 |
INTRODUCTION |
Cytosine methylation is, in eukaryotic nuclear DNA, a well
established epigenetic mechanism that controls the expression of housekeeping and possibly also tissue-specific genes (1-3), as well as
several important cellular functions such as X chromosome inactivation
and genomic imprinting (4-6), mutagenesis and tumorigenesis (7-9),
senescence, and virus latency (10-12).
Developmental changes in the methylation pattern are particularly
evident (13-16). In fact, during early embryogenesis, the original
gamete methylation pattern is erased, and most of the DNA in the
blastocyst becomes demethylated. After implantation, a de
novo methylation activity produces in the gastrula a methylation pattern characteristic of the adult animal. During the subsequent development, tissue-specific genes undergo specific demethylation events required for their transcriptional activation, according to the
general paradigm of an inverse correlation between DNA methylation and
gene expression. Knock-out experiments have highlighted the lethality
of even modest abnormal methylation patterns in the embryo (17). There
are, in addition, several lines of evidence indicating that endogenous
genes can be activated by demethylating agents and that exogenous
methylated genes are not expressed when transfected into cells but that
their expression is re-activated by demethylating agents (18-21).
Despite many years of intense studies on DNA methylation, neither the
mechanism that regulates this process nor its exact functional role in
the activation of genes has been fully clarified. Two steps need to be
considered as follows: 1) the creation of a methylation pattern in the
DNA control region of a gene, and 2) the effect of this methylation
pattern on the activity of that gene. As regards the latter step, new
light on the role of DNA methylation in gene transcription has been
shed by the three-way connection between DNA methylation, chromatin
structure, and gene activity (22), resulting from the
methyl-CpG-binding proteins and a repression multiprotein complex that
includes histone deacetylases HDAC1 and HDAC2 (23-27); a pivotal role
of methylcytosine as primary modification in establishing and
maintaining several genes in an inactive state has thus been
demonstrated. The former step is somewhat more complex. Two main
mechanisms have been proposed for the creation and maintenance of
methylation patterns. There is, on the one hand, a methylating activity
based on DNA methyltransferase enzymes that can methylate the cytosine
either in a process of reproduction of previously established
methylation patterns (maintenance methylation) or in a process of
creation of new methylation patterns (de novo methylation)
(28-30). Some distinction between several DNA methyltransferases
specific for either maintenance or de novo methylation has
emerged (31, 32). In all these methylation reactions,
S-adenosylmethionine
(AdoMet)1 has a pivotal role
as a methyl donor (33). Hypomethylation can thus arise from the absence
of methylation activity after DNA replication (passive demethylation).
On the other hand, some experimental results point, as an alternative
pathway, to some active shaping of methylation patterns and in
particular to active mechanisms of removal of methyl moiety (active
demethylation) that do not require DNA replication. So far three kinds
of such mechanisms have been proposed as follows: 1) a
dinucleotide-exchange reaction (34); 2) a glycosylase-based mechanism
that removes the 5-methylcytosine moiety, the overall demethylation
process then being completed through the involvement of mismatch repair enzymes (35); and 3) the direct removal of the methyl group from
5-methylcytosine residues in DNA, by a reaction catalyzed by a real DNA
demethylase (36), which unlike the other mechanisms would be reversible
(37).
Despite this variety of demethylating pathways, a number of aspects are
still somewhat obscure. In fact, it is not clear whether these
mechanisms, taken together or individually, can mediate not only a
stable repression of genes that have to be silenced permanently but
also a transient repression of other genes that need to be switched on
or off in response to variations in physiological conditions
(e.g. to developmental and/or environmental stimuli) and to
be expressed only in specific tissue(s) at the right time. In ~98%
of a mammalian genome, CpG dinucleotides are less frequent than would
be expected, and their cytosine moiety is almost constantly methylated.
The remaining 2% of the genome, however, contains the so-called CpG
islands (2, 38), where these dinucleotides are densely clustered, more
frequently than expected and unmethylated. It is not yet clear whether
the presence or absence of a CpG island in the control region of genes
may influence any of the previously described mechanisms. Approximately
one-half of mammalian genes, comprising the totality of housekeeping
genes and a minority of tissue-specific genes, are reported to have a
CpG island at their 5' end; the majority of tissue-specific genes,
however, are not associated with an island (39, 40). The housekeeping
genes associated with unmethylated islands are transcriptionally
active, whereas in most cell types the tissue-specific genes associated with unmethylated islands are, as a rule, transcriptionally inactive. These results raise questions as to the mechanism activating the transcription of genes in a tissue-specific manner and to how CpG sites
are methylated to affect stable and/or transient repression. The
relative contribution to the final methylation pattern of each of the
aforementioned mechanisms, and their possible interconnection, which
lead to the final expression pattern, has yet to be addressed. A
crucial point is whether these mechanisms provide the high level of
plasticity required for the regulation of the expression of tissue-specific genes.
Of the several tissue-specific transcription factors that have been
identified in mammalian cell types, the muscle regulatory factors,
which belong to the basic helix-loop-helix family (41-43) and are
involved in the commitment to myogenic fate and in muscle terminal
differentiation, are unique in terms of their ability to control a very
complex array of tissue-specific genes. The high level of structural
and functional characterization of these genes and their highly
integrated auto- and cross-activating network define the muscle as one
of the most useful cellular systems for the clarification of mechanisms
of tissue-specific gene regulation during differentiation. Of
particular interest, from this point of view, are those genes that, at
the onset of cell differentiation, show a definite on/off switch
closely connected with strict transcriptional control; a typical
example is the myogenin gene (44, 45), which plays a
central role in myogenesis. Several studies, besides dealing with the
methylation patterns of single muscle genes (46-50), have also
demonstrated a general role of hypomethylation in the induction of
muscle differentiation (51-54).
The aim of this work was to study, during muscle differentiation, the
structural and temporal variation of the methylation pattern of
myogenin. For this purpose, we used the HpaII/PCR
technique (55, 56), optimized as a multiplex assay, to study the
methylation status of a single CpG site of myogenin
5'-flanking region and of the three CpG sites of exon 1, and to relate
our findings to the transcriptional activation of this gene. We
demonstrated a strong correlation between the temporal dynamics of
demethylation of the sole CpG site of the 5'-flanking region and
myogenin expression. In vivo, the
myogenin 5'-flanking region was found to be fully methylated
in nonmuscle tissues, partially methylated in adult muscle (where no
myogenin expression was detected), and demethylated in fetal
muscle where, by contrast, myogenin was expressed at high
levels. The fact that the dynamics of hypomethylation induction in vitro did not last nearly as long as the cell cycle
supports the idea of active demethylation mechanisms. However, the
presence of 3-deaza-adenosine, an inhibitor of biological methylation
reactions based on the AdoMet metabolism (33, 57), together with
homocysteine to enhance its inhibitory effect, further shortened the
duration of the dynamics of demethylation, which suggests that
re-methylation mechanisms are also involved. For myogenin,
these findings point to the following: 1) an interplay between active
demethylation and re-methylation mechanisms in the definition of the
final methylation pattern of the gene, and 2) a physiological role, in
the definition of the transcriptional status of this gene, of the
demethylation dynamics of the CpG site present in its 5'-flanking region.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Restriction enzymes were purchased from New
England Biolabs (Mississauga, Ontario, Canada) and from Roche Molecular
Biochemicals. The Megaprime DNA labeling system was obtained from
Amersham Pharmacia Biotech. Radiochemicals were purchased from
PerkinElmer Life Sciences. Centri-Sep columns were from Princeton
Separations (Adelphia, NJ). DH5
bacteria were from Life
Technologies, Inc. The CK-NAC kit was from Abbott.
Oligo(dT)16 was from Roche Molecular Biochemicals. Moloney
murine leukemia virus-reverse transcriptase (cloned), Super
Taq (Thermus thermophilus DNA polymerase,
Hoffmann-La Roche), human placental ribonuclease inhibitor, buffer for
reverse transcription, buffer for PCR, and
X/HaeIII
marker were obtained from HT Biotechnology Ltd. (Cambridge, UK).
Oligonucleotides used as primers were synthesized by M-Medical
GENENCO (Firenze, Italy). 3-Deaza-adenosine was a kind gift from
Bioresearch Co. (Milan, Italy). All other chemicals were from Sigma.
Cell Cultures--
The experiments were performed on the C2C12
mouse muscle cell line (58) and on the clone C2T18; the latter was
selected because of its enhanced differentiative ability as described
previously (59) (see "Results"). Cells were cultured in F-14 medium
supplemented with 50 µg/ml neomycin and 10% fetal calf serum, which
favors cell growth, with only a limited amount of differentiation
(growth medium with 10% fetal calf serum (GM)) or with 1% fetal calf
serum (differentiation medium with 1% fetal calf serum (DM)), which induces differentiation with the appearance of myotubes and of creatine
kinase (CK) activity after a limited number of cell divisions (59). In
the differentiation experiments, flasks and multiwells were coated with
0.2% gelatin. 24 h after plating, the cells were either shifted
to DM or re-fed with GM according to the experimental design; the time
at which this medium shift occurred was indicated as day 0. The
demethylating drugs 3 µM 3-deaza-adenosine and 50 µM homocysteine (jointly indicated as DH) were added to
DM according to the experimental design. Cultures were re-fed every 2nd
day with the appropriate medium (with or without drugs).
Differentiation Assay--
Cells to be used for the enzymatic
test, either in GM or DM, were rinsed twice with phosphate-buffered
saline and frozen at
80 °C. After thawing, cells were scraped into
1 ml of 50 mM Tris-HCl, pH 7.2, and 1 mM
dithiothreitol, sonified for 15 s in ice, and centrifuged.
Supernatant was used for CK (EC 2.7.3.2) and total protein content
assay (60). The results, expressed as milliunits CK/µg protein ± S.E., are the average of at least three experiments.
Tissue Biopsies--
Spleen and brain tissues were isolated from
adult mice; thigh skeletal muscle (quadriceps femoris) tissue was
isolated from adult mice and from 17-day-old mouse embryos.
RNA Isolation and Expression Studies--
Total RNA extraction
was performed by the acidified phenol procedure (Ref. 61 adapted in
Ref. 62).
The cDNA of rat myogenin, excised from the plasmid
BSM13
MGN11 (45), was used as a probe for in
vitro expression studies by Northern blotting. Blots were
normalized with an 18 S ribosomal DNA mouse probe cloned in the
pBR-322 plasmid (63). Probes were labeled by random priming (64, 65)
using the Megaprime DNA labeling system with
[
-32P]dATP (3000 Ci/mmol; specific activity >1.9 × 109 dpm/µg). Radioactive probes were purified by
Centri-Sep columns.
Agarose electrophoresis of total RNA, vacuum transfer on Hybond N
membrane (Amersham Pharmacia Biotech) and cross-linking were performed
according to standard procedures (62). Each membrane was prehybridized
in a hybridization oven at 42 °C by shaking for 2 h and by
rolling for an additional 2 h in 10 ml of 50% Quick Hyb
(Stratagene), 50% formamide, and 100 µg/ml salmon sperm DNA (Sigma).
The radioactive probe was added to the prehybridization solution, and
the membranes were incubated for 18-20 h at 42 °C in the same oven.
Washes were performed with 50 ml of the following buffers: 2× SSC
(20×, 175.3 g/liter NaCl, 88.2 g/liter sodium citrate, pH 7.0), 0.1%
SDS at room temperature; 2× SSC, 0.1% SDS at 50 °C twice; 0.2×
SSC, 0.1% SDS at 55 °C twice. Autoradiographs were quantified using
a computerized densitometer (BioImage, Genomic Solutions Inc., Ann
Arbor, MI).
The expression studies in biopsies in vivo were performed by
RT-PCR. For this purpose, reverse transcription was performed on 1 µg
of total RNA by using, in each 20-µl sample, 50 mM
Tris-HCl, pH 8.3, 75 mM KCl, 10 mM
dithiothreitol, 3 mM MgCl2, and 300 µM each of dGTP, dATP, dCTP, and dTTP, 20 units of human
placental ribonuclease inhibitor, 50 pmol of oligo(dT)16,
and 50 units of Moloney murine leukemia virus reverse transcriptase at
42 °C for 1 h, followed by heat inactivation at 94 °C for 5 min.
In the subsequent amplification reactions, 2 µl of each sample were
mixed in a final volume of 50 µl that contained 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM
MgCl2, 0.1% Triton X-100, 0.01% (w/v) gelatin, and 175 µM each of dGTP, dATP, dCTP, and dTTP, with 1 unit of
Super Taq DNA polymerase and 20 pmol of each specific
primer. For mouse myogenin (GenBankTM accession
number M95800) and for mouse
-actin
(GenBankTM accession number L21996) genes, we performed 30 and 25 cycles of 1 min at 94 °C, 1 min at 62 °C, 1.5 min at
72 °C, respectively, followed by 7 min at 72 °C, using a
PerkinElmer Life Sciences Thermal Cycler model 480. For the
amplification of myogenin cDNA, we used MyoP3 (exon 1, 5'-TTCCTGTCCACCTTCAGGGCTTCG-3', nt 1686-1709) as forward primer and
MyoM2 (exon 3, 5'-TAAGGAGTCAGCTAAATTCCCTCGC-3', nt 3507-3531) as
backward primer, obtaining an amplified fragment of 808 bp. For the
amplification of
-actin cDNA, the forward primer used was MMGACTP3 (exon 5, 5'-ACCCAGGCATTGCTGACAGGATGC-3', nt
2753-2776) and the backward primer was MMGACTM2 (exon 6, 5'-CCATCTAGAAGCATTTGCGGTGGACG-3', nt 3046-3071); an amplified fragment
of 216 bp was obtained. The amplified fragments were analyzed by
standard electrophoretic procedures and ethidium bromide staining. The
specificity of amplified products was assessed by restriction analysis
and/or sequencing;
-actin was used as an
internal standard to ensure that the quantity of samples was always the same.
DNA Isolation and Methylation Studies by Multiplex
HpaII/PCR--
Genomic DNA was extracted using a standard
phenol/chloroform method followed by ethanol precipitation (62).
Genomic DNA was treated separately with the following restriction
endonucleases: (i) EcoRI, which has no recognition sites within the amplified fragments of myogenin gene; (ii)
HpaII, which has a recognition site within the 5'-flanking
region and three recognition sites within exon 1 (Fig.
1) and is methylation-sensitive (i.e. it fails to cut if the CCGG recognition sequence is
methylated at any C). Exon 3, which possesses no HpaII or
EcoRI recognition sites (Fig. 1), was used as an internal
standard. In each case, 1.5 µg of genomic DNA were digested at
37 °C, first with 5 units of enzyme overnight and then with 3 more
units for an additional 6 h, in a total volume of 40 µl of the
buffer provided by the manufacturer. The following primers were used
for the amplification of the mouse myogenin gene: for the
5'-flanking region, the forward primer MyoP1
5'-TGGAGTGGTCCTGATGTGGTAGTGG-3' (nt 1022-1046) and the backward primer
MyoM8 5'-ACCCAGAGATAAATATAGCCAACGC-3' (nt 1496-1520); for exon 1, the
forward primer MyoP3 (described above) and the backward primer MyoM7
5'-GCGCTCAATGTACTGGATGGCG-3' (nt 1990-2011); for exon 3, the forward
primer MyoP10 5'-TCCATCGTGGACAGCATCACG-3' (nt 3266-3286) and the
backward primer MyoM2 (described above). These pairs of primers were
expected to produce, from uncut DNA, the following fragments: 499 bp
for the 5'-flanking region, 326 bp for exon 1 and 266 bp for exon 3. PCR was performed, with 20 pmol of each primer, on 50 ng of DNA, in a
total volume of 50 µl containing 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1%
Triton X-100, 0.01% (w/v) gelatin, and 175 µM dNTPs with 1 unit of SuperTaq. After an initial 3 min of denaturation at 94 °C,
30 cycles (1 min at 94 °C, 1 min at 62 °C, and 4.5 min at
72 °C) and a final extension of 7 min at 72 °C were performed using a PerkinElmer Life Sciences Thermal Cycler model 480. Aliquots of
the PCR products (15 µl) were examined by electrophoresis in 1.5%
agarose gel. Each gel was scanned by a CCD camera and was acquired on
the BioImage computerized densitometer. The specificity of the
fragments was assessed by restriction analysis and/or sequencing.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of
myogenin gene. Primers used are shown as
gray boxes and HpaII sites as
arrows.
|
|
 |
RESULTS |
Differentiation--
Table I shows
the results of differentiation (measured as CK activity) of C2C12 and
C2T18 cells. The C2C12 cells showed, after 96 h in GM, a low level
of CK activity which, 96 h after the shift to DM, underwent a
7-fold increase. The C2T18 clone was characterized by higher CK
activity in GM and a further increase after the shift to DM, thereby
showing greater myogenic potentialities than C2C12. The presence of the
drugs increased the degree of differentiation of both C2C12 and C2T18;
the increase was greater in C2T18.
View this table:
[in this window]
[in a new window]
|
Table I
Differentiation pattern of C2C12 and C2T18 clone
The CK activity normalized for protein content (expressed as milliunits
of CK/µg proteins ± S.E.), 96 h after induction, in cells
cultured in GM, DM, or DM + DH, is shown.
|
|
Myogenin Expression in in Vitro Cell Cultures--
In GM, C2C12
and C2T18 showed, at early times (48 h), a very low level of
myogenin expression, which increased at later times (96 h)
(Fig. 2, A and B,
lanes 1 and 2). After the shift to DM, myogenin expression was enhanced both in C2C12 and C2T18
(Fig. 2, A and B, lane 3); the final
myogenin expression was markedly higher in C2T18 than in
C2C12 (Fig. 2C). In DM, the drugs produced an increase in
myogenin expression both in C2C12 and C2T18 (Fig. 2,
A and B, lane 4), with greater effects
exerted on the latter clone.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 2.
Myogenin expression in in
vitro cell cultures. A, expression pattern
of C2C12 in GM, DM, and DM + DH. B, expression pattern of
C2T18 in GM, DM, and DM + DH. C, comparison of expression in
DM at 96 h, between C2C12 and C2T18. Because of some probing and
exposure differences between blots, the only comparisons allowed are
within blot. For each blot, ribosomal 18 S (r18S) signals
and ethidium bromide staining (EtBr) are shown as controls;
h, hours after medium shift.
|
|
In semiquantitative densitometric assays, the signal intensity was
calculated within the blot of Fig. 2A for C2C12 and of Fig.
2B for C2T18; a densitometric comparison was obtained by scaling the relative signals using the common blot of Fig.
2C. The densitometric analysis (Table
II) confirmed the qualitative analysis of
Northern blot shown in Fig. 2. The C2T18 clone showed, in all
conditions, higher signals than the corresponding ones of C2C12. In
particular, the increase in myogenin expression of the C2T18
clone after 96 h in GM produced a signal comparable to that of
C2C12 in DM, whereas its overall increase in DM, whether with or
without drugs, produced the highest myogenin expression. In
some experiments the C2T18 signal after 96 h in GM reached an
intensity as strong as that reached in DM (data not shown). These
results further stress the higher myogenic potentiality of C2T18 when
compared with C2C12.
View this table:
[in this window]
[in a new window]
|
Table II
Densitometric evaluation of myogenin expression
Densitometric values in GM, DM, and DM + DH were normalized as
described under "Results" and are expressed as a percentage of the
highest signal (C2T18 in DM + DH, shown as 100%) ± S.E.
|
|
The results obtained by Northern blot analysis were confirmed by RT-PCR
(data not shown).
Myogenin Expression in Vivo in Biopsies of Various
Tissues--
Fig. 3 shows the expression
of
-actin, used as a positive control, and of
myogenin in adult and embryonic muscle as well as in brain
and spleen. Myogenin expression was detected in
vivo only in embryonic muscle (lane 11). Despite the
large number of cycles performed in some RT-PCR experiments, there was
no expression in any of the other adult tissues. The lack of
myogenin expression found in adult muscle is in agreement
with the findings of some authors (45) but in contrast with those of
others (44).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Myogenin expression in
vivo in biopsies of various tissues. RT-PCR signals are
shown for spleen (S, lanes 2 and 8),
brain (B, lanes 3 and 9), adult muscle (AM,
lanes 4 and 10), and embryonic muscle (EM, lanes
5 and 11) using myogenin-specific primers
(from lane 8-12) and
-actin-specific primers as positive controls
(from lanes 2-6); lanes 6 and 12 show
negative controls; lanes 1, 7, and 13 show a
X/HaeIII molecular weight marker (MM).
|
|
Optimization and Test Performance of HpaII/Multiplex PCR
Assay--
PCR amplification from EcoRI-treated DNA
produced the expected three bands (shown in all EcoRI panels
in Figs. 4-6); some particularly efficient PCRs also displayed another
product, of 990 bp, that originated in a secondary although specific
reaction from MyoP1 and MyoM7 primers (often visible in the panels in
Figs. 4-6).
The same band pattern was obtained both from uncut DNA and from DNA
treated with heat-inactivated HpaII (data not shown). In
preliminary experiments we also verified that demethylated PCR
products, digested with HpaII and re-amplified after the
cut, failed to show any band relative to the 5'-flanking region or exon
1 but produced, by contrast, an amplified product relative to exon 3. In addition, we selected a prolonged extension time (4.5 min) to obtain
a more efficient amplification of the largest PCR product (499 bp); the
result was a signal with an intensity similar to that of the other
amplified fragments, regardless of the amount of target DNA used.
Myogenin Methylation Patterns in in Vitro Cell Culture, Without
Drugs--
The HpaII/multiplex PCR assay yields an
amplified product only if the DNA fragment that is to be amplified by
the specific pairs of primers fails to be cut by a restriction
endonuclease. The analysis of the amplified products obtained from the
HpaII-treated samples allows the determination of the
methylation status of the single CCGG site in the 5'-flanking region
and of the three CCGG sites in exon 1 of the myogenin gene.
Exon 3 was used together with EcoRI-treated samples as a
positive control. The panels in Figs. 4 and 5 show the methylation
patterns of the myogenin gene at different times, after
either EcoRI digestions as positive controls (on the
left of each panel) or HpaII digestion in
methylation-sensitive experiments (on the right of each panel).
In C2C12 grown in GM, all the CCGG myogenin sites were
methylated up to 48 h (Fig.
4A), although in some
experiments a partial loss of methylation in the 5'-flanking site
occasionally occurred at 48 h (data not shown). In all the
experiments, this site was found to be unmethylated after 72 h of
culture in GM (Fig. 4B, lane 9) and remained
unmethylated at 96 and 120 h. In DM (Fig. 4C), C2C12
showed a demethylation of the 5'-flanking site 24 h after
induction of differentiation (lane 12), although this site was occasionally still methylated at 24 h (Fig. 7A,
lane 3). The 5'-flanking site was, however, always
unmethylated 48 h after differentiation induction (Fig.
4C, lane 13, and Fig. 7A, lane 4). We never observed demethylation of any of the three CCGG sites of exon 1.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 4.
Myogenin methylation patterns in
C2C12, without drugs. A, methylation pattern after
1-48 h of culture in GM; MM = /HindIII + EcoRI molecular weight marker. B, extended
methylation pattern after 2-120 h of culture in GM; MM = X/HaeIII molecular weight marker. A negative control is
shown in lane 12. C, methylation pattern after
1-48 h in DM; MM = /HindIII + EcoRI molecular weight marker. Each electrophoretic panel
shows the EcoRI digestions, used as positive controls on the
left, and the HpaII digestions, used as
methylation-sensitive experiments, on the right;
h, hours after medium shift.
|
|
All the CCGG sites of the C2T18 clone were methylated in GM up to
2 h (Fig. 5A, lane
7). In all the experiments, the CCGG site of the 5'-flanking
region was found to be demethylated after 24 h of culture in
GM (Fig. 5A, lane 8). Demethylation was
maintained for over 96 h in GM. In DM, the C2T18 clone showed
early demethylation of the 5'-flanking site 2 h after
differentiation induction (Fig. 5B, lane 8); this
pattern was maintained for over 72 h. Earlier time courses in DM
showed methylation of the 5'-flanking site of this clone up to 1 h
after differentiation induction (Fig. 5C, lane
10). In addition, there was no demethylation of the CCGG sites of
exon 1 in C2T18.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 5.
Myogenin methylation patterns in
C2T18, without drugs. A, methylation pattern after
2-96 h of culture in GM; MM = X/HaeIII
molecular weight marker. A negative control is shown in lane
12. B, methylation pattern after 2-72 h of culture in
DM; MM = /HindIII + EcoRI
molecular weight marker. C, early methylation pattern after
20 min to 8 h in DM; MM = X/HaeIII
molecular weight marker. A negative control is shown in lane
13. Each electrophoretic panel shows the EcoRI
digestions, used as positive controls on the left, and the
HpaII digestions, used as methylation-sensitive experiments,
on the right; h, hours after medium shift.
|
|
Myogenin Methylation Patterns in Biopsies of Various Tissues in
Vivo--
myogenin was found to be fully methylated in
nonexpressing tissues (spleen (S) and brain (B),
Fig. 6, lanes 7 and
8) and demethylated at the 5'-flanking site in
myogenin-expressing embryonic muscle (Fig. 6, lane
10). Adult muscle (Fig. 6, lane 9), where no
myogenin expression was found, showed partial demethylation
of the 5'-flanking region site, with a band of reduced intensity
visible in some preparations. Demethylation in vivo was also
strictly limited to the site of the 5'-flanking region.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Myogenin methylation patterns
in vivo in various tissues. The EcoRI
panel, used as a positive control is shown on the left, and
the HpaII panel, used as methylation-sensitive experiments,
is shown on the right, in spleen (S, lanes 2 and
7), brain (B, lanes 3 and 8), adult
muscle (AM, lanes 4 and 9), and embryonic muscle
(EM, lanes 5 and 10); lane 11 is a
negative control; lanes 1, 6, and 12 show a
X/HaeIII molecular weight marker (MM).
|
|
Myogenin Methylation Patterns in in Vitro Cell Culture, with
Drugs--
Methylation (up to 8 h, Fig. 4C) of the
CCGG 5'-flanking site of C2C12 in DM completely disappeared as early as
2 h after differentiation induction in the presence of DH (Fig.
7A, lane 6), even
in experiments in which some methylation had been found up to 24 h
after differentiation induction (Fig. 7A, lane
3); this demethylated status was maintained for over 72 h.
These effects were observed only on the CCGG 5'-flanking site, never on
the exon 1 CCGG sites.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 7.
Myogenin methylation patterns
in vitro with drugs. A, C2C12
methylation pattern after 2-72 h of culture in DM without drugs
(lanes 2-5) and with DH (lanes 6-9).
B, C2T18 methylation pattern after 20 min to 1 h of
culture in DM without drugs (lanes 2-4) and with DH
(lanes 5-7). Each electrophoretic panel shows
HpaII digestions, used as methylation-sensitive experiments.
MM, X/HaeIII molecular weight marker;
h, hours after medium shift.
|
|
Clone C2T18 showed a completely demethylated 5'-flanking site from 2 to
72 h, whether with (data not shown) or without DH (as previously
shown in Fig. 5B). An earlier time course (Fig. 7B) showed demethylation of this site after only 20 min, in
DM in the presence of DH, whereas controls remained methylated up to 1 h. The effect in these experiments was also strictly limited to the 5'-flanking site.
 |
DISCUSSION |
The DNA methylation patterns of muscle regulatory factors have
seldom been studied. MyoD1 plays a fundamental regulatory
role in the commitment of the cell to muscle fate; it is expressed both
in myoblasts and in myofibers and acts upstream of myogenin which, given that its expression is restricted to terminally
differentiated muscle, is instead devoted to the control of final
differentiation. This functional distinction is associated with
structural differences in the control region of the two genes, since
MyoD1 is associated with a CpG island (48, 66),
whereas myogenin is not. Some authors (67) have shown that
the density of methylated sites in the MyoD1 promoter is
inversely correlated with the ability to undergo auto- and
cross-activation and that, in vivo, the MyoD1 promoter is partially methylated in nonmuscle tissues but is
unmethylated in skeletal muscle. Other studies have indicated, however,
that the CpG island of MyoD1 is unmethylated also in normal
nonmuscle mouse tissues, that it becomes methylated during
immortalization of cell lines and in vitro transformation
(48), as well as during in vivo carcinogenesis, and that
this methylation is also correlated with the heterochromatinization of
this gene (66). The generalized in vivo hypomethylation of
the MyoD1 CpG island has stimulated the search for the real
target sequences with regulatory functions on the expression of this
gene, leading to the identification of a distal enhancer that is
completely unmethylated in myogenic cells and in somite cells but is,
on the contrary, methylated in nonmuscle cells (46, 47). Previous work
from this laboratory (52) provided evidence of the existence of a link,
following a differentiative stimulus or exposure to demethylating
agents, between earlier overall DNA hypomethylation, enhanced
muscle differentiation and myogenin expression. The dynamics
of changes in the methylation pattern during differentiation, however,
is not yet fully understood for either MyoD1 or
myogenin, nor is the site-specific pattern of methylation
yet known of the latter gene.
In this work we show that the myogenin expression pattern of
C2C12 and C2T18 is strongly correlated with the myogenic potentialities of these cells. The final differentiation levels (evaluated as CK
activity) were in fact closely correlated with the final expression levels of myogenin in DM and with the partial induction of
myogenin in GM, as well as with its enhanced expression in
C2T18. The drugs produced enhanced myogenin expression and a
concomitant increase in terminal differentiation, both effects being
highest in the C2T18 clone.
Demethylation of myogenin always preceded its activation.
The dynamics of the demethylation displayed, in both C2C12 and C2T18, a
positive correlation between early demethylation on the one hand, and
the levels of myogenin expression and muscle differentiation on the other. After the shift to DM, the 5'-flanking site of
myogenin in C2T18, i.e. in the clone with the
highest expression of myogenin and the best differentiative
ability, was methylated up to 1 h but demethylated after 2 h,
whereas in C2C12 it became demethylated only after 24 h. Although
GM allowed only limited myogenin expression and muscle
differentiation, in this culture condition there was also a good
correlation between myogenin demethylation, its expression, and muscle differentiation. In fact, higher myogenin
expression and muscle differentiation were shown in the C2T18 clone,
whose 5'-flanking site was methylated at 2 h in GM but
demethylated at 24 h, than in C2C12, which showed the lowest level
of myogenin expression and muscle differentiation in GM
combined with a very late methylated status (up to 48 h). However,
hypomethylation eventually occurred also in this condition (at 72 h), accounting for the limited effect on myogenin expression
and muscle differentiation. By comparing myogenin
methylation in DM versus GM, the demethylation of both C2C12
and C2T18 was found to be anticipated after the shift to DM.
Demethylating agents (DH) were able to further anticipate demethylation
of the 5'-flanking region site in both clones, concomitantly producing
enhanced myogenin expression and terminal differentiation.
Taken together, these findings indicate that the demethylation of
myogenin follows a highly specific time pattern;
myogenin expression and terminal differentiation were
strongly correlated with the timing, rather than with the extent, of
5'-flanking region demethylation, since the clone and conditions
showing greatest differentiation and myogenin expression
also exhibit earlier demethylation. The evidence of demethylation
dynamics occurring much faster than the cell cycle (which lasts ~24 h
in these cells), i.e. within a few hours without drugs, or
even in minutes in the presence of the drugs, strongly points to the
involvement of an active demethylation mechanism. No definite answer
can yet be given as to which of the mechanisms proposed so far (34, 35,
37) is most likely to be involved in the control of myogenin
expression; nevertheless, the myogenin/muscle system appears
to be a model that can be used to investigate the involvement of active
demethylation processes in the transcriptional control of
tissue-specific genes. The observed effect of the further anticipation
of demethylation by drugs interfering with the AdoMet metabolism is
intriguing given that these drugs should deal only with the methylation
processes and with passive demethylation. 3-Deaza-adenosine is, in
fact, both a substrate and a competitive inhibitor of AdoHcy hydrolase (EC 3.3.1.1). Administration of 3-deaza-adenosine, particularly in
the presence of homocysteine (DH), results in intracellular accumulation of AdoHcy and 3-deaza-AdoHcy, which are both inhibitors of
transmethylation processes; the accumulation of these inhibitors often
causes increased levels of AdoMet (owing to its decreased utilization)
(33, 57, 68, 69). The overall effect at the DNA level, specifically
demonstrated in an in vitro muscle system (52, 70), is
marked hypomethylation due to inhibition of methylating processes.
Since passive demethylation mechanisms are unlikely to be involved in
the dynamics of demethylation that occurs within a few minutes, some
interplay between demethylation and methylation processes must be
hypothesized. In our opinion, there may be a balance between active
demethylation processes removing the methyl moieties with very fast
dynamics on the one hand and re-methylating mechanisms acting on the
same sites on the other hand. The equilibrium between these two main
mechanisms would produce the final methylation pattern of the gene,
thereby defining its transcriptional status. As far as
myogenin is concerned, the differentiative stimulus can, by
acting on active demethylation processes, lead to early demethylation which is possibly enhanced by a concomitant
inhibition of re-methylation processes achieved by drugs acting on the
AdoMet metabolism.
The drastic results of the presence or absence of an amplified product
relative to the 5'-flanking site, without intermediate signal levels,
obtained in almost all the experiments is indicative of a fully
methylated or fully demethylated status which requires a very short
time interval for transition. This supports the hypothesis according to
which the mechanism involves a DNA demethylase which directly removes
the methyl groups from methylcytosine residues in DNA (37) and is
thought to have the characteristics of a processive enzyme, with the
enzyme landing on a molecule of DNA and proceeding to demethylate
in cis (71).
Myogenin demethylation was found to be highly controlled
also in a site-specific manner. In fact, the modulation of its
methylation was strictly limited to the sole CCGG site of the
5'-flanking region, whereas the three CCGG sites of exon 1 were, by
contrast, always methylated. Our results suggest that one site on its
own may play a role in the activation of some muscle regulatory genes. Indeed, some methyl-CpG-binding proteins show distinct requirements in
terms of methyl-CpG density needed for binding, spanning from greater
density to single methylated CpG moiety (72-74). Interactions between
the methyl-CpG density, the location of the methyl-CpG moieties, and
the promoter strength have also been reported (75-77).
The 5'-flanking region site was also demethylated in embryonic muscle
(a myogenin-expressing tissue) but was methylated in nonexpressing tissues. These findings substantiate, also in
vivo, a role of methylation of the 5'-flanking site in the
expression of myogenin. This site was (at least partially)
demethylated in adult muscle without evidence of expression, thus
confirming previous evidence that DNA demethylation is a condition that
is necessary, but is not sufficient, for the complete expression of a gene.
In conclusion, our study suggests that DNA demethylation is an
essential active mechanism in the transcriptional control of myogenin, both in vitro and in vivo,
and that it is subject to strict site- and time-specific regulation.
Demethylation dynamics results from an interplay between demethylating
and re-methylating processes and is a crucial variable in the
quantitative control of myogenin expression and in muscle
terminal differentiation.