 2003; 69:902–914.

36. Schultz RM, Montgomery RR, Belanoff JR. Regulation of mouse oocyte
meiotic maturation: implication of a decrease in oocyte cAMP and protein
dephosphorylation in commitment to resume meiosis. Dev Biol 1983; 97: 264–273.

37. Whitten WK. Nutrient requirements for the culture of preimplantation
mouse embryo in vitro. Adv Biosci 1971; 6:129–139.

38. Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved
culture medium supports development of random-bred 1-cell mouse
embryos in vitro. J Reprod Fertil 1989; 86:679–688.

39. Ho Y, Wigglesworth K, Eppig JE, Schultz RM. Preimplantation
development of mouse embryos in KSOM: augmentation by amino acids
and analysis of gene expression. Mol Reprod Dev 1995; 41:232–238.

40. Stein P, Svoboda P, Anger M, Schultz RM. RNAi: mammalian oocytes do
it without RNA-dependent RNA polymerase. RNA 2003; 9:187–192.

41. Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM,
Bartolomei MS. Selective loss of imprinting in the placenta following
preimplantation development in culture. Development 2004; 131:3727–3735.

42. Laemmli UK. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 1970; 227:680–685.

43. Tremblay KD, Duran KL, Bartolomei MS. A 50 2-kilobase-pair region of
the imprinted mouse H19 gene exhibits exclusive paternal methylation
throughout development. Mol Cell Biol 1997; 17:4322–4329.

44. Market-Velker BA, Zhang L, Magri LS, Bonvissuto AC, Mann MR. Dual
effects of superovulation: loss of maternal and paternal imprinted
methylation in a dose-dependent manner. Hum Mol Genet 2010; 19:36–51.

45. Rohde C, Zhang Y, Jurkowski TP, Stamerjohanns H, Reinhardt R, Jeltsch
A. Bisulfite sequencing Data Presentation and Compilation (BDPC) web
server–a useful tool for DNA methylation analysis. Nucleic Acids Res 2008; 36:e34.

46. Schultz RM. Regulation of zygotic gene activation in the mouse.
BioEssays 1993; 15:531–538.

47. Pan H, O’Brien MJ, Wigglesworth K, Eppig JJ, Schultz RM. Transcript
profiling during mouse oocyte development and the effect of gonadotropin
priming and development in vitro. Dev Biol 2005; 286:493–506.

48. Liang J, Wan M, Zhang Y, Gu P, Xin H, Jung SY, Qin J, Wong J, Cooney
AJ, Liu D, Songyang Z. Nanog and Oct4 associate with unique
transcriptional repression complexes in embryonic stem cells. Nat Cell
Biol 2008; 10:731–739.

49. Kim J, Kim JD. In vivo YY1 knockdown effects on genomic imprinting.
Hum Mol Genet 2008; 17:391–401.

50. Kim J, Kollhoff A, Bergmann A, Stubbs L. Methylation-sensitive binding
of transcription factor YY1 to an insulator sequence within the paternally
expressed imprinted gene, Peg3. Hum Mol Genet 2003; 12:233–245.

51. Kim JD, Hinz AK, Bergmann A, Huang JM, Ovcharenko I, Stubbs L, Kim
J. Identification of clustered YY1 binding sites in imprinting control
regions. Genome Res 2006; 16:901–911.

52. Kim JD, Yu S, Choo JH, Kim J. Two evolutionarily conserved sequence
elements for Peg3/Usp29 transcription. BMC Mol Biol 2008; 9:108.

53. Zhang Y, Ng HH, Erdjument-Bromage H, Tempst P, Bird A, Reinberg D.
Analysis of the NuRD subunits reveals a histone deacetylase core complex
and a connection with DNA methylation. Genes Dev 1999; 13:1924–1935.

54. Brunmeir R, Lagger S, Seiser C. Histone deacetylase HDAC1/HDAC2-
controlled embryonic development and cell differentiation. Int J Dev Biol
2009; 53:275–289.

55. Kaji K, Caballero IM, MacLeod R, Nichols J, Wilson VA, Hendrich B.
The NuRD component Mbd3 is required for pluripotency of embryonic
stem cells. Nat Cell Biol 2006; 8:285–292.

56. Szeto IY, Barton SC, Keverne EB, Surani AM. Analysis of imprinted
murine Peg3 locus in transgenic mice. Mamm Genome 2004; 15:284–295.

http://www.biolreprod.org/content/83/6/1027/suppl/DC1

Supplemental Figure S1:

Supplemental Figure S2:

Supplemental Figure S1: Immunocytochemical analysis of NuRD components in MTA2-depleted embryos.

Immunocytochemical detection of the NuRD components detected by immunoblotting in Figure 3 was performed. Save for MTA1, which exhibits a reduced nuclear-localized signal (see Figure 2), the NuRD components remained nuclear with no apparent change in the intensity of the signal, which is consistent with the immunoblotting data.

(A) One-cell embryos were injected with either Gfp- or Mta2-dsRNA, and cultured to the blastocyst stage at which time they were processed for immunocytochemical (A) or immunoblot.

(B) analysis for YY1. At least 20 embryos were examined for the immunocytochemical analysis and representative images are shown. The immunoblotting experiment 80 embryos were used and TUBB was used as a loading control.

In this extensive analysis of the molecular overlap between developing embryos and metastatic neoplasms,
Pengpeng Ma, Shu Lin, Marisa Bartolomei , and Richard Schultz have dissected the epigenetic controls of DNA methylation, and have demonstated that the origins of embryonic development rely on genes that are also later important in neoplastic progression,

This convergence between embryonic development and later neoplastic metastases in adults illustrates the importance of embryonic gene activation as an unfavorable factor in adult neoplasms.

Such co-occurrence has now been recognized in the findings of embryonal epithelial-mesenchymal transitions in a wide variety of human adult embryomas, and in the possible use of embryonic ribo-regulators for the therapy of such embryomas.

Additional References:

1. Zhang H, Singh RR, Talukder AH, Kumar R.
"Metastatic tumor antigen 3 is a direct corepressor of the Wnt4 pathway".
Genes Dev 2006; 20:2943–2948.

2. Brunmeir R, Lagger S, Seiser C.
"Histone deacetylase HDAC1/HDAC2-controlled embryonic development and cell differentiation".
Int J Dev Biol 2009; 53:275–289.

3. Gao Y, Schug J, McKenna LB, Lay JL, Kaestner KH, and Greenbaum LE,
"Tissue-specific regulation of mouse MicroRNA genes in endoderm-derived tissues".

4. . Meseguer S, Mudduluru G,  Escamilla JM,  Allgayer H,  and  Barettino D,
"MicroRNAs-10a and -10b Contribute to Retinoic Acid-induced Differentiation of Neuroblastoma Cells and Target the Alternative Splicing Regulatory Factor SFRS1 (SF2/ASF)".
 

1. Each cell retains all of its embryonic genes for a lifetime.

2. Controls for embryonic genes are often absent in adults.

3. Uncontrolled embryonic genes can replicate wildly.

4.  Replicating genes participate in  intra-cellular competition.

5.  The basis for gene competition is selective transcription.

6.  MicroRNAs can reprogram embryomic transcription.

7.  Gene reprogramming can produce normal phenotypes.

8.  Normal phenotypes can by-pass chromosomal lesions.

9.  MicroRNA therapy may need to be permanent.

10. Transplantation of microRNAs could be preferred.

http://www.embryomas.net/

1. Pathways within cell genomes involve a flow of information.

2. Information can flow by direct contact or by third parties.

3. Direct contact within whole genomes is difficult to regulate.

4. DNA-DNA direct contects are influenced by agents.

5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.

6. Third parties within genomes involve RNAs and proteins.

7.  RNAs and proteins are easy to regulate or reverse.

8.  Information can be shared, lost, or transformed.

9. System information can be hidden during system isolation.

10.  Local information can be permanently lost during system entropy.

http://www.cancerbiophysics.net/

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