From the Departments of Surgery,
** Microbiology and Immunology, and ¶ Biochemistry, University of
Tennessee, Memphis, Tennessee 38163 and the § Veterans
Affairs Medical Center, Memphis, Tennessee 38104
Received for publication, March 29, 2001, and in revised form, May 1, 2001
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ABSTRACT |
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Methionine adenosyltransferase (MAT) catalyzes
the biosynthesis of S-adenosylmethionine (AdoMet), a key
molecule in transmethylation reactions and polyamine
biosynthesis. The MAT II isozyme consists of a catalytic Methionine adenosyltransferase
(MAT)1
(ATP·L-methionine S-adenosyltransferase) (EC
2.5.1.6) catalyzes the biosynthesis of S-adenosylmethionine
(AdoMet) (1, 2). AdoMet is the major methyl donor in transmethylation
reactions and the propylamine donor in the biosynthesis of polyamines
(3-5). Furthermore, AdoMet participates as a co-factor in key
metabolic pathways (3-5). Most species studied to date have more than
one MAT isozyme (6). In mammals, the two major MAT isozymes are
designated MAT I/III and MAT II (7-10). The MAT I/III isozymes are,
respectively, a tetramer and dimer of a catalytic In contrast to MAT The expression of the MAT II Based on our previous results, we hypothesized that the down-regulation
of the MAT II Isolation and Genomic Organization of the MAT2B Gene--
Based
on the sequence of the previously reported MAT II Chromosomal Localization of the Human MAT2B Subunit
Gene--
Highly purified DNA was obtained from the MAT2B
P1 genomic clone 22646 using the Wizard PureFection DNA purification
system (Promega, Madison, WI). The DNA was labeled with digoxigenin
dUTP by nick translation combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from
phytohemagglutinin-stimulated peripheral blood lymphocytes in a
solution containing 50% formamide, 10% dextran sulfate, and 2× SSC.
Specific hybridization signals were detected by using
fluorescein-conjugated antidigoxigenin antibodies. The chromosomes were
counterstained with propidium iodide and analyzed.
Mapping the MAT2B Gene Transcription Start
Site--
Identification of the transcription start site was done by
primer extension analysis using poly(A)+ RNA prepared from
normal human lymphocytes. The primer extension reaction was conducted
using the avian myeloblastosis virus reverse transcriptase primer
extension system (Promega). Poly(A)+ RNA was isolated from
500 ml of human blood by the Poly(A)Tract mRNA isolation system
(Promega). Two primers, Bra1 5'-GTTCTTTCTCCCTCCCCACCAT-3' (complementary to positions +22-+1 of the open reading frame) and Bra2
5'-CAGTTCTTTCTCCCTCCCCACC-3' (complementary to positions +24-+3 of the
open reading frame), were synthesized and end-labeled with
[ Cloning of the Human MAT2B Gene Promoter--
We cloned and
sequenced 3.5 kbp of the 5'-flanking DNA of the human MAT2B
gene. The promoter was contained within 1.1 kbp. The primers
5'-WP (5'-GCTCGAGTAAGATGATCTTGGC) and 3'-WP
(5'-GAAGCTTGCCCGCCGTCTTCAC) were designed to amplify the region
Generation of Luciferase Reporter Constructs of the MAT2B
Promoter--
A 1.1-kbp XhoI/HindIII-digested
fragment of the MAT2B gene containing the 5'-flanking region
starting at position +204 from the transcription start site was cloned
upstream of the firefly luciferase reporter gene in the pGL3-Enhancer
vector (Promega). Directional insertion was verified by restriction
digestion and by sequencing the clone from both directions. Subsequent
deletion constructs were generated by PCR using sequence-specific
primers (Table I) containing the
restriction sites XhoI/HindIII as described above. The purified PCR products were cloned into the pGEM-TEasy vector
(Promega) and sequenced for verification. The cloned deletion constructs were excised from the pGEM-TEasy vector using
XhoI/HindIII and then cloned upstream from the
firefly luciferase gene into the pGL3-Enhancer vector (Promega). All
constructs were verified by sequence analysis.
In Vivo Analysis of the MAT2B Promoter Activity--
The
functional expression of the pGL3-MAT2B promoter deletion
constructs was analyzed in Cos-1 and Jurkat T cells as detailed elsewhere (27).
In Vitro Analysis of the MAT2B Promoter Activity by
Electrophoretic Mobility Shift (EMSA) and Supershift
Assays--
Double-stranded oligonucleotide probes were generated by
PCR amplification with 32P end-labeled primers. The
amplified DNA representing specific regions of the proximal promoter of
the MAT2B gene was generated using the primers listed in
Table I. Jurkat cell nuclear extracts were prepared as described
previously (27). The binding reactions were performed in a 20-µl
final volume by incubating 60 fmol (~50,000 cpm) of
32P-labeled probe with 5 µg of crude nuclear extracts
from Jurkat cells in the presence of 2 µg of poly(dI-dC), 1 µg of
salmon sperm DNA in 20 mM HEPES, pH 7.9, 50 mM
KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 6 µg of bovine
serum albumin for 40 min on ice. The reactions were conducted in the absence or presence of specific or nonspecific competitors, both added
at a 100-fold molar excess. Specific competitors were cold probes;
nonspecific competitors were unlabeled PCR amplification products of an
unrelated DNA sequence, free of sites of interest, and similar in size
to specific competitor. An Sp1-specific competitor was purchased from
Santa Cruz Biotechnology, Inc. (catalog no. SC 2502). The DNA·protein
complexes formed were analyzed by electrophoresis on nondenaturing 4%
polyacrylamide gels. The gels were pre-run for 1 h at 100 V, and
electrophoresis was conducted at a 30-mAmp constant current.
Supershift assays were performed using antibodies (Abs) to specific
factors that have corresponding recognition elements within the
MAT2B promoter region analyzed. Binding reactions were
carried out as described above in the absence or presence of 2 µg of
an Ab specific to one of the transcription factors of interest. The Ab
was premixed with the nuclear extract, incubated for 30 min at room
temperature prior to the addition of radiolabeled probe, or added after
the binding reaction was completed and incubated for 30 min on ice. The
following Abs (Santa Cruz Biotechnology, Inc.) were used in supershift
analyses: rabbit polyclonal anti-Sp1 (catalog no. SC-59X), anti-Sp2
(catalog no. SC-643X), anti-Sp3 (catalog no. SC-644X), anti-Sp4
(catalog no. SC-645X), and anti-NF1 monoclonal Ab (catalog no.
SC-870X).
Chromatin Immunoprecipitation--
Cross-linking between
transcription factors and chromatin was achieved in Jurkat cells by
following the method described by Yang et al. (32). Briefly,
formaldehyde was added to cells at a final concentration of 1% for 10 min, and 0.125 M glycine was used to stop the reaction. The
cells were washed three times with cold PBS and once with PBS
containing 1 mM phenylmethylsulfonyl fluoride and then
lysed in 2 ml of cell lysis buffer (5 mM Pipes-KOH, pH 8.0, 85 mM KCl, and 0.5% (v/v) Nonidet P-40) in the presence of
protease inhibitors (100 ng/ml leupeptin, 100 ng/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The lysates were
homogenized, and the nuclei were recovered by centrifugation at
250 × g for 10 min and resuspended in 0.2 ml of
nuclear lysis buffer (50 mM Tris, pH 8.0, 10 mM
EDTA, 1% (w/v) SDS plus the protease inhibitors. The lysate was
sonicated to shear the chromatin to an average length of <2 kb. The
samples were diluted 10-fold with the immunoprecipitation dilution
buffer (1% (v/v) Triton X-100, 16.7 mM Tris, pH 8.0, 1.2 mM EDTA, and 167 mM NaCl plus the protease
inhibitors). A 240-µl slurry of salmon sperm DNA/protein A-agarose
(Upstate Biotechnology, Inc., Lake Placid, NY) was added to reduce
nonspecific binding, and the mixture was rotated for 1 h at
4 °C then centrifuged at 500 × g for 1 min.
Precleared chromatin solutions were incubated with antibody to Sp1 (10 µg), Sp3 (10 µg) (Santa Cruz Biotechnology, Inc.) or no antibody
(negative control) and rotated at 4 °C for 12 h. Immune
complexes were collected by adding 80 µl of salmon sperm DNA/protein
A-agarose slurry for 4 h with rotation. Samples were washed four
times with 1 ml of wash buffer (0.1% (v/v) Triton X-100, 20 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM EDTA), and the immunoprecipitated material was eluted by
three successive 5-min incubations with 150 µl of elution buffer (1%
(w/v) SDS and 50 mM NaHCO3). To reverse the
formaldehyde-induced cross-linking, the eluates were pooled, NaCl was
added at a final concentration of 0.3 M, and the samples
were incubated at 65 °C for 4 h. This step was followed by
digestion in 10 µl of 2 M Tris, pH 6.8, 10 µl of 0.5 M EDTA, and 2 µl of proteinase K (20 mg/ml). The samples were incubated for 2 h at 45 °C, and then the DNA was extracted with phenol/CHCl3 followed by ethanol precipitation
and resuspension in 50 µl of sterile H2O. Five
µl of DNA solution were used as a template in PCR analysis.
Primers were designed to amplify the regions from In Vivo and in Vitro Analysis of the Effect of MAT2B
Promoter Mutations--
Several mutations were introduced into the
clones pGL3-MAT2B(
Site-directed mutagenesis was performed on pGL3-MAT2B
promoter constructs by PCR using Pfu turbo DNA polymerase
(Stratagene) and the oligos listed above following the method of Wang
and Wilkinson (33). Briefly, after the PCR reaction the product was
incubated at 37 °C with DpnI (Promega) to remove the
methylated template DNA. A portion of the digested reaction mix was
analyzed on a 1% agarose gel to verify the PCR product size. E. coli strain JM109 was transformed using 5 µl of the
PCR-amplified vector, and six clones of each transformation were
sequenced to confirm the presence of the desired mutations. The mutated
DNA products were excised from the pGL3 vector using XhoI
and HindIII and individually recloned into an unamplified
pGL3-Enhancer vector (Promega) to ensure that the vector itself was not
modified during the mutagenesis reaction. The in vitro and
in vivo activity of each mutant construct was determined as
described above. EMSA probes Genomic Organization of the MAT2B Gene--
Human genomic clone
22646 was determined to harbor the MAT2B gene and a
significant portion of its 5'-flanking sequence. A series of primers
were designed, based on the known MAT2B cDNA sequence,
to determine the structure of the MAT2B gene. The gene consisted of seven exons interrupted by six introns spanning ~6.8 kbp
of genomic DNA (Fig. 1). The sizes and
locations of the various exons and introns as well as the donor and
acceptor sequence are summarized in the inset table. All
boundaries were found to conform to the GT-AG rule (34). Exon 1 contained 203 bp of 5'-noncoding region and 63 bp of coding sequence.
Exon 7 contained 171 bp of coding sequence and 802 bp of
3'-untranslated sequence.
Chromosomal Localization of the Human MAT2B Subunit Gene--
A
total of 80 metaphase cells was analyzed as detailed under "Materials
and Methods"; 69 of those exhibited specific labeling with DNA from
clone 22646 on chromosome 5. An anonymous genomic probe, previously
mapped to 5q22 and confirmed by cohybridization with a probe from the
cri du chat locus, hybridized to the same chromosome as clone 22646, confirming the location as the long arm of chromosome 5. Ten individual
measurements of specifically labeled chromosome 5 demonstrated that the
22646 clone hybridized to a position 89% of the distance from the
centromere to the telomere of 5q, an area that corresponded to the
interface between bands 5q34 and 5q35.1 (data not shown).
Mapping the MAT2B Gene Transcription Start Site--
The
transcription start site was identified by primer extension analysis.
Two primers, Bra1 and Bra2, corresponding to +22 and +24 of the
MAT2B open reading frame were annealed to mRNA prepared
from normal human lymphocytes and extended in the presence of avian
myeloblastosis virus reverse transcriptase as described under
"Materials and Methods." The primer Bra2 yielded a 227-bp product,
and Bra1 yielded a 225-bp product (Fig.
2). Therefore, transcription was shown to
start 203 bp upstream of the MAT2B translation start
site.
Cloning and Sequencing of the Human MAT2B Gene Promoter--
The
sequence of ~1.1 kbp of the 5'-flanking region of the
MAT2B gene is shown in Fig. 3.
The sequence from In Vivo Analysis of the MAT2B Promoter Activity--
A series of
promoter deletion constructs coupled to a firefly luciferase reporter
gene in a pGL3-Enhancer vector were generated as described under
"Materials and Methods" and used to analyze functional expression
in Cos-1 and Jurkat human leukemic T cells. There was little difference
in the pattern of expression of the pGL3-MAT2B promoter
constructs in both types of cells (data not shown). Successive
deletions from Identification of Functional Sites in the MAT2B Promoter Activity
by EMSA and Supershift Assays--
Analysis of the proximal
MAT2B promoter sequence identified several putative
recognition sites for known transcription factors. These regions were
subjected to further functional analysis using EMSA, supershift assays,
and mutation of specific sites. Competition experiments showed that the
complexes that formed on a probe representing the region from
The region from
Chromatin immunoprecipitation studies showed that both the
anti-Sp1 and anti-Sp3 antibodies were independently able to pull down
the MAT2B promoter, because PCR products were obtained in reactions with primers covering the regions from Effect of Mutating Specific Sites in the MAT2B Promoter on the in
Vitro Promoter Activity--
Several mutations were also made to
probes covering the region from Effect of Mutating Specific Sites in the MAT2B Promoter on the in
Vivo Promoter Activity--
The effect of mutating several putative
factor recognition sites located in the region between The MAT II isozyme is expressed in all tissues in which it is
found as an oligomer that comprises catalytic In leukemic T cells, both the To shed light on the mechanisms underlying the differences in MAT II
The transcription factor Sp3 seems to bind to the Sp1 site at +9/+15,
although we cannot rule out that Sp1 and Sp2 are also part of the
complexes that form on this site. That Sp3 is involved in regulating
MAT2B promoter activity is particularly interesting in light
of our recent studies that showed that this transcription factor plays
a key role in regulating the MAT2A gene, which encodes the
catalytic subunit of the same enzyme. As mentioned above, stimulation
of T cells with a physiological stimulus induces the expression of 2
and a regulatory
subunit. Down-regulation of the MAT II
subunit
expression causes a 6-10-fold increase in intracellular AdoMet
levels. To understand the mechanism by which the
subunit
expression is regulated, we cloned the MAT2B gene,
determined its organization, characterized its 5'-flanking sequences,
and elucidated the in vitro and in vivo
regulation of its promoter. Transcription of the MAT2B gene
initiates at position
203 relative to the translation start site.
Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between
4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position
115 provided full activity; this was attributed to a TATA at
32. The Sp1 site at position +9 was key for
the formation of protein·DNA complexes. Mutation of both the Sp1 site
at +9 and the TATA at
32 reduced promoter activity to its minimal
level. Supershift assays showed no effect of the anti-Sp1 antibody on
complex formation, whereas the anti-Sp3 antibody had a strong effect on
protein·DNA complex formation, suggesting that Sp3 is one of the main
factors binding to this Sp1 site. Chromatin immunoprecipitation assays
supported the involvement of both Sp1 and Sp3 in complexes formed on
the MAT2B promoter. The data show that the 5'-untranslated
sequences play an important role in regulating the MAT2B
gene and identifies the Sp1 site at +9 as a potential target for
modulating MAT2B expression, a process that can have a
major effect on intracellular AdoMet levels.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 subunit that is
expressed only in liver (7, 11-13). The MAT II isozyme is expressed in all tissues including the liver (9, 14-21). Previous studies from our
group (15, 22) have determined that MAT II from human leukemic T and B
cells, as well from activated human lymphocytes, is a hetero-oligomer
that consists of
2 (53 kDa),
2' (51 kDa), and
(38 kDa)
subunits. The
2/
hetero-oligomeric composition of MAT II was also
determined in bovine brain, Ehrlich ascites tumor, and calf thymus (15,
19). The
2 subunit is responsible for the enzyme catalytic activity
and is post-translationally modified to generate
2' (15, 22-26).
Recently, Halim et al. (27) reported the characterization
and regulation of the human MAT2A gene and found it to be
remarkably similar to that of the rat and mouse MAT2A genes
(28, 29) as well as to the human MAT1A gene (30), which
encodes the liver-specific
1 subunit.
subunits, which are highly conserved throughout
evolution, the
subunit of MAT II seems to be only present in the
mammalian species (15, 19). Recently, we cloned and characterized the
human MAT II
subunit (26, 31), found that it has no catalytic
activity, and confirmed that it acts as a regulatory subunit for the
enzyme. When
associates with the
subunit it alters its kinetic
properties and renders MAT II more susceptible to product inhibition by
AdoMet (26, 31). Interestingly, the human
subunit can also interact
with the
1 subunit of MAT I/III and the Escherichia coli
MAT subunit and alter their kinetic properties as well (26,
31).
2,
2', and
subunits varies
considerably in different tissues. The
2,
2', and
subunits are constitutively expressed at high levels in leukemic cells and at
low levels in normal resting T cells. Stimulation of normal human
lymphocytes results in marked changes in the level of expression of
these subunits. Nonphysiological polyclonal mitogenic stimulation of
human T cells induces an increased expression of the
2/
2' subunits but not the
subunits (22, 25). By contrast, physiological stimulation of T cells by bacterial superantigens induces an
up-regulation of the
2/
2' subunits and a down-regulation of the
subunit (25). This results in the formation of
2 and
2' homo-
and/or hetero-oligomers (no
) with a 3-fold higher
Km for L-Met. The form of MAT II without
is resistant to product inhibition by AdoMet when compared with the
form of MAT II found in resting or leukemic T cells (includes
)
(25). Importantly, the down-regulation of the
subunit in
physiologically stimulated T cells was accompanied by a 6-10-fold
increase in intracellular AdoMet levels, presumably caused by the loss
of product inhibition of the enzyme (25). An increase in AdoMet levels
is likely to stimulate certain transmethylation reactions catalyzed by
methyltransferases with a relatively high Km value
for AdoMet.
subunit may be an important event in the
physiological stimulation of T cells, and we sought to characterize the
regulation of expression of the MAT2B gene. Here we report
the chromosomal localization and organization of the MAT2B
gene and provide a detailed characterization of the structure and
function of its promoter.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit cDNA
(31), forward and reverse primers spanning the entire open reading
frame were designed and used to amplify genomic DNA isolated from
normal human lymphocytes. Reactions yielding larger than expected
products suggested the presence of introns, and these PCR products were
cloned and sequenced to verify the authenticity of introns. Sequences
at the intron-exon boundaries of the MAT2B gene were
determined by aligning the cDNA sequence of MAT2B
cDNA with the genomic sequence. A set of primers, GSF
(5'-GATTCCTGAGTCCTGTCTTAG) and GSR (5'-GCACTTTTGGCTTTCACTCAG),
that amplified a 79-bp product that included the 3' end of intron 4 and
the 5' end of exon 5 were used to screen a human P1 genomic library
(Genome Systems, Inc., St. Louis, MO). Positive clones were partially
sequenced to ascertain the presence of the MAT2B gene. Clone
22646 was selected for further characterization and used to determine
the chromosomal location of the MAT2B gene, determine gene
organization, verify intron positions, and characterize
MAT2B promoter function.
-32P]ATP and T4 polynucleotide kinase. Free
[
-32P]ATP was removed by the QIAquick nucleotide
removal kit (Qiagen). Reverse primer extension was carried out using 3 µg of mRNA and 5 × 105 cpm of the
labeled primer. The M13mp18 sequencing ladder was prepared by
fmol sequencing (Promega) and run alongside the samples on
8% urea/polyacrylamide gels as a size marker.
998/+204 with respect to the transcription start site. The primers
introduced an XhoI site at the 5' end and a
HindIII site at the 3' end of the cloned promoter. The
Pfu-amplified fragment was cloned into the pGEM-TEasy vector (Promega), and the cloned MAT2B promoter was sequenced in
both directions with overlapping segments to verify the sequence and confirm the integrity of the cloned promoter. The cloned promoter DNA
was excised from the pGEM-TEasy vector by digestion with
XhoI and HindIII (Promega), purified, and
processed as described below.
Sequence of primers used for MAT2B promoter constructs
174 or
4 to + 52. The primer 5'-BP8 (5'-ACTCGAGAAACTCAAGGCGATCCACTT) or 5'-BP9
(5'-CCTCGAGACCGCGCGTACC) was paired with primer 3'-BP8 (5'-TCTGCCCCCAGCCCACG). The PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide staining.
174)-LUC,
pGL3-MAT2B(
115)-LUC, and
pGL3-MAT2B(
4)-LUC according to a
modification of the method of Wang and Wilkinson (33) that targets
putative factor recognition sites. Mutation of the TBF site at
111/
105 was generated using the primers 5'-MU1
(5'-GCTCGAGAATAACAAGCACTCAAATAAAATCTCC) and 3'-MU1
(5'-GAGATTTTATTTGAGTGCTTGTTATTCTCGAGCC). Another possible TBF site at
95/
91 was mutated using the primers 5'-MU2
(5'-GCACTCAAATACCATCTCCGAAACAAAACCTG) and 3'-MU2
(5'-CAGGTTTTGTTTCGGAGATGGTATTTGAGTGC). The TATA box at
32/
28 was
mutated with oligos 5'-MU3 (5'-CTTTTGTGTGTCGCTTTTTGCATCGGCGCGTG) and
3'-MU3 (5'-CACGCGCCGATGCAAAAAGCGACACACAAAAG). Mutation of the Sp1 site
at position +9/+15 was generated with the primers 5'-MU2
(5'-CAGACCGCGCGTACCTTACCTCTTTCTGG) and 3'-MU2
(5'-CCAGAAAGAGGTAAGGTACGCGCGGTCTG). The second Sp1 site at +23/+28 was
mutated using the primers 5'-MU5 (5'-CCTCTTTCTGGGTTTTCGGCGGAGCGTGGC)
and 3'-MU5 (5'-GCCACGCTCCGCCGAAAACCCAGAAAGAGG).
115/
4 and
4/+52 were generated by
using the mutated pGL3-MAT2B (115)-LUC clones as templates
and external primers 3'-BP8 (5'-TCTGCCCCCAGCCCACG) and 3'-BP9
(5'-GTTGATTGGCCACGCTCC). Results from only those mutations that
affected promoter activity are described below.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structural organization of the human
MAT2B gene. The MAT2A gene consists of
seven exons and six introns. Shaded boxes represent the open
reading frame of the MAT II subunit protein. The length of each
exon in nucleotide pairs is denoted by the numbers in the
boxes. Asterisks mark the translation initiation
and termination sites. The inset table indicates the exact
location of exons and introns and the sequence at each exon/intron
junction.
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Fig. 2.
Transcription initiation of the human
MAT2B gene as determined by primer extension
analysis. Two primers starting at MAT2B cDNA
sequence +22 (Bra1) and +24 (Bra2) with respect to the translation
initiation site were annealed to human lymphocyte poly(A)+
RNA and extended in the presence of avian myeloblastosis virus reverse
transcriptase. Extension products of 225 and 227 bases in length were
observed, indicating that transcription starts 203 bases upstream of
the translation start site. An M13mp18 sequencing reaction was used as
a size marker.
15 to + 203 in the 5'-flanking region is high in GC
content with clusters of overlapping Sp1 sites (Fig. 3). A TATA box is
located 32 bp upstream from the transcription start site.
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Fig. 3.
Sequence of the 5'-flanking region of the
human MAT2B gene. Depicted is 1.1 kb of the
MAT2B gene 5'-flanking sequence. The transcription start
site is marked with an arrow. Putative sites for known
transcription factors are underlined.
998 to
115 of the MAT2B promoter had
little effect on promoter activity; however, further deletions resulted
in a gradual decrease of promoter activity (Fig.
4). Little to no promoter activity was
seen when only the region from +93 to +204 was included in the
construct. The region from +52 to +93 provided minimal promoter
activity, and the inclusion of sequences between
4 and +52
significantly increased activity. This indicated that the 5'-noncoding
sequences of MAT2B are contributing to promoter function.
Another significant enhancement in promoter activity was seen when
sequences between positions
115 and
4 were also included.
Functional studies described below indicated that this enhancement was
conferred by the inclusion of a TATA sequence at position
32/-8. No
further enhancement was observed when additional upstream sequences
were included. Together the data indicate that the sequences between
+52 and +93 provide minimal promoter activity, whereas sequences
between
115 and +93 provide the full promoter activity.
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Fig. 4.
In vivo activity of the human
MAT2B gene promoter constructs. A series of
MAT2B promoter deletion constructs fused to the firefly
luciferase reporter were transfected into Cos-1 cells and luciferase
activity was assayed by the dual luciferase reporter assay system as
described under "Materials and Methods." The data were calculated
in relative luciferase units and expressed as a percentage of
the pGL3-MAT2B( 998) construct, which was set at 100%
activity. Data presented are mean ± S.E. of at least four
separate experiments, each performed in triplicate. An almost identical
pattern was seen when the pGL3-MAT2B constructs were
transfected into the Jurkat human leukemia cell line by the method
described previously (27).
115 to
4 were nonspecific (Fig.
5B). By contrast, strong and
specific complexes were formed on a probe representing the region from
4 to +52 (Fig. 5C). Strong complexes were also formed on
the region from +52 to +93, which is GC-rich. These complexes
were partially competed off with the nonradioactive probe covering the
same sequence (Fig. 5D).
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Fig. 5.
In vitro analysis of the MAT2B
proximal promoter by EMSA. Probes used for EMSA and
supershift analyses with Jurkat nuclear cell extracts are indicated,
and only those that show relevant patterns are shown. For each
autoradiogram, lane 1 is probe alone, lane 2 is
probe plus Jurkat nuclear cell extract, and lane 3 is probe
and Jurkat nuclear cell extract plus a 100-fold molar excess of the
relevant nonradioactive probe.
4 to +93 has several Sp1 and NF1 sites; however,
neither the Sp1 nor the NF1 antibodies caused supershift of the
complexes. Although the anti-Sp2 antibody induced a slight supershift,
the anti-Sp3 antibody had the strongest effect, causing the complete
disappearance of complex II (Fig. 6,
A and B). The data suggest that Sp3 is one of the
main factors involved in complex formation in this region; other
members of the Sp1 family may also be involved in protein-DNA
interaction on this region of the MAT2B promoter.
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Fig. 6.
Involvement of different members of the Sp1
family of transcription factors in protein·DNA complexes formed on
the MAT2B promoter. A and
B, the pattern of supershift with antibodies to NF1 and
different members of the Sp1 family of transcription factors.
Lane 1 is probe alone, lane 2 is probe plus
Jurkat nuclear cell extract, and lanes 3-6 are supershift
with anti-Sp1, -Sp2, -Sp3, and -Sp4 Abs, respectively. Lane
7 is supershift with an anti-NF1 antibody. Complexes are
designated I and II in order of mobility. C, the in
vivo interaction of Sp1 and Sp3 with the MAT2B promoter
using chromatin immunoprecipitation. Shown is an ethidium
bromide-stained 1.5% agarose gel showing PCR analysis of complexes
immunoprecipitated with Sp1 or Sp3 antibodies. PCR was performed with
the immunoprecipitates as described under "Materials and Methods."
Lane a is a positive control for PCR reaction and represents
amplification with primers for the region from 174 to
4 using the
pGL3-MAT2B plasmid as template; lane b is a
negative control consisting of samples processed through the chromatin
immunoprecipitation protocol (CHIP) without the
primary antibody; lanes c and d are samples
processed through the chromatin immunoprecipitation
protocol with either an Sp1 or Sp3 antibody. Primers were designed to
amplify the regions from
174 or
4 to + 52.
174 to
4 and
4
to +52 (Fig. 6C). Thus, even though Sp3 seems to be a major factor that binds to the proximal MAT2B promoter, the
binding of Sp1 to this site in vivo cannot be ruled out.
115 to +93. Mutation of the Sp1 site
at position +9 completely abolished protein·DNA complex formation on
probes representing sequences from
115 to +52 or from
4 to +52
(Fig. 7). By contrast mutation of other
Sp1 sites in the region from
115 to +52 had no effect on complex
formation. Similarly, mutation of the three TBF sites located between
115 and
4 had no effect on complex formation. Together the data
suggest that the Sp1 site at +9 is key for protein-DNA interaction on
this region of the promoter. The sequence from +52 to +93 is too
heavily GC-rich to mutate in a meaningful way.
View larger version (67K):
[in a new window]
Fig. 7.
Effect of mutating specific factor
recognition sites on MAT2B promoter on EMSA. The
position of each mutation is indicated by underlines, and
the resultant sequence is shown in the inset table.
Indicated wild-type (WT) or mutated (Mu) probes
were incubated with Jurkat cell nuclear extract and subjected to EMSA
analysis as detailed in the Fig. 5 legend. The data show the dramatic
effect of mutating the Sp1 site at +9/+15. P, probe alone;
NE, nuclear extract.
115 and +52 on
the in vivo activity of the MAT2B promoter was
tested; however, only two mutations affected promoter activity (Fig.
8). The TATA at
32 and the Sp1 site at
+9 were individually or simultaneously mutated on the pGL3-MAT2B(
115)-Luc or pGL3-MAT2B(
4)-Luc
reporter construct. Mutation of the Sp1 site at position +9 reduced
promoter activity by 35-50%, whereas mutation of the other Sp1 sites
located in the region from
115 to +52 had no effect on activity.
Mutation of the TATA at
32 reduced in vivo activity of the
MAT2B promoter by only 25%. However, when both the TATA at
32 and the Sp1 site at +9 were mutated simultaneously, promoter
activity was reduced by almost 60%, reaching a level that is
comparable with that driven by the GC-rich sequence from +52 to +93.
Together the data indicate that GC-rich sequences in the region from
+52 to +93 can drive MAT2B gene expression up to 25-30% of
it full activity, whereas the presence of the Sp1 site at +9/+15 and
the TATA sequence at
32/
28 are required for 100% activity.
View larger version (22K):
[in a new window]
Fig. 8.
Effect of mutating specific factor
recognition sites on MAT2B promoter on in
vivo activity. The position of each mutation is
indicated in the line diagram. The reporter
pGL3-MAT2B( 115)-Luc or pGL3-MAT2B(
4)-Luc
constructs with and without TATA and/or Sp1 mutations were transfected
into Cos-1 cells and assayed for luciferase activity as described in
the Fig. 4 legend. The activity of the indicated wild-type
(WT) or mutated (Mu) constructs is expressed as a
percentage of respective wild-type construct activity. The data are
from at least three experiments performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 (53 kDa) and
2'
(51 kDa) subunits complexed with the
regulatory subunit (38 kDa)
(15, 22, 31). The
subunit lowers the Km value of
the enzyme for L-Met and confers susceptibility to product inhibition by AdoMet (26, 31). Interestingly, we have not been able to
detect the
protein in E. coli or yeast extracts (26, 35)
despite the presence of a high level of homology between this protein
and enzymes that catalyze the reduction of thiamine
diphosphate-linked sugars in bacteria (36). Therefore, the
unique role and significance of the
protein in mammalian cells
represents an intriguing area of study, particularly because
is
differentially expressed in normal and leukemic T cells and subsequently affects AdoMet levels.
2 and
subunits of MAT II are
constitutively expressed at a high level. Nonphysiological polyclonal mitogenic stimulation of primary human lymphocytes induces an increased
expression of MAT II
2 subunit only, whereas physiological stimulation via the T cell receptor results in a down-regulation of the
subunit (25). This is accompanied by a 6-10-fold increase in
AdoMet levels (25). Thus the pattern of expression of the MAT2B gene may be an important mechanism for regulating
intracellular levels of AdoMet.
subunit expression in different cells, we cloned and characterized
the MAT2B gene and its 5'-flanking sequence. Promoter activity was very similar in Cos-1 cells and Jurkat human leukemic cells. This is consistent with our previous observations that the
subunit is constitutively expressed in both cell types (37). Minimal
promoter activity is contained between position +52 and +93, which is
rich in GC content. However, full promoter activity was achieved when
sequences from
115 to +52 were included. The Sp1 site located within
the 5'-noncoding region of the gene (+9/+15) seems to play a key role
in this enhancement inasmuch as mutation of this site abolished
DNA-protein interactions and significantly reduced promoter activity
in vivo. It is possible that the GC-rich region of the
proximal promoter drives the residual activity when this Sp1 site is
mutated. Further enhancement of MAT2B promoter activity is
conferred by the TATA at
32/
28. When the Sp1 at +9 and the TATA at
32 are mutated simultaneously, promoter activity is reduced by
60-70%.
2
and down-regulates the expression of the
subunit, whereas in
leukemic T cells both subunits are expressed at a high level (22, 37).
Sp3 is a bifunctional protein that can both activate and repress the
transcription of genes (38, 39). Internal isoforms of this protein
containing activation and/or repressor domains have been described
(40). It is conceivable that Sp3 may enhance MAT2A and
MAT2B gene expression in leukemic T cells while enhancing
MAT2A and suppressing MAT2B in normal T cells.
Studies of the role of the Sp1 family of transcription factors in
regulating MAT II
2 and
subunit expression in normal and
leukemic T cells are ongoing in our laboratory. The identification of
an Sp1 site on the promoter for both subunits that is key for driving
promoter activity puts us closer to our goal to elucidate the
differential regulation of MAT II subunits in normal and leukemic T
cells. Achieving this goal will allow us to design targeted therapeutic
strategies for potentiating intracellular AdoMet levels that may lead
to the control of malignant growth.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-54892-09 and Merit Review Award funds from Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY033822.
Both authors contributed equally to this work.
To whom correspondence should be addressed: University of
Tennessee, 956 Court Ave., Suite A-202, Memphis, TN 38163. Tel.: 901-448-7247; Fax: 901-448-7208; E-mail: Mkotb@utmem.edu.
Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M102816200
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ABBREVIATIONS |
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The abbreviations used are: MAT, methionine adenosyltransferase; AdoMet, S-adenosylmethionine; PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s); EMSA, electrophoretic mobility shift assay; Ab, antibody; Pipes, 1,4-piperazinediethanesulfonic acid; LUC, luciferase.
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