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Division of Gastroenterology and Liver Diseases, USC Liver Disease Research Center, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine USC, Los Angeles, California 90033, USA
1Correspondence: Division of Gastrointestinal and Liver Diseases, HMR Bldg. 415, Department of Medicine, USC School of Medicine, 2011 Zonal Ave., Los Angeles, CA 90033, USA. E-mail: shellylu{at}hsc.usc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: methionine adenosyltransferase • transcriptional regulation • HCC • S-adenosylmethionine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
In mammals, two different genes, MAT1A and MAT2A, encode for two
homologous MAT catalytic subunits, 
1 and 2 (3
4
5)
.
MAT1A is expressed only in liver and encodes the 1 subunit found in
two native MAT isozymes, which are either a dimer (MAT III) or tetramer
(MAT I) of this single subunit (5)
. MAT2A encodes for a
catalytic subunit (2) found in a native MAT isozyme (MAT II), which
is associated with a catalytically inactive regulatory subunit (ß) in
lymphocytes encoded by yet a third gene (5
, 6)
. MAT2A is
widely distributed (3
4
5)
. MAT2A also predominates in the
fetal liver and is progressively replaced by MAT1A during liver
development (7
, 8)
. In adult liver, increased expression
of MAT2A is associated with rapid growth or de-differentiation of the
liver. We showed a switch in the gene expression from MAT1A to MAT2A in
human liver cancer (9)
from 12 to 24 h after partial
hepatectomy in the rat (10)
and after treatment with
thioacetamide (11)
. Using a cell line model that differs
only in the type of MAT expressed, we demonstrated that the type of MAT
expressed by the cell significantly influences the rate of cell growth
(12)
. Specifically, MAT1A expression is associated with
the lowest rate of cell growth whereas the opposite is true of MAT2A
expression (12)
. Thus, the switch in MAT expression in
liver cancer plays an important pathogenetic role by facilitating liver
cancer growth. Little is known about transcriptional regulation of
MAT2A. To better understand the mechanism for transcriptional
regulation of human MAT2A, we cloned the human MAT2A promoter
(13)
. Recently we confirmed that up-regulation of MAT2A in
liver cancer occurs at the transcriptional level (14)
.
Sequential deletion analysis of the human MAT2A promoter revealed
several regions that are important for promoter activity in HepG2 cells
(13
, 14)
. The human MAT2A promoter construct -571/+60
produced the maximal promoter activity whereas -270/+60 produced half
of the maximal promoter activity. Inclusion of additional upstream
sequences to -1329 did not alter luciferase activity significantly
(14)
. In the current work, we examined regions of the
MAT2A promoter implicated in positive regulation and identified two
important cis-acting elements and corresponding
trans-activating factors that are involved in the
transcriptional up-regulation of MAT2A in human hepatocellular
carcinoma (HCC). |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Source of normal and cancerous liver tissue
Normal liver tissue was obtained from normal liver included in
the resected liver specimens of five patients with metastatic colon or
breast carcinoma. Cancerous liver tissue was obtained from five
patients undergoing surgical resection for primary HCC. Written
informed consent was obtained from each patient. The contamination of
HCC samples with noncancerous tissue was less than 5% as determined by
histopathology. These tissue were immediately frozen in liquid nitrogen
for subsequent isolation of RNA and nuclear proteins as described
below.
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by University of Southern California School of Medicine’s human research review committee.
Cell culture
HepG2 and Drosophila melanogaster Schneider L2 (SL2)
cells were obtained from the Cell Culture Core of the USC Liver Disease
Research Center and grown according to instructions provided by the
American Type Culture Collection (Rockville, MD). HepG2 cells were
cultured in Earle’s minimal essential medium supplemented with 10%
fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin
mixture. SL2 cells were cultured in Drosophila cell medium
supplemented with 12% fetal bovine serum at room temperature in the
dark.
Recombinant plasmids
Recombinant expression vectors were created by cloning
restriction fragments isolated from the 5'-flanking sequence of the
human MAT2A into pGL-3 enhancer vector (Promega) or introducing
synthetic oligonucleotides into pGL3-promoter vector (Promega) for
determination of enhancer activity. The human MAT2A promoter constructs
-571/+60 and -270/+60 (13)
were subcloned in the sense
orientation upstream of the luciferase coding sequence of the pGL-3
enhancer vector (Promega). Mutant human MAT2A promoter constructs
-571/+60 and -270/+60 were generated by using polymerase chain
reaction (PCR). MAT2A promoter constructs mutated in either the
putative c-Myb site (-350 to -333) (from 5'-CGGCCAACGGTCGGAAGG-3' to
5'-CGCGTGCTAGCCCGGGCT-3')
or Sp1 site (-58 to -48) (from 5'-GGGGCGGGGC-3' to
5'-GGCTAGCCCC-3') were subcloned into the same
pGL-3 vector. Mutant sequences were confirmed by dideoxy sequencing. To
assess the functionality of these elements in a heterologous promoter,
fragments of the MAT2A promoter that contains the putative c-Myb
binding site (-355 to -324, wild-type or mutant, where AA at
positions -344 and -345 were mutated to TT) or the Sp1 binding site
(-64 to -32, wild-type or mutant, where GG at positions -55 and -56
were mutated to TT) were subcloned into the pGL-3 promoter-luciferase
vector (Promega).
The c-Myb expression vector pMTHMb, driven by a mouse metallothionein-I
promoter, was kindly provided by Dr. Michael Kuehl (15)
.
The Sp1 expression vector pPacSp1 was previously described
(16)
and kindly provided by Dr. Richard Rippe (University
of North Carolina, Chapel Hill, NC).
Nucleic acid extraction
RNA was isolated from frozen liver specimens, HepG2, and SL2
cells according to the method of Chomczynski and Sacchi
(17)
. RNA concentration was determined
spectrophotometrically before use and the integrity was checked by
electrophoresis with subsequent ethidium bromide staining.
Northern blot analysis
Northern blot analysis was performed on total RNA using specific
MAT2A cDNA probe as described (9)
. After digestion with
XhoI, the Sp1 cDNA probe was obtained from the Sp1
expression vector (16)
. The 580 bp human c-myb cDNA probe
corresponding to nucleotides 380 to 961 of the published human c-myb
sequence (18)
was obtained by reverse transcription and
subsequent amplification by PCR. Primary PCR (forward primer and
reverse primer are 5'-TCGAACAGATGTGCAGTGCCAG-3' and
5'-GTTCTGTGTTGGTAGCACCTGC-3', respectively. Cycle parameters are
94°C, 30 s; 60°C, 1 min; 72°C, 1 min; 25 cycles) and
secondary PCR (forward primer and reverse primer are
5'-GCTCATCAAGGGTCCTTGGACC-3' and 5'-GTCTCTGAATGGCTGCGGCTG-3',
respectively. Cycle parameters (94°C, 30 s; 65°C, 1 min;
72°C, 1 min; 28 cycles) were used to amplify the 580 bp human c-myb
cDNA probe. To ensure equal loading of RNA samples and transfer in each
of the lanes, the same membranes were also rehybridized with
32P-labeled ß-actin probe as described
(19)
. All cDNA probes were labeled with
[32P] dCTP using a random-primer kit (Primer-It
II Kit; Stratagene, La Jolla, CA). Autoradiography and densitometry
(Gel Documentation System, Scientific Technologies, Carlsbad, CA, and
NIH Image 1.60 software program) were used to quantitate relative RNA.
Results of Northern blot analysis were normalized to
ß-actin.
Reverse transcription-polymerase chain reaction (RT-PCR) for c-Myb
Total RNA (1 µg as determined spectrophotometrically) was
subjected to reverse transcription and subsequently amplified by PCR.
The human c-myb primers used were 5-CTCATCAAGGGGTCCTTGG-3'
and 5'-GTCTCTGAATGGCTGCGG-3'. The size of the amplified fragment
corresponding to the human c-myb was 580 bp. The same volume of the
reversed transcription product was subjected to PCR for 25 cycles under
identical conditions using primers for internal control, 18S (primers
and competitors were obtained from Ambion, Austin, TX). The PCR
conditions were verified to be in the linear range for both c-myb and
18S. The PCR products were then analyzed by electrophoresis on the same
2% agarose gel.
In vitro DNase I footprinting analysis
Two 32P end-labeled fragments of the
5'-flanking region of human MAT2A gene were generated by digestion with
restriction endonucleases. DNase I footprinting analysis was performed
using double-stranded fragments corresponding to nucleotides -494 to
-152 and -148 to +64 of the human MAT2A gene as described (13
, 20)
. Singly end-labeled fragments were generated by filling
5'-protruding ends with [-32P] dCTP (3000
Ci/mmol) using the exo-klenow enzyme. Labeled probes were purified by
electrophoresis with 2% agarose gel. Approximately 5 x
104 cpm of end-labeled DNA fragments were
incubated with 0 to 45 µg of nuclear protein from normal human liver,
HCC, or HepG2 cells. After 30 min incubation on ice,
CaCl2 and MgCl2 were added
to give a final concentration of 0.5 mM and 1 mM, respectively. DNase I
digestions were performed at room temperature for 1 min. Upon phenol
extraction and ethanol precipitation, DNA fragments were resolved by
electrophoresis in a denaturing 8% acrylamide sequencing gel.
In vivo footprinting by ligation-mediated PCR (LM-PCR)
In vivo footprinting by LM-PCR was performed as described with
minor modifications (21
, 22)
. For in vivo DNase I
digestions, HepG2 cells (5x106 cells) were
permeabilized by treatment with 0.05% lysolecithin in buffer A (150 mM
sucrose, 80 mM KCl, 35 mM HEPES, pH 7.4, 5 mM
K2HPO4, 5 mM
MgCl2, 0.5 mM CaCl2) for 1
min at 37°C. After this, cells were centrifuged, washed once with
buffer A without lysolecithin, and treated at room temperature for 5
min with DNase I (75 Kunitz units/ml) in buffer A. The DNase I treated
cells were centrifuged and lysed in 10 mM Tris-HCl, pH 8.0, 85 mM NaCl,
12.5 mM EDTA, 0.5% SDS, 300 µg/ml proteinase K, 100 µg/ml RNase A
by incubation at 37°C for 7 h. The reaction was
phenol/chloroform extracted and the DNA ethanol precipitated.
Naked DNA controls (in vitro DNase I treated), which is essential for interpreting the in situ data, were prepared by digestion of DNA (35 µg) purified from HepG2 with DNase I (10 Kunitz units/ml) at room temperature in buffer A for 10 min. The in vitro treated DNA was phenol/chloroform extracted and precipitated with ethanol. The in vivo and in vitro digested DNAs were resuspended in buffer (40 mM Tris-HCl, pH 7.7, 25 mM NaCl, 6.7 mM MgCl2), denatured at 95°C for 3 min, cooled for 1 min on ice, and the free 3'OH groups were blocked by incubation of the DNA with T7 DNA polymerase (5 units, Sequenase version 2.0; US Biochemical) in the presence of 5 µM ddNTPs for 20 min at 45°C. The reaction mixtures were heated at 94°C for 3 min, cooled, and adjusted to a final concentration of 200 mM potassium cacodylate, pH 7.0, 1 mM 2-mercaptoethanol. Terminal transferase (30 units, New England Biolabs, Beverly, MA) was added to each reaction mixture and the incubation continued for an additional 35 min at 37°C. The DNA fragments were phenol/chloroform extracted and recovered by ethanol precipitation with 2 M ammonium acetate. Precipitated DNA was resuspended in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and used in LM-PCR reactions with MAT2A promoter-specific primers.
LM-PCR using Vent DNA polymerase (New England Biolabs) was conducted as
described (22)
. To DNA (2 µg in 5 µl in TE buffer) was
added 25 µl of first-strand synthesis mix [40 mM NaCl, 10 mM
Tris-HCl, pH 8.9, 5 mM MgSO4, 0.01% gelatin, 0.3
pmol gene-specific primer 1 (c-Myb, 5'-AATGCAGACGCGAGGAGACGTG-3', which
is reverse and complementary to -246 to -225 of MAT2A sequence
(13)
, Sp-1, 5'-GCTGCGGACAGCGTTCTACTC-3', which is reverse
and complementary to +40 to +60), 0.2 mM dNTPs, and Vent DNA polymerase
(0.5 U, New England Biolabs)]. The reactions were denatured for 5 min
at 95°C, annealed for 30 min at 60°C, and extended for 10 min at
76°C. Each reaction was mixed with 20 µl of ligase dilution
buffer (10 mM Tris-HCl, pH 7.5, 17.5 mM MgCl2, 50
mM dithiothreitol, 125 µg/ml bovine serum albumin) and 25 µl of
ligase mix [10 mM MgCl2, 20 mM dithiothreitol, 3
mM ATP, 50 µg/ml bovine serum albumin, 100 pmol linkers
(LM-PCR1: 5'-GTGACCCGGGAGATCTGAATTC-3'; LM-PCR2: 5'-GAATTCAGATC-3')].
T4 ligase (3 U) was added and the reactions incubated overnight at
17°C. Samples were iced and 9.4 µl of precipitation solution (0.1%
yeast tRNA, 2.7M sodium acetate, pH 7.0) and 220 µl of ethanol were
added. The samples were placed at -20°C for > 2 h and
then spun for 15 min at 4°C in a microcentrifuge. The pellets were
washed with 75% ethanol. Samples were resuspended in 70 µl of water
at room temperature and placed on ice. After addition of 30 µl of
amplification mix [3.33x amplification buffer (133 mM NaCl, 67 mM
Tris-HCl, pH 8.9, 17 mM MgSO4, 0.03% gelatin,
0.3% Triton X-100) with 670 µM each dNTP, 10 pmol of gene-specific
primer 2 (c-Myb: 5'-ATTGCTCTTCCTTGTTTGATGTGG-3' (reverse and
complementary to -274 to -251); Sp-1: 5'-AGCGTTCTACTCGTAGCAGGC-3'
(reverse and complementary to +31 to +51)], 10 pmol of linker primer
(5'-GTGACCCGGGAGATCTGAATTC-3'), and 3 µl (3 U) of Vent polymerase,
samples were overlaid with 90 µl mineral oil and subjected to PCR.
The PCR reactions for performed for 18 cycles: the first denaturation
was 4 min at 95°C, and subsequent ones for 1 min; annealing was 2 min
at 66°C, and extension was 3 min at 76°C. For each cycle, an extra
5 s was added to the extension step. The final extension was
allowed to proceed for 10 min. The secondary PCR reactions were
performed by adding 5 µl of end-labeling mix [1x amplification
buffer, 2.3 pmol end-labeled gene-specific primer 3 (c-Myb:
5'-GAGAAAAAGCGACTGGGGCTTG-3', reverse and complementary to -296 to
-275; Sp-1: 5'-GCGGAGCGAACGAAGCAGCG-3', reverse and complementary to
+4 to +23)], 2 mM dNTPs and Vent DNA polymerase (1 U) using two rounds
of PCR (1 cycle: 95°C/4 min, annealing temperature 2 min, 76°C/10
min; 1 cycle: 95°C/1 min, annealing temperature 2 min, 76°C/10
min). Each reaction was terminated by adding 295 µl stop solution
(260 mM NaOAc, pH 7.0, 10 mM Tris-HCl, pH 7.5, 4 mM EDTA, pH 8.0, 68
µg/µl yeast tRNA). Samples were phenol/chloroform extracted and
ethanol precipitated. Precipitated DNA was dissolved in 28 µl of gel
loading buffer [80% deionized formamide in 45 mM boric acid, 45 mM
Tris base, 1 mM EDTA, 0.05% (w/v) bromphenol blue, 0.05% (w/v) xylene
cyanol]; an aliquot (7 µl) of each sample was denatured by heating
and loaded onto the denaturing sequencing gel.
Electrophoretic mobility shift assay (EMSA) and supershift
assay
EMSAs for the putative binding sites were performed as described
(20)
. Ten or 15 µg of nuclear protein from normal human
liver, HCC, or HepG2 cells was preincubated with 2 µg of poly(dI-dC)
in a buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 0.1 mM EDTA, 1
mM dithiothreitol, 5 mM MgCl2, and 10% glycerol
for 10 min on ice. 32P-end labeled
double-stranded DNA fragments (-354 to -328 and -60 to -40) were
then added with or without a 100-fold excess of unlabeled specific
probe as competitor. Mixtures were incubated for 20 min on ice, loaded
on a 4% nondenaturing polyacrylamide gel, and subjected to
electrophoresis in 50 mM Tris, 45 mM borate, and 0.5 mM EDTA (pH 8.0).
Further confirmation of the identity of the binding proteins was
conducted by antibody supershift assays for c-Myb and Sp1
(Biotechnology, Lake Placid, NY, or Santa Cruz Biotechnology, Santa
Cruz); 1.5 µl of these antibodies was added to respective samples
after 20 min of incubation with the labeled probe, and all samples were
further incubated for another 45 min on ice before
electrophoresis. Gels were dried and subjected to autoradiography.
Western blot analysis for Sp1
Western blot analysis was performed on nuclear proteins
extracted from normal or cancerous liver tissues using antibodies
against Sp1 as described (11)
.
Transfection assays
To study the effect of mutation of putative binding sites on the
transcriptional activities of the MAT2A promoter fragments, HepG2 cells
(1x106 cells in 4 ml medium) were transiently
transfected with 8 µg of wild-type or mutant MAT2A promoter
luciferase gene constructs or promoterless pGL3-enhancer vector (as
negative control) and 2 µg of a ß-galactosidase expression plasmid
(as an internal standard of transfection efficiency) using the calcium
phosphate precipitation method (23)
. After 24 h,
cells were harvested and lysed in 1 ml of reporter lysis buffer
(Luciferase Assay System, Promega). The luciferase assay was performed
on 20 µl of the cleared lysate and 100 µl of luciferase assay
reagent using a TD-20/20 Luminometer (Promega). The ß-galactosidase
assay was conducted according to the supplier’s instructions
(ß-Galactosidase Enzyme Assay System, Promega) using 150 µl of the
cell lysate. Protein concentration was determined using the Bio-Rad
Protein Assay (Bio-Rad, Hercules, CA). The luciferase activity of each
transfection was expressed as luciferase activity/ß-galactosidase
activity/protein concentration.
To confirm the effect of the regulatory elements and transcription
factors, HepG2 cells were transfected with chimeric constructs
containing -355 to -324 of the MAT2A (wild-type or mutant where AA at
positions -344 and -345 were mutated to TT) linked to the pGL-3
promoter-luciferase vector. Some cells were also cotransfected with the
c-Myb expression vector in the presence or absence of
ZnCl2 (120 µM for 15 h) as described
(15)
. SL-2 cells were transfected with chimeric constructs
containing -64 to -32 of the MAT2A (wild-type or mutant where GG at
positions -55 and -56 were mutated to TT) linked to the pGL-3
promoter-luciferase vector. Some cells were also cotransfected with the
Sp1 expression vector (16)
, except that transfection was
performed using the Superfect Transfection Reagent according to
protocol provided (Qiagen, Valencia, CA).
Statistical analysis
Data are given as mean ± SE. For changes in
mRNA levels, ratios of c-myb or Sp1 to ß-actin or 18S densitometric
values were compared. Statistical analysis was performed using ANOVA,
followed by Fisher’s test for multiple comparisons and unpaired
Student’s t test for comparisons between normal and
cancerous liver. Significance was defined as P<0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows results of the DNase I footprinting analysis using
double-stranded fragment corresponding to nucleotides -494 to -152 of
the human MAT2A gene. One protected site was identified using nuclear
proteins from HCC but not from normal liver on both strands. Note that
the same protected site was also seen using increasing amount of
nuclear proteins from HepG2 cells. Figure 2
shows results of the DNase I footprinting analysis using
double-stranded fragment corresponding to nucleotides -148 to +64 of
the human MAT2A gene. Two protected sites were identified using nuclear
proteins from HCC and HepG2 cells, but not from normal liver on both
strands. One of the protected sites (+35 to +60) is downstream of the
transcriptional start site. We concentrated our initial effort on the
two protected sites upstream of the transcriptional start site
(designated +1).
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Since in vitro DNase I footprinting analysis may not reflect the
situation in vivo, we also examined whether these sites are protected
using LM-PCR in vivo footprinting. HepG2 cells were used for these
studies since MAT2A is induced in these cells as compared to normal
differentiated hepatocytes (9)
and in vitro DNase I
footprinting analysis yielded identical results using nuclear proteins
from HCC and HepG2 cells (Figs. 1
, 2)
. Figure 3
shows that the same sites (-352 to -314 and -70 to -30) are
protected after in vivo DNase I treatment whereas the in vitro digested
DNA show no protection.
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Figure 4
shows the sequence of the MAT2A promoter protected on both strands of
the DNA fragments and the consensus binding sites present for
transcription factors. There are potential binding sites for many
transcription factors, including nuclear factor 1 (NF1), c-Myb, nuclear
factor-erythroid 2-related factor (Nrf-2), nuclear factor kappa B
(NF-
B), and activator protein 1 (AP1) in the first protected region,
-354 to -312, of the human MAT2A. In the region -73 to -28,
potential binding sites include NF1, Sp1, and activator protein 2
(AP2). Two probes were designed for subsequent EMSA and supershift
analysis: the first probe spans the MAT2A sequence -354 to -328; the
second probe spans the MAT2A sequence -60 to -40.
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EMSA and supershift analysis
Figure 5
shows the results of EMSA using the probe that spans -354 to -328.
Two distinct bands can be seen on the gel shift in the presence of
nuclear proteins from normal liver or HCC, which disappeared when
unlabeled specific oligonucleotides were added in 100x excess. There
is increased binding activity of both bands in HCC. The top band is due
to c-Myb binding as demonstrated by supershift analysis (Fig. 5)
.
Figure 6
shows the results of EMSA using the probe that spans -60 to -40. In
normal liver, one distinct band can be seen that disappeared when
unlabeled specific oligonucleotides were added in 100x excess. In HCC
there is a second strong intensity band that supershifted in the
presence of anti-Sp1 antibodies (Fig. 6)
, indicating the identity of
the protein as Sp1.
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Expression of c-Myb and Sp1 in normal liver and HCC
To see whether increased binding of c-Myb and Sp1 to the MAT2A
promoter in HCC occurs because of increased expression of these
transcription factors, we examined the expression of c-myb by RT-PCR
and Sp1 by Northern blot analysis. c-Myb mRNA was not detectable in
normal or cancerous liver on routine Northern blot analysis (data not
shown). However, on RT-PCR, there is a clear increase in the expression
of c-myb in HCC (276±4% of normal liver by densitometric analysis;
P<0.05 by unpaired t test) (Fig. 7
). There is also a significant increase in the mRNA level of Sp1 in HCC
(230±27% of normal liver by densitometric analysis;
P<0.05 by unpaired t test) (Fig. 8A
). This translated to increased levels of nuclear Sp1
protein in HCC (Fig. 8B
).
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Effect of mutation of putative binding sites on promoter activity
To examine the contribution of these putative
cis-acting elements on the MAT2A promoter activity, HepG2
cells were transfected with MAT2A constructs that are mutated in either
the putative c-Myb site (from 5'-CGGCCAACGGTCGGAAGG-3' to
5'-CGCGTGCTAGCCCGGGCT-3')
or Sp1 site (from 5'-GGGGCGGGGC-3' to
5'-GGCTAGCCCC-3'). Figure 9
shows that luciferase activity driven by MAT2A promoter constructs
containing the mutated c-Myb or Sp1 site was reduced by 67% and 50%,
respectively.
|
MAT2A regulatory elements in heterologous promoter and effect of
overexpression of transcription factors c-Myb and Sp1
To further demonstrate the functionality of the
cis-acting elements and the transcription factors, HepG2
cells were transfected with chimeric constructs containing -355 to
-324 of the MAT2A (wild-type or mutant, where AA at positions -344
and -345 were mutated to TT) linked to the pGL-3 promoter-luciferase
vector. Some cells were also cotransfected with the c-Myb expression
vector in the presence or absence of ZnCl2 (120
µM for 15 h) (15)
. Figure 10
shows the effect of transfection with the c-Myb expression vector on
the mRNA levels of c-myb and MAT2A as well as binding activity of c-Myb
to the MAT2A promoter. After transfection with the c-myb expression
vector (in the presence of ZnCl2), c-myb mRNA
increased in a time-dependent fashion (178% of control at 8 h,
222% of control at 12 h, 253% of control at 16 h) (Fig. 10A
). The endogenous MAT2A mRNA level also increased (145%
of control at 8 h, 180% control at 12 h, 228% of control at
16 h) (Fig. 10B
). After 20 h of transfection, the
binding activity of c-Myb to MAT2A promoter fragment increased (Fig. 10C
). Table 1
summarizes the results of cotransfection of HepG2 cells with chimeric
constructs and the c-Myb expression vector. The MAT2A sequence -355 to
-324 increased the luciferase activity driven by the pGL-3 promoter by
80%. Mutation of two of the bases resulted in a slight decrease in the
induction. When cells were cotransfected with the c-Myb expression
vector in the presence of ZnCl2, the luciferase
activity driven by the pGL-3 promoter increased 116-fold.
Overexpression of c-Myb increased the luciferase activity driven by the
chimeric construct containing wild-type MAT2A sequence -355 to -324
by 273-fold. When two of the bases were mutated, this increase was
largely blocked.
|
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The putative Sp1 element and the role of Sp1 were assessed using the
SL2 cells as they lack endogenous Sp1 activity (16)
. After
transfection of SL-2 cells with the Sp1 expression vector, Sp1 mRNA
level increased in a time-dependent manner (Fig. 10D
). As
shown in Table 1
, the wild-type Sp1 MAT2A element had little influence
on pGL-3 promoter activity in SL-2 cells since there is no Sp1
expressed. Cotransfection of the Sp1 expression vector increased the
luciferase activity driven by pGL-3 promoter by 23-fold. Overexpression
of Sp1 increased the luciferase activity driven by the chimeric
construct containing wild-type MAT2A sequence -64 to -32 by 157-fold.
When two of the bases were mutated, this increase was also largely
blocked.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In mammals, two distinct
genes encode for the enzyme MAT (3
4
5)
. MAT1A is a
liver-specific gene expressed in the liver shortly before birth and
becomes the major form of MAT as the liver matures (7
, 8)
.
It is a marker for the differentiated or mature liver phenotype. In
contrast, MAT2A is expressed in all nonhepatic tissues as well as
during periods of rapid liver growth (3
, 6
7
8
9
10
11
12)
. Although
the two MAT genes are highly homologous, the enzymes they encode for
are different in the kinetic profiles and regulatory properties
(24)
. Due to these differences, the type of MAT expressed
by a cell can influence the cell’s steady-state SAM level and
methylation status. Using a cell line model that differs only in the
type of MAT expressed, cells that expressed MAT1A had the highest
intracellular SAM level and DNA methylation whereas cells that
expressed MAT2A showed the opposite (12)
. Cells that
expressed MAT2A grew faster than cells that expressed MAT1A
(12)
. Thus, the switch in MAT expression in liver cancer
is pathogenetically important since it offers the cancerous cell a
growth advantage.
Despite the importance of MAT2A, little is known about its
transcriptional regulation. MAT2A gene expression is influenced by the
cell cycle as evident by its induction during liver regeneration,
malignant liver transformation, and T lymphocyte activation (10
, 14
, 25)
. In all cases the mechanism involved in part increased
transcription (10
, 14
, 25)
. It has been speculated that
the induction in MAT2A and MAT II may be a mechanism for the cell to
provide an increased supply of SAM, the precursor to polyamine
synthesis required for cell growth (25)
. We have studied
transcriptional regulation of MAT2A, particularly the molecular
mechanism for its up-regulation in human liver cancer. In the current
work we tested the hypothesis that one of the mechanisms of the
transcriptional up-regulation of MAT2A in HCC is due to a difference in
transcription factor binding to important cis-acting
elements of the MAT2A promoter.
We had previously shown that the region that is important for MAT2A
promoter activity lies between -571 to +60 (13
, 14)
.
DNase I footprinting analysis was first performed to see whether there
is any difference between normal and cancerous liver in the binding of
proteins to this region of the MAT2A promoter. We observed a striking
difference in protein binding to the MAT2A promoter between normal and
cancerous liver. There is no protection observed on DNase I
footprinting analysis when nuclear proteins from normal liver is used
for any of the fragments examined. Protection was noted in the presence
of increasing amount of nuclear proteins from either HCC or HepG2
cells. The protected sites identified on both strands were -354 to
-312, -73 to -28 and +35 to +60. The first two protected sites
contain consensus binding sites for several transcription factors. The
third site is downstream of the transcriptional start site. It may
represent binding of TFIID or part of its complex, which has been shown
to yield DNase I protection downstream from the transcriptional
start site (26)
. More work will be necessary to see
whether this is indeed the case.
In vitro DNase I footprinting analysis may not reflect the situation in vivo; thus, LM-PCR in vivo footprinting analysis was carried out using HepG2 cells. DNA digested in vitro served as control for comparison. These studies confirmed that the same sites (-354 to -314 and -70 to -30) were protected in vivo in HepG2 cells.
The region -354 to -314 contains consensus binding sites for NF1
(-359 to -342), IK2 (-357 to -342, -338 to -327, and -314 to
-303), c-Myb (-350 to -333), NRF2 (-341 to -332), 
EF1 (-334 to
-324), NFB (-334 to -325), AP1 (-327 to -317), SREBP1 (-323 to
-313), and MZF1 (-314 to -317). The region -73 to -30 contains
consensus binding sites for NF1 (-75 to -58), AP2 (-62 to -51, and
-36 to -25) and Sp1 (-58 to -48). Candidate factors unlikely to be
important for transcriptional up-regulation of MAT2A in HCC are EF1,
a zinc finger protein that is expressed in lens, mesodermal tissues,
and the nervous system (27)
, and MZF1, the myeloid zinc
finger gene that has been shown to repress transcription in
nonhematopoietic cells and activate transcription in hematopoietic
cells (28)
. For our initial study, we focused on c-Myb and
Sp1.
c-Myb is a transcription factor encoded by the c-Myb proto-oncogene and
is best known as a regulator of cell growth and differentiation in
hematopoietic cells (29)
. c-Myb has been detected in
normal colon mucosa, developing neural tissue, and certain tumors
including colon, lung, and breast carcinomas (30)
. It is
also induced in activated human stellate cells (30)
.
However, c-Myb expression has not been described in hepatocytes or in
liver cancer. Indeed, the c-myb mRNA level was low and detectable only
with RT-PCR. Sp1 is a ubiquitous transcription factor, but its level
varies with the cell’s phenotype and is induced with stellate cell
activation (31)
. However, whether Sp1 expression is
altered in HCC is also unknown.
Using EMSA and supershift analysis, we demonstrated that there is
increased c-Myb and Sp1 binding to these regions of the MAT2A promoter
in HCC. Using the probe -354 to -328, another band is increased in
HCC (Fig. 5
, second band from the top). The nature of the binding
protein is unclear, but is likely to be one of the candidate
transcription factors such as NF1 (-359 to -342), IK2 (-357 to
-342, -338 to -327), NRF2 (-341 to -332), or NF-B (-334 to
-325). More work will be necessary to elucidate this. Using the probe
-60 to -40, there is a band that is present only in HCC samples. This
turned out to be Sp1 based on supershift analysis (Fig. 6)
.
Increased transcription factor binding to the MAT2A promoter in HCC can
be due to increased levels of the transcription factors and/or
increased access to the binding site. There is clear evidence of
increased expression of both c-myb and Sp1 in HCC. We have also
observed a difference in MAT2A promoter methylation between normal
liver and HCC (14)
. Specifically, the MAT2A promoter is
hypomethylated in HCC as compared to normal liver (14)
.
Methylation of these sites would prevent access of transcription
factors. Whether this may also play a role remains to be determined. In
HepG2 cells, these sites are clearly accessible to transcription factor
binding as evident on in vivo footprinting analysis.
Increased binding of transcription factor to the promoter does not
prove functional significance of either the cis-acting
element or the transcription factor. To prove functional importance of
the putative cis-acting elements, we evaluated the effect of
mutation (-348 to -333 and -56 to -49) on MAT2A promoter activity.
These mutations resulted in a 50–67% loss of promoter activity (Fig. 9)
, confirming their importance. However, in these mutational analyses,
many bases were mutated, which could have affected binding of
transcription factors other than c-Myb and Sp1. To definitively
establish the importance of the regulatory elements and the role of
c-Myb and Sp1, the ability of the regulatory element to increase
reporter activity driven by a heterologous promoter in the presence or
absence of overexpression of the trans-activating factor was
critically examined. Thus, the regulatory element that contains the
putative c-Myb or Sp1 binding site increased the reporter activity of a
heterologous promoter, especially when c-Myb or Sp1 was overexpressed.
In these experiments, we are confident of the role of these
transcription factors as the induction observed were largely eliminated
when only two bases were mutated, which was unlikely to affect binding
other than by c-Myb or Sp1.
In summary, the present work presents several novel data. First, the human MAT2A promoter contains functional c-Myb and Sp1 binding sites that are important for the overall promoter activity. Second, there is increased expression of c-myb and Sp1 at the mRNA level in HCC. Third, increased binding of these transcription factors to their corresponding cis-acting elements present in the MAT2A promoter occurs in HCC and is partly responsible for the transcriptional up-regulation of MAT2A in HCC.
|
ACKNOWLEDGMENTS |
|---|
Received for publication January 19, 2001. Accepted for publication March 12, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-S-adenosyl-L-methionine synthetase gene expression during T-lymphocyte activation. Biochem. J. 319,929-933
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