From Departments of Surgery, ** Microbiology and
Immunology, and
Biochemistry, and § The Veterans
Affairs Medical Center, The University of Tennessee, Memphis, Tennessee
38163
Received for publication, March 15, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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Mammalian methionine adenosyltransferase II (MAT
II) consists of a catalytic Methionine adenosyltransferase
(MAT)1 is a key enzyme in
metabolism, because it catalyzes the synthesis of
S-adenosylmethionine (AdoMet), the major methyl group donor
(1, 2). Methylation is essential for the synthesis and modification of
proteins, lipids, RNA, and DNA. It is well established that DNA
methylation can control the expression of certain genes (reviewed in
Ref. 3). Moreover, AdoMet is essential for polyamine biosynthesis and
regulates essential metabolic pathways (4, 5). Inasmuch as the levels of AdoMet vary in different tissues and at different stages of differentiation, it is believed that the regulation of AdoMet synthesis
and utilization is important for normal cell development and function
(6-11). Delineating the mechanism by which the MAT enzymes are
regulated will, therefore, be a key to understanding the control of
AdoMet level and its contribution to cell growth and differentiation.
The catalytic subunit of MAT, designated In 1985, a human 185-kDa MAT II enzyme was purified to homogeneity from
leukemic T cells (22). It was demonstrated to consist of two related
53- and 51-kDa The present study reports functional analysis of 2.4 kbp of the
MAT2A promoter, which identified regions with positive and negative regulatory activities that were not reported in previous studies investigating the rat MAT2A genes and a segment of
the human MAT2A promoter (26, 27). Detailed functional
analysis of the proximal promoter of the human MAT2A gene,
reported here, reveal a region that acts as a nucleation site for
formation of protein-DNA complexes and that appears to be essential for
promoter activity in vivo. We believe that this information
may lead to a better understanding of the differential regulation of
MAT II expression and AdoMet levels in different cell types as well as in normal and leukemic human T lymphocytes.
Isolation and Genomic Organization of the MAT2A
Gene--
Forward and reverse primers based on the sequence of the
previously reported MAT II Mapping the MAT2A Gene Transcription Start
Site--
Poly(A)+ RNA was isolated from 500 ml of human
blood by the Poly(A)Ttract mRNA isolation system (Promega)
and used for primer extension analysis. Two primers, PE-1 and PE-2,
complementary to nucleotides 5'(+10)-GTCCGTTCATGTTGGTG-3' and
nucleotides 5'( Cloning of the Human MAT2A Gene Promoter--
We cloned and
sequenced ~2.4 kbp of the 5'-flanking DNA of the human
MAT2A gene. PCR primers were designed to amplify the region
+102/ Generation of Luciferase Reporter Constructs of the MAT2A
Promoter--
A 2.4-kbp NheI/XhoI-digested
fragment of the MAT2A gene containing the 5'-flanking region
starting at position In Vivo Analysis of the MAT2A Promoter Activity--
Functional
expression of deletion constructs of the MAT2A promoter was
analyzed in Cos-1 and Jurkat T cells. Transient transfection of Cos-1
cells was performed by the TransFast method (Promega) according to
manufacturer instructions. Cos-1 cells (1 × 105) were
cultured in DMEM + 10% fetal calf serum until roughly 70% confluency
was reached and transfected with the LUC reporter pGL3-MAT2A promoter deletion constructs along with cytomegalovirus
Renilla luciferase vector (Promega) pRL-CMV DNA (40:1 ratio)
as an internal control. Briefly, 400 µl of transfection media (DMEM
without fetal bovine serum) containing 600 ng of LUC reporter plasmid
were added to the semi-confluent Cos-1 cells along with 15 ng of the
pRL-CMV DNA and 4 µl of TransFast. The plates were incubated for
2 h, then 2 ml of DMEM + 10% fetal calf serum was added and
incubated for another 48 h at which time the cells were harvested,
and luciferase activity was assayed using the dual luciferase reporter
assay system (Promega). After normalization to the Renilla
luciferase control, the activity of each MAT2A promoter
construct was calculated and expressed as relative light units (RLU).
All samples were analyzed in triplicate, and experiments were performed
at least four times. The data are presented as mean ± S.E. of RLU
of at least four different experiments as well as percent RLU relative to the activity of the largest construct used,
pGL3-MAT2A(
Initial attempts to transfect human Jurkat T cells by electroporation
yielded low levels of transfection efficiency. However, an almost
300-fold increase in efficiency of transfection of Jurkat cells with
the pGL3-MAT2A plasmid was achieved by the method of Keller
et al. (29), with a few modifications. Briefly, 2.5 × 105 Jurkat cells were seeded into 12-well tissue culture
plates that had been coated with an adherent monolayer of Cos-1 cells
at ~70% confluency. The cells were co-cultured overnight to allow
the Jurkat cells to adhere to the Cos-1 cell monolayer. The next day, the wells were rinsed twice with Hanks' balanced salt solution, and
then the pGL3-MAT2A promoter deletion constructs (1.25 µg) were added to the adherent cells along with the control
Renilla luciferase vector pRL-CMV DNA (31.25 ng;
i.e. a 40:1 ratio) in a final volume of 400 µl of
serum-free DMEM:RPMI medium (1:1) containing 7.7 µl of TransFast
(Promega). After incubation at 37 °C for 4-5 h, the transfection
mixture was removed, and the cells were further incubated for 36 h
in DMEM:RPMI medium (1:1) plus 10% fetal calf serum. The adherent
cells were washed with 1× Hanks' balanced salt solution and then
recovered by trypsinization for 5 min followed by the addition of
serum-containing medium to inhibit trypsin activity. The cell mixture
was incubated for 2 h in plastic tissue culture plates to separate
the adherent Cos-1 cells from the nonadherent Jurkat cells. The Cos-1
cells adhered readily, and the nonadherent Jurkat cells were recovered by gentle pipetting. Jurkat cells isolated in this fashion were subjected to two additional cycles of plastic adherence to remove any
residual Cos-1 cells. The purity of the recovered Jurkat cells was
>93.0% as analyzed by flow cytometry using a PerCP-Cy5.5-labeled monoclonal anti-human CD3 antibody (Becton and Dickinson, clone SK7).
The adherent Cos-1 cells were also recovered, the presence of Jurkat T
cells was tested for by flow cytometry using the anti-CD3 mAb, and the
population was shown to be 100% pure. Luciferase activity was assayed
in extracts of the recovered Jurkat and Cos-1 cells as described above.
In Vitro Analysis of the MAT2A 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 MAT2A gene were generated using the primers listed in
Table I. Cos-1 and Jurkat cell nuclear extracts were prepared by
washing cell pellets in 1× Hanks' phosphate-buffered saline
and resuspending them in a hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl,
0.2 mM PMSF, and 0.5 mM DTT). After
centrifugation for 5 min at 3000 rpm, the pellet was resuspended in the
same buffer and allowed to swell on ice for 10 min, then homogenized.
Nuclei were harvested by spinning for 15 min at 4000 rpm, resuspended
in hypotonic buffer, and homogenized. An equal volume of high salt
buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT) was added, and the extracted nuclei were pelleted by
centrifugation for 30 min at 14,500 rpm at 4 °C. Supernatants were
dialyzed for 1 h against 2 liters of dialysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl,
0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT). Aliquots were stored at
Binding reactions were performed in a 20-µl final volume by
incubating 60 fmol (
Supershift assays were performed using antibodies (Abs) to specific
factors that have corresponding recognition elements within the
MAT2A promoter region analyzed. Binding reactions were
carried out as described above in the absence or in the presence of 2 µg of 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 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: mouse anti-Sp1 mAb (catalogue no. sc420X),
anti-Sp2 (catalogue no. sc-643X), anti-Sp3 (catalogue no. sc-644X),
anti-Sp4 (catalogue no. sc-645X), anti-PEA3 mAb (catalogue no. sc113X),
rabbit polyclonal anti-AP-2 (catalogue no. sc184X), anti-c-Myb
(catalogue no. sc7874X) Abs, and goat polyclonal anti-GATA1 (catalogue
no. sc1233X).
Western Blot Analysis of Proteins in Protein-DNA
Complexes--
32P-Labeled probe G representing sequences
between In Vivo and in Vitro Analysis of the Effect of Mutations in the
Sp1-1, Sp1-2, and Sp1-3 Sites--
Sp1 site mutations of clone
pGL3-MAT2A (110)-LUC were generated by site-directed
mutagenesis (23) using the following mutagenic oligos: 5'mSp1-1F, CCT
CCCGCT GCT TCG TTC; 3'mSp1-1R, CAG CGA ACG AAG CAG CGG GAG GTG CG;
5'mSp1-2F, TCG CGG GCG CTG CTC TA; 3'mSp1-2R, TTA TAG AGC AGC GCC CGC
GAG C; 5'mSP1-3F, AGG TGC GTG GCC CGA GC; and 3'mSp1-3R, GCT CGG GCC
ACG CAC CT. Primers 5'CD3 and 3'WP, which flank the 3 Sp1 sites, were
used to amplify the whole region (see Table I). PCR products were
cloned into pGEM-T-Easy (Promega). The resultant mutagenized fragments
were confirmed by sequence analysis, excised using KpnI and
XhoI, and individually cloned into the pGL3-Enhancer vector
(Promega). The in vitro and in vivo activity of
each of the three Sp1 mutants was determined as described above. EMSA
probes were generated using the mutated pGEM-T-Easy clones as templates
and outside primers 5'CD3 and 3'WGS (Table
I).
Statistical Analysis--
Paired t tests were used to
test for significant differences between the in vivo
activity of the MAT2A promoter constructs. All constructs
were analyzed in triplicate, and experiments were performed at
least four times.
Genomic Organization of the MAT2A Gene--
Human P1 genomic clone
2007, encompassing the entire MAT2A gene, was used to
characterize the gene and its promoter. The human MAT2A gene
consisted of nine exons separated by eight introns, contained within
~6 kbp of genomic DNA (31) (Table II).
The exons ranged from 78 (exon 2) to 1639 bp (exon 9) in size (Fig. 1). Exon 1 contained 91 bp of coding
sequence and 120 bp of 5'-untranslated region, and exon 9 contained 100 bp of coding sequence and 1539 bp of 3'-untranslated region (Fig. 1).
The overall organization of the human MAT2A gene was very
similar to the rat MAT2A (26) and the human MAT1A
genes (32), however, important differences in the promoter were found
as detailed below.
Mapping the MAT2A Gene Transcription Start
Site--
Identification of the transcription start site was
determined by primer extension as detailed under "Materials and
Methods" using poly(A)+ RNA from normal human lymphocytes
and primers PE-1 and PE-2 complementary to nucleotides starting at
positions +10 and
The sequence of 2.4 kbp of the 5'-flanking region of the
MAT2A promoter is shown in Fig.
3. Sequence analysis identified the presence of recognition sequences for a large number of transcription factors. They include the canonical TATA box at In Vivo Analysis of the MAT2A Promoter--
A series of
5'-MAT2A 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. The
pattern of the MAT2A promoter activity was very similar in
both cell types (Fig. 4). Promoter
sequences contained between
In this study, we focused on the proximal promoter of the
MAT2A gene (up to bp 285 relative to transcription start
site) and hereby describe detailed characterization of this region.
In Vitro Analysis of the Proximal MAT2A Promoter Activity by
Electrophoretic Mobility Shift (EMSA) and Supershift
Assays--
Analysis of the proximal MAT2A promoter
sequence identified several recognition sites for known transcription
factors. These regions were subjected to further functional analysis
using EMSA and supershift assays. A series of double-stranded
32P-labeled oligonucleotide probes were generated by PCR
amplification (Table I and Fig. 5). The
amplified DNA representing the region from
In competition experiments, probes D-G, which together
cover the region from
Based on consensus sites for known transcription factors, initial
supershift analyses were performed with antibodies to these putative
factors, and they included anti-Sp1, anti-PEA3, anti-AP2, anti-GATA1,
anti-CBF-C, and anti-c-Myb; however, only the anti-Sp1 antibody
produced a supershift with probes A, C, and D, which contain all three
Sp1 sites but not with probe E (
In contrast to the anti-Sp1 antibodies, anti-Sp2, -Sp3, and -Sp4
antibodies caused supershift with probe G (
Together the above data suggest that Sp1 may not be directly involved
in these complexes and that Sp2 and Sp3 (and possibly Sp4) are involved
in complex formation on the region from Effect of Mutating Sp1 Sites in the Proximal Region of the MAT2A
Promoter on the in Vitro and in Vivo Promoter Activity--
Taken
together, the above data from EMSA and supershift assays indicate that
the Sp1-3 site acts as a nucleation site for complex I-III formation
and that Sp2 and Sp3 (and possibly Sp4), but not Sp1, are part of these
complexes. To further investigate this notion, the three Sp1 sites
contained within probe D were individually mutated as described under
"Materials and Methods." Mutation of the Sp1-1 and Sp1-2 sites had
little effect on complex formation, promoter activity, or supershift
with anti-Sp1, -Sp2, -Sp3, or -Sp4 antibodies (Fig.
9). By contrast, mutation of the Sp1-3
site significantly diminished complex formation, and reduced promoter
activity by 85% (Fig. 9). These results underscore the importance of
the Sp1-3 site in controlling the expression of the MAT2A
gene.
The level of expression of the MAT2A gene varies in
different tissues and in different stages of differentiation or
malignant transformation of the same tissue. Our previous studies have
shown that MAT II is differentially expressed in normal and leukemic T
lymphocytes (23-25). Variation in the expression of MAT II affects intracellular levels of AdoMet (24, 25, 34-42), and this is likely to
have an effect on a number of biological reactions involving this
pivotal compound (9). To delineate the underlying mechanisms involved
in regulating MAT2A gene expression, an in-depth
characterization of the gene and its promoter was needed. Previous
studies by Hiroki et al. (26) suggested that the first 343 bp of the 5'-flanking region of the rat MAT2A promoter
contained consensus sequences for cell-type-specific promoter elements;
however, no studies of the interaction between specific nuclear factors
and these elements were reported. Similarly, Mao et al. (27)
reported a partial sequence of the human MAT2A promoter, but
many of the regulatory aspects of this gene reported here were not
included in their study.
In this study, we performed a detailed characterization of the proximal
promoter of the MAT2A gene, and this has led to the identification of a site that is essential for its activity
and can potentially be targeted for modulation of the expression of the MAT II Detailed analyses of the interaction of nuclear proteins from Cos-1
cells and the Jurkat human T cell leukemia cell line with the proximal
promoter of the human MAT2A gene revealed a very similar
pattern of promoter activity and complex formation in both cell types.
Three complexes were formed with a probe representing the region from
A significant (p < 0.05) drop in activity was seen
when sequences between Three Sp1 recognition sites are located within the region from Supershift with the anti-Sp1 antibody only occurred when all three Sp1
sites were present on the same DNA fragment and an Sp1 competitor
reduced but did not abolish complex formation. These data suggested
that Sp1 is probably not directly involved in binding to the Sp1-3
site. Sp1 is a member of a large family of related transcription
factors (43, 44). Sp3 and Sp4, and to a lesser extent Sp2, share
extensive structural and sequence homology with Sp1 and to other
members of the Sp family that share similar cognate DNA binding sites.
Sp1 and Sp3 are ubiquitously expressed in cells, whereas the
distribution of Sp2 and Sp4 appears to be more limited and
cell-specific. The data presented here indicate that Sp2, Sp3, and
possibly Sp4 are involved in complexes formed on the region between
In summary, detailed characterization of the MAT2A gene and
its promoter revealed novel regions that exert positive and negative effects on gene expression. The Sp1-3 site located 69 bases upstream from the transcription start site acts as a nucleation site for complex
formation. The DNA-protein complexes appear to involve several
members of the Sp family of transcription factors, namely Sp2 and Sp3.
The results of this study suggest that the Sp1-3 site is essential for
promoter activity and can be targeted for potentiation of
MAT2A expression. It should be mentioned that, in a recent
study2 the pattern of
complex formation with nuclear extracts from normal resting and
activated human T cells was starkly different from that seen with
nuclear extracts from Jurkat leukemic T cells or from lymphocytes from
a patient with acute lymphocytic leukemia. Interestingly, mutation of
the Sp1-3 site abolished the formation of smaller complexes in normal
cells while it resulted in the formation of a new major complex not
seen in either fresh or established leukemic T cells. We believe that
these findings support our hypothesis that differences in
MAT2A gene regulation in normal and leukemic T cells exist
and that this may account for the differential regulation of MAT II in
these cells. Delineating the regulation of this key gene should shed
light on the molecular basis for the differential expression of the
2/
2' and a regulatory
subunit.
Up-regulation of
2 subunit expression is associated with increased
intracellular levels of S-adenosylmethionine, the major
methyl group donor and a key compound in cell metabolism and polyamine
synthesis. Previous studies have shown that expression of the
2
subunit is differentially regulated in normal and malignant cells. To
delineate the molecular basis for the differential regulation of
2
subunit expression, we cloned and characterized the human
MAT2A gene and its promoter and defined regions that
contain enhancer and repressor elements. Detailed functional
characterization of the proximal promoter of the MAT2A gene
revealed the formation of three major protein-DNA complexes with
probes containing three Sp1 sites (Sp1-1 at
14, Sp1-2 at
47, and
Sp1-3 at
69). Competition with a probe copying sequence between
76
and
54, which contains the Sp1-3 site only, or mutation of this site,
abolished complex formation. Furthermore, mutation of the Sp1-3 site,
but not the Sp1-1 or Sp1-2 sites, inhibited the in vivo
promoter activity by ~85%. Supershift assays showed that the
transcription factors Sp2 and Sp3 are part of the complexes formed at
the Sp1-3 site, and that Sp1 does not appear to be directly involved.
The data indicate that complex formation is initiated at site Sp1-3,
which appears to be essential for promoter activity. However, other
regions of the proximal promoter may also contribute to the regulation
of MAT2A gene expression. These studies may lead to the
delineation of the molecular basis for the differential regulation of
MAT2A expression in normal and leukemic T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, is highly conserved among
species. In mammals, the two major MAT isozymes are designated MAT
I/III and MAT II, and their catalytic subunits are designated
1 and
2, respectively (12). The MAT1A gene encodes the
1
protein and the MAT2A gene encodes the
2 protein (12). Despite the high degree of homology (84% identity at the amino
acid level), the mammalian MAT isozymes differ considerably in their
physical and kinetic properties as well as in their tissue distribution
(12-17). Expression of
1 is confined to liver, whereas
2 is
expressed in all tissues, including liver. In addition to the normal
differences in the tissue distribution of the
1 and
2 subunits,
expression of these proteins is altered in liver cancer. In hepatic
tumors,
1 is markedly down-regulated while
2 expression is
increased (18-21). Molecular mechanisms responsible for the
differential expression of MAT
1 and
2 subunits in normal and
malignant tissues are not well defined.
2 and
2' subunits, complexed with a structurally
unrelated 38-kDa protein, which was designated the
subunit (22).
Ensuing studies showed that the regulation of the MAT II
2 and
subunits expression differs among normal resting, activated, and
malignant T cells (23-25). Physiological activation of normal human T
cells induces the up-regulation of
2/
2' and the down-regulation
of the
subunit; by contrast, expression of both
2/
2' and
is constitutively high in leukemic B and T cells (23-25).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 subunit cDNA (28) spanning the
entire open reading frame were designed and used to amplify genomic DNA isolated from normal human lymphocytes. The presence of introns was
indicated by PCR products larger than predicted from coding sequence,
and these products were cloned and sequenced. Intron-exon boundaries of
the MAT2A gene were determined by aligning the cDNA sequence of MAT2A cDNA with the genomic sequence. Probes
of 80-150 bp spanning exon/intron junctions were used to screen a
human P1 genomic library (Genome Systems Inc., St. Louis, MO), and
clone 2007 was selected for further characterization. Sequence and PCR analyses of the human MAT2A locus identified the location
and sizes of the exons and introns.
1)-GTTGGTGTCGGTGTGCGGAAGT-3' of the human MAT II
2
cDNA were synthesized and end-labeled with [
-32P]ATP using T4 polynucleotide kinase. Free ATP was
removed by the nucleotide removal kit (Qiagen), and labeled primers
(500,000 cpm) were hybridized to 2-4 µg of poly(A)+ RNA.
Extension was performed in the presence of 15 units/µg avian myeloblastosis virus reverse transcriptase (Promega). Excess labeled primer was removed using a QIAquick PCR purification system (Qiagen). The eluted cDNA was concentrated and dissolved in 3 µl of loading buffer containing 80% formamide, 1× TBE buffer (90 mM Tris borate with 2 mM EDTA), xylene cyanol,
and bromphenol blue, heated at 95 °C for 3 min, and analyzed on 6%
polyacrylamide 7 M urea sequencing gels. An M13mp18
sequencing ladder was used as a size marker.
2374 (see Table I) in the presence of Pfu DNA
polymerase (Promega), and the product was cloned into the
pGEM-TEasy vector (Promega). The primers introduced a
NheI site at the 5'-end and a XhoI site at the
3'-end of the cloned promoter. MAT2A promoter sequence was
confirmed by sequencing from both directions with overlapping segments
by the fmol sequencing system (Promega).
19 from the translation start site was cloned
upstream of a firefly luciferase (LUC) reporter gene in the pGL3
Enhancer vector (Promega). Insertion orientation was verified by
restriction enzyme digestion and sequencing from both ends. The
pGL3-MAT2A plasmid was treated with T4 DNA ligase (Promega)
to repair any nicks. The 5'-deletions were generated by exonuclease III
treatment using KpnI/NheI-digested
pGL3-MAT2A and the Erase-a-base kit (Promega). Briefly, the
vector was digested with NheI to provide 5'-overhangs for
digestion with exonuclease III and with KpnI to provide a
protected 3'-overhang. The exonuclease III reaction was incubated at
37 °C, and at specific time points aliquots were removed and added
to tubes containing S1 nuclease to digest single-stranded DNA.
Resultant deletion constructs were blunt-ended with Klenow and ligated
to the pGL3 Enhancer vector with T4 DNA ligase (Promega). Further
deletion constructs were generated by taking advantage of the
SacII, ApaI, PstI, and StuI restriction sites within the promoter sequence, or were generated by
amplification using specific primers (Table I). All constructs were
confirmed by sequence analysis.
2374)-LUC, which was set at 100%.
80 °C.
50,000 cpm) of 32P-labeled probe
with 5 µg of crude nuclear extracts from Cos-1 or 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 DTT,
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., Santa Cruz, CA (catalogue no. SC2502). The DNA-protein complexes
formed were analyzed by electrophoresis on nondenaturing 4%
polyacrylamide gels. The gels were prerun for 1 h at 100 V, and
electrophoresis was done at 30 mA of constant current.
76 and
54 was incubated with nuclear extracts from Jurkat T
cells and separated on native 4% acrylamide gels as described above.
The region containing the radiolabeled protein-DNA complexes formed was
excised from the gel and boiled in SDS sample buffer for 5 min and
loaded onto 10% SDS-polyacrylamide gel electrophoresis gels, followed
by immunoblotting as described previously (30) using antibodies to
members of the Sp1 family of transcription factors that were used in
the supershift analysis.
Sequence of primers used to generate promoter deletion constructs and
probes for EMSA
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MAT2A gene exons and introns
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Fig. 1.
Structural organization of the human
MAT2A gene. The intron-exon structure of the
MAT2A gene is diagrammed. Shaded boxes represent
the open reading frame of the MAT II 2 protein, and open
boxes represent untranslated sequence. The length of each exon in
bp is denoted by the numbers in the boxes.
Asterisks mark the translation initiation and termination
sites.
1, respectively, relative to the translation start
site. As shown in Fig. 2, transcription begins at position
120 relative to the translation start site. These
data are in conflict with data by Mao et al. (27) who reported that the transcription of the human MAT2A gene
start site is located at
132 upstream of the translation start site, but are in close agreement with Hiroki et al. (26) who
demonstrated that the transcription initiation site for the rat
MAT2A gene is located 123 bp upstream of the translation
start codon.
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Fig. 2.
Transcription initiation of the human
MAT2A gene as determined by primer extension
analysis. A primer starting at MAT2A cDNA sequence
+10 (PE-1) with respect to the translation initiation site,
was annealed to human lymphocyte poly(A)+ RNA and extended
in the presence of avian myeloblastosis virus reverse transcriptase. An
extension product of 130 bases in length was observed (lane
PE-1). An M13mp18 sequencing reaction was used as a size
marker.
32 and an inverted CAT box at
88, and three Sp1 sites, which we designated Sp1-1 (
14/
9), Sp1-2 (
47/
39), and Sp1-3 (
69/
63). The Sp1-3 site is
also the GC box. Putative binding sites for several transcription factors were found, and they included an AP-2 site at
104, two PEA3
sites at
119 and
273, a GATA1 site at
212, and an NF-1 site at
446 relative to the transcription start site. A series of deletion
constructs were used to test the functionality of the various sites, as
discussed below.
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Fig. 3.
Sequence of the 5'-flanking region of the
human MAT2A gene. A total of 2382 bases of
5'-flanking region and 120 bases of 5'-untranslated region of the
MAT2A gene are depicted. The transcription initiation site
is marked with an arrow, and various putative transcription
factor recognition sequences as well as the ATG translation start site
are underlined.
87 and
58 from the transcription start
site harbor most of the necessary elements required to drive
transcription of the MAT2A gene (Fig. 4); however, sequences
between
325 and
128 significantly contributed to enhanced promoter
activity. Sequential deletions gradually reduced promoter activity with
a significant (p < 0.05) drop in activity when
sequences between
128 and
110 were deleted (Fig. 4), suggesting
that this region contains elements that enhance promoter activity. The
inclusion of the region from
1281 to
423 significantly reduced
promoter activity (p < 0.001), suggesting the presence
of repressor elements particularly in the region from
423 to
325.
This region contains recognition sites for a c-Myb motif at
359 (
),
an AP-2 site at
396 (
), and an Sp1 site at
406. Interestingly, a
marked and highly significant enhancement of promoter activity was
observed when sequences between
1881 and
1281 were included
(p < 0.001). This enhancement, which was seen in five
different experiments, was significantly suppressed by the inclusion of
5'-flanking sequences from
2374 to
1881 (p < 0.001). The latter region contains the following putative transcription
factors recognition sites: an AP-2 site at
1886, an NF-Y site at
2025, a c-Myb site at
2309 and two GR sites at
2281 and
2347, respectively.
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Fig. 4.
Promoter activity of the human
MAT2A gene in Cos-1 cells and Jurkat human leukemic T
cells. A series of MAT2A promoter deletion constructs
fused to the firefly luciferase reporter gene are depicted in
A. Constructs were transfected into Cos-1 cells
(A), or Jurkat and Cos-1 cells (B) by the method
of Keller et al. (23). Luciferase activity was assayed by
the dual luciferase reporter assay system as described under
"Materials and Methods." The data are expressed in RLU, and are
presented as a mean ± S.E. of at least four separate experiments.
All activities are presented as a percentage of the longest construct
used, pGL3-MAT2A( 2374), which was set at 100%
activity.
285 to +102 sequences was
designated probe A. When Jurkat or Cos-1 cells nuclear extracts were
incubated with probe A and analyzed by EMSA, three complexes I, II, and
III were formed (Fig. 5). An additional complex IV was seen with
nuclear extracts from Cos-1 cells. The shift pattern observed with
probes C and D was very similar to that obtained with probe A. No
complexes were formed with probe B (
16 to +102), whereas probe F
(
110 to
45) and probe G (
76 to
54) formed complexes that were
very similar to probes A-D, except that complexes I and II seemed to
comigrate (Fig. 5).
View larger version (54K):
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Fig. 5.
In vitro analysis of the
MAT2A promoter by EMSA. Probes used for EMSA and
supershift analysis with Cos-1 or Jurkat cell extracts are
diagrammed in the upper left-hand corner and are
designated A-G. A summary of the protein-DNA complexes
formed with nuclear extracts for Jurkat and Cos-1 cells is provided in
the inset table. Complexes are designated I,
II, III, and IV in order of mobility.
For each autoradiogram, lane 1 is probe alone, lane
2 is probe plus Jurkat cell extracts, and lane 3 is
probe plus Cos-1 cell extracts.
110 to +5, abolished all complex formation
(Fig. 6). Oligonucleotides representing
sequences from
59 to +3, which contain the Sp1-1 site plus the Sp1-2
site, or representing sequences
59 to
29, which contain the Sp1-2
site alone, had no effect on the formation of complexes I, II, or III.
In addition, a consensus Sp1 oligonucleotide, used as a competitor,
reduced but did not completely block the formation of complexes. By
contrast, when probe G (
76 to
54 bp), which contains the Sp1-3 site
only, was used as a competitor, all complex formation was abolished
(Fig. 6b). No competition was seen with sequences
representing the region from
93 to
74 bp, which is upstream of the
Sp1-3 site. Therefore, the data suggest that the major complexes are
formed on the region from
76 to
54 bp, which contains the Sp1-3
site.
View larger version (63K):
[in a new window]
Fig. 6.
Competitive inhibition of complexes with
various oligonucleotides and probes. Probe D was incubated with
nuclear extracts from Jurkat T cells in the absence or presence of
various competitors. Competition was conducted as detailed under
"Materials and Methods." The competition pattern indicates that the
major complexes are formed on the region between 76 and
54, which
contains the Sp1-3 site (a and b). However, an
Sp1 competitor reduced but did not abolish complex formation
(c).
110 to
20), which contains sites
Sp1-2 and Sp1-3), or with probe G (
76 to
54 bp), which contains
site Sp1-3 (Fig. 7). These data, together with the finding that an anti-Sp1 competitor reduced but did not abolish complex formation (Fig. 6c) suggested that factors
other than Sp1, possibly other members of the Sp1 family, are binding to this region of the MAT2A promoter.
View larger version (72K):
[in a new window]
Fig. 7.
Pattern of supershift with antibodies to
different members of the Sp1 family of transcription factors. The
effect of antibodies to members of the Sp1 family of transcription
factors on complexes formed with the indicated probes and nuclear
extract from Jurkat cells is shown. The sequence represented by the
various probes is shown in Fig. 4. Lane 1 is probe alone,
lane 2 is probe plus Jurkat leukemic T cells nuclear
extracts, and lanes 3-6 are supershift with anti-Sp1, -Sp2,
-Sp3, -Sp4 Abs, respectively.
76 to
54) that contains
the Sp1-3 site only. The anti-Sp2 antibody induced a strong supershift
when probes D-G were used, and in some experiments complex II appeared
to be supershifted by the antibody. In the presence of the anti-Sp3
antibody, complexes I and III disappeared; complex III was either
inhibited or shifted to comigrate with complex II. Thus, Sp3 appears to
be involved in complex I and III formation. Supershift with the
anti-Sp4 antibody was considerably less than with either anti-Sp2 or
anti-Sp3 antibodies.
76 to
54. To further
test this possibility, the area containing complexes I-III was excised
from the EMSA gel, boiled in SDS buffer, and analyzed by Western blots
using antibodies to various members of the Sp1 family of transcription
factors. As shown in Fig. 8, Sp1 protein
was not detected in complexes formed with probe E, whereas Sp2, Sp3,
and Sp4 proteins were found in these complexes (Fig. 8). The anti-Sp3
antibody reacted with at least three major bands on the blot. Inasmuch
as the nuclear extract included a mixture of protease inhibitors, it is
less likely that the multiple bands are proteolytic products of Sp3;
instead, it is possible that they represent internal isoforms of Sp3
that have been reported by others (33).
View larger version (58K):
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Fig. 8.
Analysis of proteins bound to complexes
I-III. 32P-Labeled probe G representing sequences
between 76 and
54 was incubated with nuclear extracts from Jurkat T
cells and separated on native 4% acrylamide gels as described in the
legend to Fig. 4. The region containing the radiolabeled protein-DNA
complexes formed was excised from the gel and boiled in SDS sample
buffer, loaded onto 10% SDS-polyacrylamide gel electrophoresis gels,
and analyzed by Western blots using anti-Sp1, -Sp2, -Sp3, and -Sp4
antibodies.
View larger version (51K):
[in a new window]
Fig. 9.
Effect of mutation of Sp1 sites on
MAT2A promoter activity Cos-1 cells and
electrophoretic mobility shifts in Jurkat cells. The three Sp1
sites found within the pGL3-MAT2A ( 110)-Luc reporter
construct were individually mutated as listed in the inset
table. Nucleotide changes between the wild type (WT)
and mutant (M) Sp1 sites are underlined. Reporter
pGL3-MAT2A (
110)-Luc constructs with and without Sp1 site
mutations were transfected into Cos-1 cells and assayed for luciferase
activity by the dual luciferase reporter assay system (right-hand
panel) as described in the legend to Fig. 4. The individual
mutations listed in the inset table were introduced into
probe D and used for EMSA and supershift analyses with Jurkat cell
extracts (left-hand panel). Control, unmutated
probe D; Sp1-1M, probe D with mutation of the Sp1-1 site;
Sp1-2ML, probe D with mutation of the Sp1-2 site;
Sp1-3M*, probe D with mutation of the Sp1-3 site and a
longer exposure of autoradiogram. Lane 1 is probe alone,
lane 2 is probe plus Jurkat cell extracts, and lane
3 is probe plus Cos-1 cell extracts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 subunit in leukemic T cells. The organization of the
human MAT2A gene was found to be quite similar to that of the human MAT1A gene (32) and the rat MAT2A gene
(26). However, important differences in promoter activity and
regulation were noted in this study. Sequences located 207 bases
upstream from the translation start site or 87 upstream from the
transcription start site were sufficient to drive promoter activity.
Significant enhancement of activity was seen when sequences from
128
to
110, relative to the transcription start site, were included. In
contrast to the rat gene and a recent report by Mao et al.
(27), we found evidence for strong repressor elements contained between
the region from
1281 and
325 of the human gene. This repressor
activity was overcome by addition of sequences from
1881 to
1281.
285 to +102. However, sequential deletions of this region showed that
sequences between
76 and
54 are responsible for the major complexes seen.
128 and
110 were deleted, suggesting that
this region contains elements that enhance promoter activity. This region harbors a putative PEA3 site at
119, but antibodies to this
factor did not affect complex formation and failed to cause a
supershift. Similarly, the region from
110 to +5 contained many sites
for known transcription factors, including AP2, AP1, PEA3, AP-2, Sp1,
GATA-1, CBF-C, c-Myb, and CAT-binding proteins, however, only
antibodies to the Sp1 family of transcription factors affected the
pattern of complex formation. Although this may indicate that putative
sites for the other known factors are not functional, the possibility
that other proteins are involved in these complexes or that functional
interactions between known factors and their cognate sites may only
take place in vivo should not be excluded and will be
addressed in future studies.
110 to
+5. However, complex formation was completely abolished either by
competition with a probe representing sequences between
76 and
54
or by mutation of the Sp1-3 site (at
69). Mutation of either the
Sp1-1 site (at
14) or the Sp1-2 site (at
47) did not affect complex
formation and only slightly reduced promoter activity; whereas mutation
of site Sp1-3 reduced promoter activity by ~85%. These data indicate
that site Sp1-3 is important for driving MAT2A promoter activity.
76 and
54, which contains the Sp1-3 (GC box) site. Complex II
appeared to be supershifted by anti-Sp2 antibodies, and complexes I and
III disappeared in the presence of the anti-Sp3 antibody. Together, the
findings that anti-Sp3 antibodies have the strongest effect on
protein-DNA complexes and that this protein was found at a relatively
high amount in these complexes suggest that it may play an important
role in regulating MAT2A gene expression. Sp3 is a
bifunctional protein that can both activate and repress transcription
of genes (45, 46). Internal isoforms of this protein containing
activation and/or repressor domains have been described previously
(33). Our data showed three major bands were recognized by the anti-Sp3 antibodies in Western blots of proteins bound to complexes formed on
site Sp1-3. It is conceivable that several isoforms of Sp3 may be
interacting at this site, but this remains to be determined. Studies
are ongoing in our laboratory to further define the role of specific Sp
proteins in regulating MAT2A gene expression, to identify
other proteins that may be involved in complex formation at the Sp1-3
site, and to determine whether the DNA context of the Sp family cognate
site confers specificity as well as affect the ability of the Sp
proteins to regulate MAT2A gene expression (44).
2 subunit of MAT II in normal and leukemic T cells.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Harry Jarrett and William Taylor for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant GM-54892-09 from the National Institutes of Health (to M. K.) and by Merit Review Award Funds from The Veterans Affairs Merit Award Fund (to M. K.).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) BankIt375639 and AF329366.
¶ 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; Mkotb@utmem1.utmem.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M002347200
2 A.-B. Halim, H. L. LeGros, M. E. Chamberlin, A. M., Geller, and M. Kotb, manuscript submitted.
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ABBREVIATIONS |
---|
The abbreviations used are: MAT, methionine adenosyltransferase; AdoMet, S-adenosylmethionine; kbp, kilobase pair(s); PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; RLU, relative light unit(s); mAb, monoclonal antibody; Ab, antibody; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; bp, base pair(s).
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