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* División de Hepatología y Terapia Génica, Departamento de Medicina Interna, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain;
Servicio de Hepatología, Hospital Clínico y Provincial, 08036 Barcelona, Spain;
Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, Burjassot, 46100 València, Spain; and
§ Center for Liver Disease Research and Division of Gastrointestinal Liver Diseases, Department of Medicine, University of Southern California School of Medicine, Los Angeles, California 90033, USA
2Correspondence: División de Hepatología y Terapia Génica, Departamento de Medicina Interna. Facultad de Medicina. Universidad de Navarra. 31008, Pamplona, Navarra, Spain. Tel: 34–948-425678. Fax: 34–948-425677. E-mail: jmmato{at}unav.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: S-adenosylmethionine synthesis • DNA methylation • tissue-specific expression • liver cirrhosis • hepatocarcinoma
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. MAT activity in mammals is the product of two genes,
MAT1A and MAT2A, which display a tissue-specific
pattern of expression. MAT1A is expressed only in the adult
liver whereas MAT2A shows a wider distribution, being
responsible for AdoMet synthesis in the extrahepatic tissues (reviewed
in ref 2
). In addition, MAT2A is expressed in the fetal
liver, where it is replaced by MAT1A upon maturation of this
organ (3)
. Similarly, in the transformed hepatocyte, a
switch in MAT gene expression is observed and the dedifferentiated
hepatoma cell mimics the fetal pattern by expressing MAT2A
in place of the liver-specific MAT1A (4)
. This
switch in MAT gene expression appears to confer a growth advantage to
the tumor cell (5)
.
Although all this evidence clearly establishes that MAT1A is
a tissue-specific gene silenced outside the liver and in situations
such as development and malignant transformation, the mechanisms behind
this tightly controlled pattern of expression are not known. CpG
methylation in mammalian DNA has been linked for many years to the
establishment of tissue-specific patterns of gene expression (6
, 7)
. However, the molecular mechanisms underlying
methylation-dependent repression of gene expression have only recently
been elucidated. The recruitment of histone deacetylase, by means of
methylated DNA binding proteins such as MeCP2, seems to be critical for
assembly of the methylated sequences into a condensed state (8
, 9)
. This situation renders the promoter regions inaccessible to
the transcription machinery and thus can block transcription
(10
11
12)
. In light of these recent developments, we have
searched for the molecular mechanisms that may explain the tissue- and
differentiation stage-specific expression of MAT1A.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The human hepatoma cell line HepG2 was grown in DMEM
medium supplemented with 10% fetal calf serum, glutamine, and
antibiotics. Treatments were performed on exponentially growing
cultures. Total RNA from cells and tissues was obtained by the
guanidinium thiocyanate method. Aliquots (20 µg) of total RNA were
size fractioned by electrophoresis in agarose gels under denaturing
conditions, blotted, and fixed on Nytran (Schleicher and Schuell,
Keene, N.H.) membranes using standard procedures. Blots were
prehybridized and hybridized as described (13)
. Human and
rat MAT1A cDNAs probes (14
, 15)
were labeled
with [
-32P]dCTP (Amersham Pharmacia Biotech,
Amersham, U.K.) by random priming. Hybridization with a probe for 18S
rRNA was performed as loading control. Membranes were then exposed to
X-ray films. Genomic DNA was prepared as described previously
(16)
. Aliquots of high molecular weight DNA (10 µg) were
digested to completion with restriction enzymes (Boehringer Mannheim,
Mannheim, Germany), loaded on agarose gels, electrophoresed, and
blotted onto Nytran membranes as described (17)
.
Hybridizations were carried out with a 1.4 kb
SspI-SmaI probe derived from the rat
MAT1A promoter (probe 1) (18)
or with the human
MAT1A full-length cDNA (15)
labeled with
[-32P]dCTP. Membranes were then exposed to
X-ray films.
Induction of cirrhosis
Male Wistar rats (250 g) were fed ad libitum a
standard laboratory diet and received humane care according to our
institution’s guidelines for the use of laboratory animals in
research. Cirrhosis was induced by intraperitoneal injections of
CCl4 (0.5 ml administered twice a week) as
described previously (19)
. The establishment of cirrhosis
was monitored by histological examination of liver sections from
treated animals (19)
. Rats were killed after 9 wk of
treatment by cardiac puncture and exsanguination.
Nuclei isolation
Liver and kidney from Wistar rats were placed in buffer A (0.25M
sucrose, 10 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 0.2%
Triton X-100, 2 mM p-aminobenzamidine, 1 mM PMSF, 2.5
µg/ml leupeptin, 1.5 µg/ml chymostatin, 50 mM Tris-HCl, pH 7.5) at
a ratio of 5 ml buffer per gram of tissue. All the steps were carried
out at 4°C. Tissues were homogenized on ice; after filtration of the
homogenates through 200 and 100 µM nylon filters, nuclei were
collected by centrifugation at 1500 x g for 5 min and
the sediment was washed twice in buffer B (buffer A without Triton and
EDTA). Nuclear pellets were resuspended in the same buffer and placed
on a discontinuous sucrose gradient (0.75 M/1.75 M) in buffer B. After
centrifugation at 10,000 x g for 30 min, nuclei were
collected from the 1.75 M sucrose cushion and washed twice with buffer
B.
Micrococcal nuclease digestion
Nuclei were resuspended in digestion buffer (0.25 M sucrose, 10
mM NaCl, 3 mM MgCl2, 1 mM
CaCl2, 10 mM sodium butyrate, 2 mM
p-aminobenzamidine, 1 mM PMSF, 2.5 µg/ml leupeptin, 1.5
µg/ml chymostatin, 50 mM Tris-HCl, pH 7.5) at a concentration of 5 mg
DNA/milliliter. Nuclei from liver were digested with micrococcal
nuclease (Worthington, Freehold, N.J.) at 15 u per mg DNA and
nuclei from kidney with 20 u per mg DNA for 10 min at 37°C. The
digestion was stopped by adding EDTA to a final concentration of 5 mM
and the samples were cooled on ice and centrifuged at 12,000 x
g for 10 min. The supernatant was saved and the pellet was
resuspended in lysis buffer (0.25 mM EDTA, 10 mM sodium butyrate, 2 mM
p-aminobenzamidine, 1 mM PMSF, 2.5 µg/ml leupeptin, 1.5
µg/ml chymostatin, 50 mM Tris-HCl, pH 7.5) incubated 10 min on ice
and recentrifuged as above. The first and second supernatants were
pooled and analyzed on agarose gels to determine the extent of the
micrococcal digestion.
Production of antisera to hyperacetylated histone H4 and antibody
purification
Polyclonal antisera to hyperacetylated H4 were raised by
immunization with a synthetic peptide corresponding to residues 2–19
of histone H4 and acetylated in the 
-amino groups of lysines 5, 8,
12, and 16. The acetylated peptide was custom-synthesized by Genosys
and coupled to keyhole limpet hemocyanin (20)
. The
immunoglobulin (IgG) fraction was purified by HiTrap protein-A FPLC
chromatography according to the manufacturer (Amersham Pharmacia
Biotech).
Immunofractionation of nucleosomes
Immunofractionation of nucleosomes obtained by micrococcal
nuclease digestion of nuclei were performed essentially as described
previously (21)
. FPLC-purified antibody (500 µg of IgG)
dissolved in fractionation buffer (50 mM NaCl, 10 mM sodium butyrate, 1
mM EDTA, 0.2 mM p-aminobenzamidine, 0.1 mM PMSF, 2.5 µg/ml
leupeptin, 1.5 µg/ml chymostatin, 10 mM Tris-HCl, pH 7.5) was
incubated with 100 mg of protein A Sepharose prewashed with
fractionation buffer for 3 h at 4°C under gentle rotation. The
protein A Sepharose-bound antibody was mixed with 2 mg of soluble
chromatin in a final volume of 3 ml of fractionation buffer and
incubated overnight with gentle rotation at 4°C. The immunocomplexes
were collected by centrifugation (6500 x g, 1 min) on
a 0.65 µM Ultrafree filter unit (Millipore) and the filtered through
fraction was removed and stored on ice. Protein A Sepharose beads were
washed eight times with 500 µl of fractionation buffer as before and
filtrates were pooled together (unbound fraction). Antibody-bound
fraction was eluted from protein A Sepharose by addition of 400 µl of
1.5% sodium dodecyl sulfate (SDS) in fractionation buffer and rotated
for 15 min at room temperature. After centrifugation as before, the
eluate was saved and protein A Sepharose was reincubated for another 15
min period with 400 µl of 0.5% SDS in fractionation buffer. The two
eluates (bound fraction) were pooled. DNA from all chromatin fractions
(input, unbound, and bound) was purified and quantitated by
fluorescence with PicoGreen dye (Molecular Probes, Eugene, Oreg.).
Slot-blot and hybridization analysis
DNA samples were denatured in 0.5 M NaOH, 1.5 M NaCl for 10 min
at 37°C, followed by 1 min at 95°C and then kept on ice. 150 ng of
DNA from each fraction were loaded through a slot-blot manifold
(Bio-Rad, Hercules, Calif.) on a Biodyne B membrane (Pall) prewetted in
2x SSC. The filters were immersed in denaturation solution for 5 min,
neutralized in 1.5 M NaCl, 1 mM EDTA, 0.5 M Tris-HCl, pH 7.5 for
30 s and the membranes were dried by baking at 80°C for 30 min.
Hybridizations were carried out using a 750 bp
MspI-SmaI probe derived from MAT1A
promoter (probe 2). The probe was labeled by random priming. Filters
were prehybridized for 2 h and hybridized overnight using Quick
Hyb solution (Stratagene, San Diego, Calif.) at 65°C in the presence
of 200 µg of heat-denatured salmon sperm DNA. Filters were rinsed in
2x SSC, 0.1% SDS and washed sequentially twice with 2x SSC, 0.1%
SDS at 65°C for 5 min, once with 2x SSC, 0.1% SDS at 65°C for 30
min, and once with 0.2x SSC, 0.1% SDS at 65°C for 20 min. The
radioactivity present in the filters was initially measured with an
InstantImager (Packard, Downers Grove, Ill.) and the filters were then
autoradiographed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Two
MspI/HpaII restriction sites are present in the
reported 5'-flanking region of rat MAT1A (18)
(Fig. 1A
). Genomic DNA samples from rat liver and kidney were
sequentially digested with the methylation-insensitive enzyme
SspI and either MspI or HpaII.
Southern blot analysis was then carried out using a 1.4 kb
SspI-SmaI probe from rat MAT1A
promoter (probe 1 in Fig. 1A
). DNA from liver was
unmethylated as indicated by the presence of two fragments of 0.64 and
0.75 kb, respectively, consistent with cleavage at the
MspI/HpaII sites by both enzymes (Fig. 1B
). In the case of kidney DNA, MspI treatment
resulted in the same pattern obtained in liver, but HpaII
could not cleave with the same efficiency observed in hepatic tissue
and generated higher molecular weight bands (Fig. 1B
). These
results are consistent with hypomethylation of the cytosines located at
positions -13 and -755 of MAT1A promoter in liver and
enhanced levels of methylation of these two sites in kidney DNA. Other
tissues examined, such as spleen and heart, generated the same
restriction pattern observed in the kidney (data not shown).
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The mechanism of gene silencing by means of cytosine methylation has
been recently worked out through the implication of histone
deacetylation (8
, 9)
. We wanted to ascertain the degree of
acetylation displayed in vivo by the histones associated
with MAT1A promoter in the liver and kidney and whether this
covalent modification correlated with the degree of promoter
methylation. Mononucleosomes obtained from both tissues were
immunoprecipitated with an antibody mainly specific to acetylated
histone H4, as described in Materials and Methods. DNA was extracted
from the input, unbound, and bound fractions; equal amounts from each
fraction were immobilized onto nylon membranes by slot-blot and
hybridized with a 750 bp probe derived from the 5'-flanking region of
rat liver MAT1A gene, close to the transcription initiation
site (probe 2 in Fig. 1A
). The intensity of signal from the
antibody-bound slot relative to the intensity from the input slot gives
the enrichment generated by the antibody selection. Our data show a
strong enrichment (10- to 15-fold) in the bound fraction compared to
the input fraction in liver, where the gene is actively transcribed.
However, no preferential selection by the antibody was observed in the
kidney, indicating the absence of hyperacetylation in this region of
MAT1A in a tissue where the gene is not expressed.
We have also analyzed MAT1A promoter methylation in
developing rat hepatocytes. Genomic DNA was isolated from fetal rat
hepatocytes corresponding to gestational day 19, when MAT1A
expression is negligible (Fig. 2
) (3)
, and then digested with SspI and
MspI or HpaII. The restriction pattern obtained
with probe 1 indicated that in the developing hepatocyte the
MAT1A promoter is hypermethylated, as evidenced by the
appearance of high molecular weight bands in the
HpaII-treated samples (Fig. 2)
. This pattern was identical
to that obtained in extrahepatic tissues, where MAT1A is not
expressed.
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A significant number of cirrhotic patients develop hepatocellular
carcinoma, thus, the cirrhotic liver could be considered to be in a
precancerous condition (23
, 24)
. This notion along with
our previous observation of MAT1A silencing in transformed
liver (4)
led us to evaluate MAT1A expression
in a rat model of CCl4-induced cirrhosis. In
agreement with our previous results (19)
, no large changes
were observed in MAT1A mRNA levels, although a significant
trend toward a reduction in the hepatic content of this messenger was
observed (Fig. 3A
). MAT1A promoter methylation was evaluated in
control and cirrhotic livers using the same strategy described above.
High molecular weight bands were detected in the
HpaII-digested samples from cirrhotic rat liver, which is
consistent with enhanced methylation of the
MspI/HpaII sites in MAT1A promoter
(Fig. 3B
).
|
As mentioned before, transformed hepatic cells lose MAT1A
expression (4)
. We have tested whether the silencing of
MAT1A in this condition is accompanied by changes in
MAT1A methylation. Since the human MAT1A promoter
has not been cloned, we performed MspI/HpaII
restriction analysis using a full-length human MAT1A cDNA
clone as a probe (15)
. As observed in Fig. 3A
,
HpaII treatment of normal human liver yielded low molecular
weight bands that were absent in the transformed hepatic cells. This
result indicates hypermethylation at the internal C of CCGG sites of
MAT1A in HepG2 cells. Similar results consistent with
hypermethylation of MAT1A were obtained in Hep3B cells,
another human hepatoma cell line in which MAT1A is also
silenced (data not shown). To test whether this epigenetic modification
could play a role in MAT1A silencing, we tried to modify the
methylation status of this cell line by treatment with the
demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR)
(25)
. For this purpose, HepG2 cells were incubated with
0.1 µM 5-Aza-CdR for 24 or 48 h and MAT1A expression
was assessed by Northern blotting. Under these conditions,
MAT1A expression was induced after 48 h of treatment
(Fig. 4B
). It has recently been established that
methylation-dependent gene silencing involves histone deacetylation
(8
, 9)
. These observations led us to evaluate the effect
of the histone deacetylase inhibitor trichostatin A (26)
on MAT1A expression in HepG2 cells. In this case, 24 h
of treatment with 3 µM trichostatin A was sufficient to induce
MAT1A mRNA as detected by Northern blotting (Fig. 4B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This function is met in part by the
presence of a liver-specific MAT enzyme, which is the product of the
MAT1A gene expressed only in this organ (2)
. We
have recently reported the cloning and functional analysis of rat liver
MAT1A promoter (18)
. We observed that this
promoter was able to drive the expression of a reporter gene not only
in liver-type cells such as HepG2, but also in the nonhepatic cell line
CHO (18)
. These observations raised the possibility that
the silencing of MAT1A outside the liver could be the
consequence of situations known to repress transcription, such as DNA
methylation and chromatin compaction (7
, 25)
, rather than
to the lack of liver-specific transcription factors.
In the present work we have first demonstrated that MAT1A
promoter methylation is tissue specific, being hypomethylated in liver
but hypermethylated in nonexpressing tissues such as the kidney. We
have observed that MAT1A promoter is also hypermethylated in
the developing hepatocyte, in which we had previously demonstrated that
this gene is not expressed and MAT activity derives from the expression
of the extrahepatic MAT2A gene (3)
. Thus,
cytosine methylation in the 5' region of MAT1A conforms to a
pattern of hypermethylation, where the gene is inactive, and
hypomethylation, where the gene is active. DNA methylation has been
generally associated to an inactive chromatin conformation; however,
the mechanisms behind the transcriptional repression dictated by methyl
groups in the 5-position of cytosine residues had not been exposed
until recently. The recruitment of histone deacetylases to methylated
5'CpG dinucleotides through methylcytosine binding proteins such as
MeCP2 can lead to a condensed and inactive chromatin (8
, 9)
. These observations linking DNA methylation to histone
deacetylation led us to evaluate the degree of acetylation of histones
associated with MAT1A promoter. Immunoprecipitation of
mononucleosomes with antibodies specific to acetylated histone H4
indicated that in liver MAT1A is enriched in the highly
acetylated mononucleosome fraction, whereas in the kidney
MAT1A is not preferentially associated with acetylated
histones. The histone acetylation pattern of MAT1A promoter
in liver corresponds to that of an actively transcribing gene; however,
in the kidney this pattern is that of a silent gene. Similar patterns
of histone acetylation have been reported for active and silent genes,
respectively (28)
. MAT1A promoter methylation
profile correlates with the local acetylation status of histones. Thus,
in liver, where MAT1A promoter is hypomethylated, elevated
levels of histone acetylation would result in a chromatin structure
accessible to transcription factors whereas in the kidney we find the
opposite situation, which may be consistent with a condensed and
inactive chromatin state (8
, 9
, 29)
. These observations
may be relevant to the mechanisms by which tissue-specific
MAT1A expression is achieved in vivo.
Hepatic methionine metabolism is altered in various chronic liver
disorders as well as in the transformed liver (2
, 30)
. We
wanted to know whether the MAT1A methylation pattern would
be preserved in situations of hepatocellular damage or abnormal
proliferation. We first examined the methylation status of
MAT1A in an experimental model of liver cirrhosis, trying to
reproduce a pathological condition that in many cases precedes the
neoplastic conversion of the liver (23
, 24)
. Our data
indicate that enhanced levels of MAT1A promoter methylation
can be detected in the liver of cirrhotic rats. Although no marked
changes in liver MAT1A expression occur in this model, a
significant reduction (30% compared to controls) in steady-state
MAT1A mRNA levels was detected. Although global DNA
hypomethylation has been observed in this model of cirrhosis
(31)
, it is known that DNA methyltransferase expression is
higher in liver tissues that show chronic hepatitis or cirrhosis
(32)
. In this regard, de novo methylation and
reduced gene expression in cirrhosis have been reported for certain
loci such as the D17S5 locus, in which the candidate tumor suppressor
HIC-1 was identified (33)
. Our present findings
indicate that MAT1A expression in diseased liver is
compromised and that hypermethylation of its promoter region could
participate in this process.
As we previously mentioned, MAT1A expression is lost upon
neoplastic transformation of the hepatocyte (4)
.
MAT1A silencing is accompanied by the induction of
MAT2A, a situation that reproduces the pattern of expression
found in the fetal hepatocyte (3)
. In addition,
MAT2A is also induced under conditions of active
proliferation in the liver parenchyma as happens during liver
regeneration after partial hepatectomy (34)
. Thus, it
appears that proliferating cells may find an advantage in switching MAT
genes (5)
whose products behave differently from a kinetic
and regulatory point of view (2)
. Although global
hypomethylation is a characteristic of many transformed cells, certain
genomic sequences such as that of tumor suppressor and tissue-specific
genes appear to be hypermethylated and repressed in cancer
(7)
. We next tested MAT1A methylation status in
the human transformed hepatic cell line HepG2, in which
MAT1A is silenced. Our data indicate that hypermethylation
of MAT1A gene accompanies loss of expression. This cellular
model has allowed us to directly assess the role of cytosine
methylation and histone acetylation on MAT1A expression
regulation. We therefore treated HepG2 cells with 5-Aza-CdR, a
demethylating agent well known for its ability to reactivate the
expression of genes silenced by de novo methylation
(7
, 35)
. In the presence of 5-Aza-CdR, MAT1A
expression was induced after 48 h of treatment; this observation
suggests that methylation may indeed play a role in the transcriptional
regulation of this gene. A slower rate of cell growth accompanied
5-Aza-CdR-mediated induction of MAT1A expression (data not
shown). Growth arrest of tumor cells has previously been observed in
response to 5-Aza-CdR, and the reactivation of genes such as the cell
cycle regulatory p16 gene, among others, may explain the
effect of this drug (35)
. However, it is worth mentioning
that when MAT1A is transfected into HepG2 cells, slower
rates of cell growth and DNA synthesis are observed (5)
.
As mentioned above, recent findings have demonstrated that
transcriptional repression associated with cytosine methylation relies
to a significant extent on histone deacetylation (8
, 9)
.
These observations prompted us to test whether this mechanism could
also participate in the silencing of MAT1A in the
transformed HepG2 cells. Our findings indicate that treatment with the
histone deacetylase inhibitor trichostatin A (26)
for
24 h restored MAT1A expression in HepG2 cells. Taken
together, these observations suggest that MAT1A silencing
during transformation is mediated by de novo cytosine
methylation and the recruitment of histone deacetylase, a situation in
which a condensed and inactive chromatin state is favored. These
results also support a role for MAT1A promoter methylation
and histone acetylation in the maintenance of a tissue-specific
expression profile, which may be progressively lost as the transition
from a healthy to a diseased and, finally, neoplasic liver
proceeds.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 20, 1999. Revised for publication August 2, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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