Gene 272 (2001) 199±208

www.elsevier.com/locate/gene

DNA (cytosine-5) methyltransferase turnover and cellular localization in

developing Paracentrotus lividus sea urchin embryo
Rossella Di Giaimo a, Annamaria Locascio b, Francesco Aniello b, Margherita Branno b,
Rosanna del Gaudio a, Nicoletta Potenza a, Giuseppe Geraci a,*
a
Department of Genetics, General and Molecular Biology, University of Naples Federico II, Via Mezzocannone 8, 80134 Naples, Italy
b
Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica A. Dohrn, Villa Comunale, 80121 Napoli, Italy
Received 4 February 2001; received in revised form 23 April 2001; accepted 16 May 2001

Abstract
The turnover and localization of the enzyme DNA (cytosine-5) methyltransferase (Dnmt1) were studied during Paracentrotus lividus sea
urchin embryo development using antibody preparations against the NH2 and COOH-terminal regions of the molecule. The antibodies reveal,
by Western blots and whole-mount analyses, that the enzyme is differently required during embryonic development. The changeover point is
at blastula stage, where a proteolytic mechanism hydrolyses the enzyme present in all embryonic cells by removing a peptide of about 45 kDa
from the amino terminal region of the 190 kDa enzyme initially synthesized on maternal transcripts. The resulting 145 kDa enzyme shows
modi®ed catalytic properties, different antibody reactivity and is rapidly destroyed in the few hours before gastrulation. At more advanced
stages of development the enzyme is newly synthesized but only in particular cell types, among which neurons. The data show that Dnmt1 is
removed from embryonic cells before gastrulation to be synthesized again at different levels in different cell types, indicating that the
concentration of Dnmt1 is critical for the various differentiated cells of the developing sea urchin embryo. q 2001 Elsevier Science B.V. All
rights reserved.
Keywords: DNA methylation; Dnmt1 immunolocalization; Dnmt1 expression; Dnmt1 in neurons; Embryonic development

1. Introduction enzyme forms more recently identi®ed in mammals

Methylation of eukaryotic DNA at position 5 of cytosines
of CpG dinucleotides is an epigenetic mechanism with
important roles in diverse chromosomal activities such as
control of developmental processes (Monk, 1990), gene
expression (Razin and Cedar, 1991), chromatin packaging
(Adams, 1995), labeling of parasitic sequence elements
(Bestor and Tycko, 1996) and transcriptional silencing
(Walsh and Bestor, 1999). Methylation of DNA is
performed by the family of enzymes DNA methyltrans-
ferases. The best studied and most abundant form of these
is Dnmt1, responsible for the maintenance of methylation
sites in the newly replicated DNA (Leonhardt et al., 1992)
while methylation of previously unmethylated CpGs, indi-
cated as `de novo' activity, is considered performed by
Abbreviations: Dnmt1, DNA methyltransferase 1; Dnmt3a, DNA
methyltransferase 3a; PAGE, polyacrylamide gel electrophoresis; 5mC,
5-methyl-cytosine; SAM, S-adenosyl-methionine
* Corresponding author. Tel.: 139-081-2535191; fax: 139-081-
2535000.
E-mail address: geraci@dgbm.unina.it (G. Geraci).
(Okano et al., 1998; Okano et al., 1999) important for chro-
mosome stability (Xu et al., 1999), tumor growth (Robert-
son et al., 1999) and human genetic disorder (Bestor, 2000).
These different functional roles for the DNA methyltrans-
ferases have found con®rmation in experiments on trans-
genic ¯ies where Dnmt1 was unable to perform `de novo'
methylation, but when co-expressed with Dnmt3a, methyla-
tion patterns were established and maintained ef®ciently in
that organism (Lyko et al., 1999).
Dnmt1, the enzyme involved in genomic imprinting and
chromosome X inactivation (Li et al., 1993), has been
demonstrated to be necessary for embryonic development
but not for cell survival because targeted inactivation of
Dnmt1 in the mouse resulted in lethality for mutant embryos
while had no effects on the viability of embryonic stem cells
(Li et al., 1992). This interesting ®nding in the mouse
provided the ®rst evidence that methylation of DNA is an
absolute requirement for embryonic development. Indeed,
the patterns of DNA methylation change dramatically in the
developing mouse embryo. A generalized demethylation
occurs before the implantation as shown by the ®nding

0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0378-111 9(01)00539-X
200 R. Di Giaimo et al. / Gene 272 (2001) 199±208
that the 5mC content of DNA decreases sharply between the
8-cell and blastocyst stages (Monk, 1990; Chaillet et al.,
1991). Interestingly, in studies of enzyme activity during
mouse embryonic development, DNA methylation and
Dnmt1 activity have been found to be not correlated to
each other because enzyme activity is high when DNA
methylation actually decreases (Monk et al., 1987; Monk,
1990). Also in the zebra®sh uncoupling between level of
DNA methylation and of DNA methyltransferase activity
has been observed (Martin et al., 1999). A clue to under-
stand the reason of the apparent uncorrelation between level
of DNA methylation and of DNA methyltransferase activity
was provided by the evidence that enzyme localization in
the cell is regulated during mouse oocyte and embryonic
development (Carlson et al., 1992). Similarly, in X. laevis,
Dnmt1 is initially localized in the nuclei during oocyte
maturation then its presence is con®ned to germinal vesi-
cles. After fertilization, in the 1±2-cell embryo, it is loca-
lized in the cell cytoplasm, near the plasma membrane, and
only after the 8-cell stage it is translocated in the nuclei
(Kimura et al., 1999). It has been shown also that a regula-
tory domain is present in the NH2-terminal region of
Dnmt1, that is necessary and suf®cient to retain the enzyme
in the cytoplasm during mouse early development (Cardoso
and Leonhardt, 1999).
Methylation of DNA CpG cytosine in the sea urchin
embryo Paracentrotus lividus was initially reported by
Scarano (Scarano et al., 1965). The relevance of that modi-
®cation for proper embryonic development of that organism
was later documented (Branno et al., 1993). It was shown
that interference with DNA methylation altered the normal
embryonic development, but only if perturbation occurred
in one of the initial four cell division periods. Perturbation
of embryos had no effect on proper embryonic morphology
and proper expression of some regulated genes at 32 cell or
at later stages of development. Similar results have been
reported for the zebra®sh embryo where morphological
defects were reported to occur only if DNA methylation
was perturbed in the period centered around 2±3 h after
fertilization, just prior to midblastula transition. The in
situ hybridization experiments showed that while the mater-
nal transcripts for zebra®sh Dnmt1 are ubiquitously present
at high levels in the early embryo, the enzyme concentration
decreases after blastula stage; in the more advanced embryo
it is found predominantly in particular cell structures
(Martin et al., 1999). More recently, it has been reported
that in X. laevis embryos Dnmt1 contributes to chromatin
transcription silencing until midblastula transition when
zygotic transcription begins. Depletion of Dnmt1 by anti-
sense RNA during cleavage stages causes early expression
of genes leading to body plan defects (Stancheva and
Meehan, 2000). In the sea urchin, the mRNA for DNA
methyltransferase was cloned and sequenced and the mole-
cule was found to code for an enzyme form highly homo-
logous to Dnmt1 (Aniello et al., 1996). In this embryo,
uncorrelation between Dnmt1 activity and the correspond-
ing enzyme mRNA was observed during initial develop-
ment. Enzyme activity shows a peak value at the blastula
stage after which, in the few hours before gastrulation, it
decreases to a very low level that remains nearly constant at
prism and pluteus stages (Tosi et al., 1988). In contrast, the
concentration of sea urchin Dnmt1 mRNA is highest at the
four and eight-cell stages, as typical of maternal transcripts,
and at the blastula stage, where enzymatic activity is maxi-
mal, the mRNA could not be detected (Aniello et al., 1996).
It appeared that some post-translational mechanism had to
be operative to produce the uncoupling between the peak
value of mRNA concentration and that of enzyme activity.
For these reasons, it was decided to investigate on the
enzyme turnover and localization during embryonic devel-
opment, using immunolocalization and enzymatic analyses.
To this aim the NH2-terminal and the COOH-terminal
regions of sea urchin Dnmt1 were expressed in vitro and
polyclonal antibodies against those two protein regions
were prepared in the rabbit.
The results of these studies, presented in this paper, add
the new evidence that the enzyme concentration is regulated
by two independent mechanisms, a negative one eliminat-
ing, at blastula stage, the enzyme synthesized on maternal
transcripts present in all embryonic cells and a positive one,
operative at more advanced stages of development, that
differentially controls the level of enzyme activity in the
different cell types. A hypothesis is presented on the rele-
vance of DNA methylation for the proper development of
embryonic cells at different stages of determination and
differentiation.
2. Materials and methods
2.1. Collection of eggs, embryo culture and enzyme
extraction
Paracentrotus lividus sea urchins were collected in the
bay of Naples by the Fishery Service of the Zoological
Station of Naples. Collection of gonads and preparation of
embryos at different stages of development were as
described (Branno et al., 1983). Total and nuclear protein
extracts were prepared from P. lividus eggs and embryos at
different stages of development as reported (Tomlinson et
al., 1990). Brie¯y, eggs, embryos or puri®ed nuclei (Branno
et al., 1983) were collected by centrifugation, the pellets
were suspended in ten volumes of 100 mM KCl, 15 mM
HEPES pH 7.9, 3 mM MgCl2, 1 mM DTT, 0.1 mM EDTA,
0.1 mM PMSF and sonicated. Ammonium sulfate at 0.36 M
®nal concentration was added to the resulting homogenate,
the sample was left for 30 min at 48C under stirring and
centrifuged at 35,000 rev./min in Ti60 Beckman rotor for
30 min at 48C. The supernatant was recovered and ammo-
nium sulfate (0.25 g/ml) was added and left for 1 h at 48C
under stirring. The pellet was recovered by centrifugation at
18,000 rev./min in SS34 Sorval rotor for 30 min at 48C and
 R. Di Giaimo et al. / Gene 272 (2001) 199±208
solubilized in 100 mM KCl, 20 mM HEPES pH 7.9, 20%
glycerol, 2 mM MgCl2, 1 mM DTT, 0.2 mM EDTA, 0.5 mM
PMSF buffer. Soluble proteins were dialyzed against the
same solubilization buffer until ammonium sulfate was
removed. Dialyzed extracts were cleared by centrifugation
and stored in aliquots at 2808C. Protein concentration was
determined by the BioRad Protein Assay.
2.2. Determination of enzyme activity and substrate DNA
speci®city
The enzymatic activity of Dnmt1 was determined by
measuring the incorporation of 3H from S-adenosyl[-
methyl- 3H]- methionine (SAM) (Amersham, 15 Ci/
mmole) into DNA-5mC as described (Tosi et al., 1988).
Substrate ss and ds Micrococcus luteus DNAs were puri®ed
as described (Tosi et al., 1988), poly (dIdC) and poly
(dCdG) were from Sigma. Each data point for activity
measurement is the average of two independent determina-
tions with values differing less than 10%. The activity
values of the immunoprecipitated samples were determined
(Tosi et al., 1988) by suspending each sample, prepared as
described in Section 2.4, in 50 ml of solubilization buffer,
using 10 ml for each test, corresponding to 1/5 of the amount
prepared in each batch experiment.
2.3. Preparation of polyclonal antibodies
Polyclonal antibodies against the NH2 and COOH-term-
inal regions of P. lividus Dnmt1 were prepared as already
reported for the identi®cation of Chaetopterus variopedatus
DNA methyltransferase (del Gaudio et al., 1999). In brief,
both peptides were prepared in vitro as fusion proteins
downstream Maltose Binding Protein in the plasmid
pMAL-c2 (Biolabs, New England). The protein construct
containing amino acid residues 1±100 of Dnmt1, was
produced as a soluble component in the bacterial cell extract
and was puri®ed by af®nity chromatography on an amylose
column according to the manufacturer's instructions. The
fusion product with amino acid residues 1073±1612 of
Dnmt1 was present in inclusion bodies that were solubilized
in SDS from which it was isolated by electroelution after
SDS PAGE fractionation. Three aliquots of 400 mg of each
fusion protein in 500 ml of saline solution were mixed with
an equal volume of complete Freund's adjuvant and injected
at 15 days interval in rabbits to promote antibody production
following standard procedures (Sambrook et al., 1989).
Antibodies were isolated from the antisera of immunized
rabbits by precipitation with ammonium sulfate at 33%
saturation followed by DEAE column chromatography of
the precipitated fraction.
2.4. Western blot analysis and immunoprecipitation of the
enzyme
Protein samples prepared from eggs and embryos at the
reported stages of development, as described in Section 2.1,
201
were fractionated by SDS PAGE using 7.5% acrylamide
concentration and acrylamide-bisacrylamide ratio 19:1 to
improve the separation of high molecular weight compo-
nents, as reported (del Gaudio et al., 1999). The protein
molecular weight markers were from FMC (Rockland,
ME, USA). After electrophoretic fractionation on acryla-
mide gel, the proteins were electrophoretically transferred
to a nitrocellulose membrane (Sartorius). Blots were stained
with Ponceau Red to assess the protein content on the ®lters
and con®rm equal loading and transferring in the different
lanes. The immunoenzymatic procedure to localize Dnmt1
on the ®lters was performed using the polyclonal antibodies
against the NH2-terminal or COOH-terminal regions of the
enzyme, as indicated, and goat anti-rabbit secondary anti-
bodies conjugated with horseradish peroxidase (Sigma±
Aldrich).
Dnmt1 was immunoprecipitated as reported (del Gaudio
et al., 1999). In short, 4 mg of Staphylococcus aureus
protein A-Sepharose CL-4 beads (Pharmacia), were
suspended and equilibrated in 1 ml of 100 mM Tris pH
7.5, 10 mM EDTA, 0.5 mM PMSF, 10 mM DTT. The
swollen beads were sedimented and the pellet was mixed
with 35 mg of the appropriate polyclonal antibody. Two-
hundred mg of proteins of the dialyzed ammonium sulfate
fraction of four-blastomere embryos were added and the
volume was brought to 300 ml with the equilibration buffer.
The suspension was kept under gentle stirring for 2 h at 48C
and the beads were separated with a 20 sec centrifugation
pulse at 48C in an Eppendorf microfuge. The immunode-
pleted supernatant was saved for Western blot analysis of
the unbound protein and to test for the presence of enzy-
matic activity. The pellet of sedimented beads was washed
two times by centrifugation with 300 ml of equilibration
buffer, suspended in 50 ml of the same buffer and divided
into 10 ml aliquots that were used directly for enzymatic
assays or suspended in SDS buffer to analyze by SDS
PAGE and Western blot the proteins dissociated from the
protein A-Sepharose CL-4 beads.
2.5. Immunolocalization of Dnmt1 in whole mount embryos
Eggs and embryos were collected by sedimentation and
®xed for a period of time between 30 min and 2 h in 3.7%
formaldehyde in 0.8 M glucose, 0.1 M KCl, 2 mM MgCl2, 5
mM CaCl2, 19 mM MOPS, pH 6.7 at room temperature. The
®xed embryos were washed very gently three times with the
same buffer and once with 1 M glycine. They were then
permeabilized treating for 5±10 min in 0.1% Triton X-100
in saline solution and washed once with 0.1% BSA in saline
solution. The samples were incubated overnight with differ-
ent dilutions of anti-Dnmt1 NH2-terminal antibodies
washed twice for 5 min with 0.1% Triton X-100, 0.1%
BSA in saline solution and incubated for 1 h with ¯uores-
cein-conjugated goat af®nity puri®ed antibodies to rabbit
IGG (Cappel) diluted from a minimum of 1/100 to a maxi-
mum of 1/500 in 0.1% BSA in saline solution, depending on
202 R. Di Giaimo et al. / Gene 272 (2001) 199±208
the sample, as indicated in each particular experiment. After
washing with 0.1% BSA in saline solution, the embryos
were examined with a Zeiss Axiophot microscope equipped
with Nomarsky and epi¯uorescence optics, using 20 £ plan
apochromat and plan-neo¯uor lenses. Pictures were taken
with Kodak Ektachrome 320 T ®lm. For Dnmt1 immunolo-
calization in embryos at 2 and 4 blastomere stages, it was
necessary to remove the fertilization membrane. To this
aim, immediately after fertilization, eggs were treated
with 1% Sodium thioglycolate and 0.05% Protease E pH 9
in sea water for 2±3 min and washed several times in sea
water, pipetting up and down to completely remove the
fertilization membranes.
3. Results
3.1. Speci®city of antibody preparations and enzyme
immunoprecipitation
The speci®city of the anti-NH2-terminal antibody
preparation against Dnmt1 was studied using the enzyme
present in the protein extract of four-blastomere embryos
(Fig. 1). As apparent from Western blot of the embryonic
protein extract, of the supernatant of the immunoprecipi-
tated sample (immunodepleted preparation) and of the
Fig. 1. Western blot analysis of the proteins immunoprecipitated from the
extract of four-blastomere sea urchin embryos. SDS gel electrophoresis in
7.5% acrylamide. PR, Ponceau Red staining of the ®lter after electroblot-
ting of the fractionated proteins. W, same ®lter after immunoreaction with
anti-Dnmt1-NH2-terminal antibodies. Twenty-®ve mg of proteins in each
sample. Lane 1, initial sample; lane 2, proteins present in the supernatant
after the immunoprecipitation step; lane 3, proteins eluted from the immu-
noprecipitated pellet. For details see text.
proteins that are dissociated from the immunoprecipitated
complex (Fig. 1, PR and W, lanes 1, 2 and 3, respectively),
the antibody preparation interacts and removes from the
sample solution only a protein component of about 190
kDa, that corresponds to the molecular weight of the sea
urchin enzyme as derived from the cDNA analysis (Aniello
et al., 1996). In fact, lanes 1 and 2 show identical electro-
phoretic protein compositions by Ponceau-red staining
while Western blot analysis shows an immunoreactive
component in the untreated sample, lane 1, but not in the
immunodepleted solution, lane 2. The immunoreactive
component is present in lane 3, where the molecules eluted
from the immunoprecipitated pellet are analyzed. The
enzyme bound to this antibody preparation in the Sephar-
ose-proteinA immunoprecipitate, is still enzymatically
active, as expected if the catalytic domain is in the
COOH-terminal region of the molecule (Bestor et al.,
1988; Aniello et al., 1996). The results of determinations
of enzyme activity on the different fractions of the immu-
noprecipitation experiment (Table 1) further con®rm the
correlation between the 190 kDa immunoreactive compo-
nent and the Dnmt1 enzyme. In fact, DNA methyltransfer-
ase activity is present in the initial sample, it is not present in
the immunodepleted solution and is present in the immuno-
precipitated pellet. These results are in line with the notion
that the catalytic activity is present in the COOH-terminal
domain of the molecule, and is not affected by the binding of
antibodies to the NH2-terminal domain of the protein.
3.2. Immunodetection of Dnmt1 in the developing sea urchin
embryo
Protein extracts, prepared from unfertilized eggs, from
embryos at the four-blastomere, at morula, blastula,
gastrula, prism and pluteus stages and from blastula nuclei,
were fractionated by SDS gel electrophoresis and assayed
by Western blot with antibodies anti NH2 or anti COOH-
terminal domain of P. lividus Dnmt1. The results of a typi-
cal set of experiments (Fig. 2) show the time course of
immunodetected enzyme molecules at the different stages
of embryonic development. Both antibody preparations
interact in all samples with a protein band, with a mobility
corresponding to about 190 kDa (Fig. 2A,B, Panels W), as
already observed in the experiment on the protein extract
Table 1
Identi®cation of Dnmt1 activity in immunoprecipitated complex a
Sample Activity (pmole/mgDNA/h)
Ammonium sulfate fraction 150
Immunodepleted supernatant 0
Immunoprecipitated complex 120
a
The ammonium sulfate fractions from sea urchin embryos at four-blas-
tomere stage was immuprecipitated with antibodies anti-NH2-terminal
region of Dnmt1 bound to protein A-SepharoseCL-4 beads. The Dnmt1
activity was determined using M. luteus ss DNA as substrate. For details
see text.
 R. Di Giaimo et al. / Gene 272 (2001) 199±208 203

Fig. 2. Western blot analysis of 25 mg protein extracts of sea urchin egg and embryos at different stages of development. SDS gel electrophoresis in 7.5%
acrylamide. Panel A. PR, Ponceau Red staining of the ®lter after protein electroblotting; W, the same ®lter after immunoreaction with anti-Dnmt1-NH2-
terminal antibodies. Panel B. Same as Panel A, but proteins are solubilized in 8 M urea before addition of the SDS electrophoretic buffer and the immunor-
eaction is performed with anti-Dnmt1-COOH-terminal antibodies. U, unfertilized eggs. 4b, four blastomere embryo; MT, morula; BT, blastula; BN, blastula
nuclei; GT, gastrula; Pr, prism; Pl, pluteus. Panel C, electrophoresis of BT and BN samples in 12.5% acrylamide, to show the occurrence of a 45 kDa
component immunoreactive with anti-Dnmt1-NH2-terminal antibodies not present at the other stages of development.

from four-blastomere embryos (Fig. 1). Some additional
minor components are present in the lower range of mole-
cular weight when anti-NH2-terminal antibodies are used
(Fig. 2A, Panel W). These are similar to those observed in
the eluted fraction from the immunoprecipitated pellet (Fig.
1, Panel W, lane 3) and probably correspond to shorter
enzymatic forms missing the carboxy terminal region,
since they do not react with anti-COOH-terminal antibodies
(Fig. 2B, Panel W), as expected for unterminated translation
products. Equal amounts of proteins were loaded in each
lane for the electrophoretic separation and Ponceau-red
staining (Fig. 2A,B, Panels PR), indicated that transfer of
molecules had occurred regularly. The intensity of the
immunoreacted band decreases from that of egg and 4 blas-
tomere embryos to a minimum value at blastula stage. The
190 kDa component is not observed in the blastula nuclei
and is present again, although at lower intensity, in the
extracts of gastrula, prism and plutei (Fig. 2A, Panel W).
This electrophoretic analysis was performed at 7.5% acry-
lamide concentration to improve the mobility and separation
of high molecular weight proteins. For this reason, in order
to show a new component of low molecular weight, detected
by anti-NH2-terminal antibodies in the nuclear fraction of
the sea urchin blastula, the SDS electrophoretic analysis for
this sample was performed also at 12.5% acrylamide
concentration (Fig. 2C). It is apparent that, in the protein
extract of total blastula, a high molecular weight component
is visible in the blot immunoreacted with anti-NH2-terminal
antibodies (Fig. 2C, Panel W, lane BT), corresponding to
the band observed at 190 kDa in the 7.5% acrylamide elec-
trophoresis while no immunoreactive component is appar-
ent in the lower molecular weight region. At a variance, in
204 R. Di Giaimo et al. / Gene 272 (2001) 199±208
Fig. 3. Enzymatic activity of the two different forms of the Dnmt1. Ammo-
nium sulfate fractions from four blastomere embryos and from blastula
nuclei were used in activity assay with different DNA substrates. Activities
determined using ds M. luteus DNA, poly(dIdC) and poly(dCdG). Results
have been normalized with respect to ss DNA. For details, see Section 3.
Activity from four-blastomere embryos (striped box) and from blastula
nuclei (dotted box) on the different substrate DNAs. The speci®c activity
of the enzyme from 4 blastomere embryos and that of the enzyme from
blastula nuclei were, respectively, 6 and 15 pmole/50 mg DNA/mg prot./h.
the extract of blastula nuclei anti NH2-terminal antibodies
interact with a component at a position corresponding to
about 45 kDa while no reaction is apparent in the high
molecular weight region (Fig. 2C, Panel W, lane BN).
Anti-COOH terminal antibodies, on the same sample,
show a new immunoreactive band in the high molecular
weight region, corresponding to about 145 kDa (Fig. 2B,
Panel W, lane BN). It should be noted that, when proteins
are solubilized in 8 M urea, as is the case of the experiments
reported in Fig. 2, immunoreaction of anti-COOH terminal
antibodies reveals, in addition to the 190 kDa component, a
145 kDa protein band not revealed by the anti N-terminal
antibodies. The 145 kDa band is the main component in the
blastula nuclei sample. In total blastula sample the main
component is a 170 kDa band with two minor ones at 190
kDa and 145 kDa, respectively. This indicates that the cata-
lytic carboxy terminal region of the 190 kDa protein has no
exposed epitopes for anti-COOH-terminal antibody
preparation when solubilized in the presence of SDS and
that the epitopes become available after solubilization in 8
M urea. Comparative analysis of the intensity of immunor-
eacted bands with the two different antibody preparations
(compare Fig. 2A,B, Panels W) show that immunoreaction
is less intense with anti-COOH terminal antibodies that
require previous solubilization in 8 M urea. In conclusion,
Western blot analyses using both antibody preparations
show only the presence of the 190 kDa enzyme form in
all embryonic samples except blastula nuclei where the
presence of a 145 kDa enzyme form, alternative to the
190 kDa enzyme, is observed. This molecule is very likely
produced by modi®cation of the 190 kDa enzyme rather
than by translation of a new message. Indeed, the 190 kDa
component is no more present while a new 45 kDa protein
component is reactive to anti-NH2-terminal antibodies. In
addition, previous data have demonstrated that no Dnmt1
mRNA is apparent by Northern blot analysis in embryos at
the blastula stage (Aniello et al., 1996).
3.3. Analysis of the DNA substrate speci®city of the 190 kDa
and of the 145 kDa enzyme forms
Western blots of protein extracts of embryos at the inves-
tigated stages of development (Fig. 2) show that the enzyme
has a molecular weight of 190 kDa except in the blastula
nuclei where that component is not observed and two differ-
ent bands are apparent, a 145 kDa component directly
immunoreactive with anti-COOH-terminal antibodies and
a 45 kDa component, immunoreactive with anti-NH2-term-
inal antibodies. The data show also that the immunoreactive
component decreases in the intensity in the extracts from the
level observed in the embryo at the four-blastomere to that
at the blastula stage, in contrast with the maximum value of
enzymatic activity observed at the blastula stage (Tosi et al.,
1995). For these reasons it was considered of interest to
compare the catalytic properties of the enzyme of embryos
at the four-blastomere stage with that of the enzyme of
blastula nuclei. To this aim, extracts were prepared from
these two samples and the methyltransferase activity was
determined using as substrates ds and ss M. luteus DNA,
poly(dIdC) and poly(dCdG). The results of these analyses,
performed on equivalent amounts of proteins (Fig. 3), show
that indeed the enzyme from blastula nuclei and that from
four-blastomere embryos show different activity for the
different types of substrate DNA. In the experiments of
Fig. 3 the speci®c activity of the enzyme from blastula
nuclei on ss M. luteus DNA is about three fold higher
than that of the enzyme from four-blastomere embryos.
To better compare the activity of the two enzyme forms
on the different substrates, the incorporation values were
normalized with respect to those obtained on ss DNA. It is
apparent that the ratio of incorporated methyl groups per
unit time between ssDNA and dsDNA, is about 3.7 for the
enzyme from blastula while it is 1.3 for the enzyme from
four-blastomere embryos. The differences between the two
enzyme forms are even more evident using poly(dIdC) and
poly(dCdG) as substrates. The rate of methylation, using
poly(dIdC) as substrate, is about ®ve times higher for the
enzyme from blastula nuclei than from the four-blastomere
embryos. When poly(dCdG) is the substrate, the value of the
relative rates of enzymatic methylation are inverted. Taken
together, these results seem to indicate that the four-blasto-
mere enzyme is a bona ®de `de novo activity' since it
methylates with similar ef®ciency ss DNA, ds DNA and
poly(dCdG) while has only about 20% activity on poly(-
dIdC), that mimics hemimethylated DNA. The removal of
the N-terminal region of the enzyme, as occurs for the 145
kDa form, gives rise to a molecule that methylates with
higher ef®ciency ss DNA than ds DNA and poly(dCdG)
and has even higher activity on hemimethylated DNA, as
typical of enzymes with maintenance activity. It remains to
be established if the programmed degradation of Dnmt1 in
 R. Di Giaimo et al. / Gene 272 (2001) 199±208 205

P. lividus sea urchin embryos, giving rise to a transient form
of the enzyme endowed of peculiar speci®c activity, has a
functional role in development, performing `de novo' and
maintenance activity at different embryonic stages, after
proper molecular modi®cation.
3.4. Immunolocalization of the enzyme in the whole-mount
embryo
The presence of the enzyme in the cells of sea urchin
embryos at different stages of development was studied by
whole-mount immunolocalization on ®xed specimens. The
enzyme was studied also in the oocyte and in the mature egg
because the enzymatic activity was clearly present in those
cells (Tosi et al., 1995). In the oocyte, (Fig. 4A) the enzyme
is present almost exclusively in the nucleus. In the mature
egg (Fig. 4B), immunoreaction is clearly evident both in the
nucleus and in the cytoplasm of the cell. Immediately after
fertilization, the immuno¯uorescence is present essentially
in the nuclei, and this location is maintained until blastula
stage (Fig. 4C,D). At gastrula stage, immunoreaction
becomes dif®cult to be detected. The concentration of anti-
body solution had to be increased ®ve-fold in order to
observe immuno¯uorescence. In these conditions (Fig.
4E,F), there is a general low level of ¯uorescence in all
cells, with the exception of a group on the tip of the arch-
enteron, corresponding to secondary mesenchyme cells
where the ¯uorescence intensity is appreciably higher
(Fig. 4E,F, arrow sm). This overall decrease of immunor-
eactivity is in agreement with the decrease of enzyme activ-
ity reported to occur at gastrula stage (Tosi et al., 1995) and
with the decrease of the immunoreactive component in the
protein extracts of gastrula stage (Fig. 2A,B, Panels W,
lanes GT). At more advanced stages of development, immu-
noreactivity is observed only in particular cell types and the
antibody concentration required to observe immuno¯uores-
cence in those cells is less than two times that used at the
blastula stage, indicating the presence of an increased
enzyme concentration. At pluteus stage (Fig. 4H±L) the
differences in enzyme concentration in the different cells
are evident and at least three levels of ¯uorescence intensity
can be distinguished. Cells that are non ¯uorescent, cells
with a low level and cells with a particularly high level of
¯uorescence intensity. Plutei in different orientations are
presented in panels H±K (Fig. 4). Inspection of the plutei
shows that the ¯uorescence intensity is high in the cells that

Fig. 4. Immunolocalization of Dnmt1 in whole mount sea urchin embryos at different stages of development. (A) oocyte; (B) fertilized egg; (C) two blastomere
embryo; (D) blastula; (E,F) gastrula; (G) embryo at the pluteus stage, observed with Nomarsky optics; H±L, pluteus embryos in different orientations. Arrows
indicate cells of: sm, secondary mesenchyme; e, epaulette; cp, coelomic pouches; n, apical plate neurons. Samples immunoreacted with anti-Dnmt1-NH2-
terminal antibodies with the following dilutions: A±D, 1/500; E,F, 1/100; G±L, 1/300. Secondary labeling, with goat anti-rabbit IGG antibodies, conjugated
with ¯uorescein isothiocyanate. Photographs taken with a Zeiss Axiophot ¯uorescence microscope using 20 £ lens.
206 R. Di Giaimo et al. / Gene 272 (2001) 199±208
are descendants of the secondary mesenchyme cells such as
the cells of the two coelomic pouches (cp arrows in Fig. 4H±
J) and lower, but still clear, in cells of the digestive appa-
ratus detailing the contour of the digestive tract. Immuno-
¯uorescence is high in cells of the marginal lateral
ectoderm, particularly in those of the epaulettes between
the postoral and anterolateral arms of the pluteus (e arrows
Fig. 4H±J), with intensity decreasing towards the arm tips.
Immunoreaction is evident also in neurons, as shown by the
twelve cells clearly evident in the apical plate of the pluteus
(n arrow Fig. 4L), corresponding to the serotonergic neuro-
blasts located in that position and in the cells that are distrib-
uted in a regular array along the embryo (Fig. 4H,I,K). No
¯uorescence is appreciable in cells of the aboral ectoderm,
of oral ectoderm or of other regions of the embryo. An
immunoreacted sea urchin pluteus, observed under
Nomarsky optics, is shown as a reference embryo (Fig. 4G).
4. Discussion
In the study reported here we have followed the time
course of Dnmt1 in the developing P. lividus sea urchin
embryos, analyzing the enzyme size, its catalytic properties
and the relative concentration at the different stages and in
the different cell types. There is a good correlation between
the reported time course of Dnmt1 mRNA concentration in
the developing sea urchin embryo (Aniello et al., 1996) and
the relative signal intensity of the protein identi®ed by
Western blot analysis in the electrophoretic patterns of
proteins extracted from embryos at increasing stages of
development (Fig. 2). Reaction with antibodies against the
NH2-terminal region of Dnmt1 shows that the 190 kDa
enzyme molecule synthesized on maternal transcripts
decreases in relative amount during development, is modi-
®ed in a peculiar manner at blastula stage and is almost
completely removed from the cells in the few hours before
gastrulation. At more advanced stages of development a
new enzyme activity appears in some speci®c cell types.
This new enzyme is synthesized on embryonic transcripts,
as revealed by the time course of the Dnmt1 mRNA present
in the embryo at different stages of development (Aniello et
al., 1996). It results that the enzyme is removed from the
cells even if it is required again at later stages indicating that
Dnmt1 cannot be tolerated when not necessary so that the
embryo has to remove it from the cells to eventually
produce exactly the amounts required by each cell type. It
is interesting that DNA methylation in transgenic D. mela-
nogaster expressing Dnmt1 and Dnmt3a, causes lethality at
pupal stages or altered phenotype in the viable individuals
(Lyko et al., 1999).
The 145 kDa enzyme form present in the nuclei at blas-
tula stage appears to be a transient intermediate molecule in
the elimination of the 190 kDa enzyme from the embryonic
cells before gastrula stage. In fact, it appears produced by
removal of a peptide of about 45 kDa from the N-terminal
region of the 190 kDa enzyme, as revealed by the ®nding of
a 45 kDa component that interacts with anti-NH2-terminal
antibodies that do not reveal anymore the 190 kDa form in
samples from blastula nuclei. In agreement with this inter-
pretation, the results of Northern blot of the time course of
Dnmt1 mRNA concentration at different stages of develop-
ment show that Dnmt1 mRNA is undetectable at blastula
while it is again present at gastrula and at later stages of
development as a new transcript (Aniello et al., 1996). It is
interesting that the 145 kDa enzyme form, missing the
amino terminal region of the 190 kDa enzyme, is about
four fold more active on ss M. luteus DNA providing a
rationale for the apparent increase of enzyme activity
observed in the blastula nuclei when the protein concentra-
tion is actually decreased. In addition, also the antibody
reactivity of the 145 kDa enzyme is changed. In fact,
while the COOH-terminal domain of the 190 kDa enzyme
is not reactive with antibodies anti COOH-terminal region,
unless the molecule is previously dissolved in 8 M urea, it is
directly reactive in the 145 kDa form. It appears that the
NH2-terminal region of the enzyme has structuring effects
on the molecule as already proposed for the mouse enzyme
(Cardoso and Leonhardt, 1999).
At gastrula stage immunoreaction with antibodies shows
a minimum ¯uorescence in all cells except those of the
secondary mesenchyme on the top of the archenteron, that
show a higher value. It seems that this condition of higher
expression is conserved in the progeny of those cells since,
among cells showing a high enzyme concentration at
pluteus stage, are those of the two lateral coelomic pouches
that directly derive from the secondary mesenchyme cells.
In addition to these, also the cells of the epaulettes, those of
the lateral marginal ectoderm and cells corresponding to
neurons, particularly those of the apical plate, show high
¯uorescence labeling. The high enzyme concentration in
neurons is in line with the results in the mice (Monk et
al., 1991; Carlson et al., 1992; Inano et al., 2000) and in
the zebra®sh (Martin et al., 1999). In mice the highest
expression of Dnmt1 is observed in neural tissues and in
the adult brain, and this has been attributed to the DNA
repair activity existing in those cells. In the zebra®sh, the
highest expression is again observed in neural tissues and in
developing somites.
The published data on Northern blot analyses on mRNA
(Aniello et al., 1996) and those here reported on the time
course of the protein molecule at increasing stages of devel-
opment (Fig. 2) together with its immunolocalization in
whole-mount embryos (Fig. 4), strongly suggest that
Dnmt1 present in the initial and in later stages of develop-
ment are produced on different mRNAs. In fact, it has
already been shown that in the sea urchin, several enzymes
appear synthesized on maternal transcripts between fertili-
zation and blastula stage. This is indicated by the insensi-
tivity of their production to Actinomycin-D inhibition until
blastula stage while at later stages of development the
enzyme activity is decreased by that drug indicating that
 R. Di Giaimo et al. / Gene 272 (2001) 199±208
the translation of the molecule is now dependent on newly
synthesized zygotic transcripts (Giudice, 1986). The differ-
ent levels of Dnmt1 in the different cells of the sea urchin
embryo at different stages of development, clearly apparent
in the data reported here, provide a rationale to the different
effects that were caused by perturbation of DNA methyla-
tion in the developing sea urchin embryo (Branno et al.,
1993). The proper embryonic development could be
deranged only when DNA methylation was perturbed in
one of the four initial cell division periods, with speci®c,
reproducible and different effects in each of them. After the
fourth cell division DNA perturbation could not affect any
more the proper embryo development. Recently, similar
results have been reported to occur when DNA methylation
is perturbed during zebra®sh embryo development. In this
case the period of sensitivity is centered around 2±3 h after
fertilization, just prior to midblastula transition (Martin et
al., 1999). In both cases perturbation of DNA methylation
may be or not effective on embryonic development, strictly
depending on the time at which it is performed.
The different Dnmt1 levels present in the various cell
types of the sea urchin embryo at pluteus stage suggest
that DNA methylation may not be equally relevant for
further differentiation for cells at different stages of devel-
opment. It appears that DNA methylation is critical to ®nal
destiny only for cells in which the process of reorganization
of the chromatin is undergoing. Once this has occurred, and
the required chromatin organization has been reached, the
cell might be no more sensitive to perturbation of DNA
methylation because other mechanisms, such as those of
DNA repair, might be involved in maintaining the clonal
memory of the cell.
Acknowledgements
The work was supported in part by Italian MURST and in
part by CNR contract No. 98.1108.CT14; No.
99.2490.CT04; No. 99.1238.CT14.
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