Regulation of the Human MAT2B Gene Encoding the Regulatory beta  Subunit of Methionine Adenosyltransferase, MAT II*

Leighton LeGrosDagger §||, Abdel-Baset HalimDagger §||, Margaret E. ChamberlinDagger §, Arthur Geller**, and Malak KotbDagger §DaggerDagger

From the Departments of Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 alpha 2 and a regulatory beta  subunit. Down-regulation of the MAT II beta  subunit expression causes a 6-10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the beta  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.


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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 alpha 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 alpha 2 (53 kDa), alpha 2' (51 kDa), and beta  (38 kDa) subunits. The alpha 2/beta hetero-oligomeric composition of MAT II was also determined in bovine brain, Ehrlich ascites tumor, and calf thymus (15, 19). The alpha 2 subunit is responsible for the enzyme catalytic activity and is post-translationally modified to generate alpha 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 alpha 1 subunit.

In contrast to MAT alpha  subunits, which are highly conserved throughout evolution, the beta  subunit of MAT II seems to be only present in the mammalian species (15, 19). Recently, we cloned and characterized the human MAT II beta  subunit (26, 31), found that it has no catalytic activity, and confirmed that it acts as a regulatory subunit for the enzyme. When beta  associates with the alpha  subunit it alters its kinetic properties and renders MAT II more susceptible to product inhibition by AdoMet (26, 31). Interestingly, the human beta  subunit can also interact with the alpha 1 subunit of MAT I/III and the Escherichia coli alpha  MAT subunit and alter their kinetic properties as well (26, 31).

The expression of the MAT II alpha 2, alpha 2', and beta  subunits varies considerably in different tissues. The alpha 2, alpha 2', and beta  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 alpha 2/alpha 2' subunits but not the beta  subunits (22, 25). By contrast, physiological stimulation of T cells by bacterial superantigens induces an up-regulation of the alpha 2/alpha 2' subunits and a down-regulation of the beta  subunit (25). This results in the formation of alpha 2 and alpha 2' homo- and/or hetero-oligomers (no beta ) with a 3-fold higher Km for L-Met. The form of MAT II without beta  is resistant to product inhibition by AdoMet when compared with the form of MAT II found in resting or leukemic T cells (includes beta ) (25). Importantly, the down-regulation of the beta  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.

Based on our previous results, we hypothesized that the down-regulation of the MAT II beta  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.

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Isolation and Genomic Organization of the MAT2B Gene-- Based on the sequence of the previously reported MAT II beta  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.

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 [gamma -32P]ATP and T4 polynucleotide kinase. Free [gamma -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.

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 -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.

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.

                              
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Table I
Sequence of primers used for MAT2B promoter constructs

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 -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.

In Vivo and in Vitro Analysis of the Effect of MAT2B Promoter Mutations-- Several mutations were introduced into the clones pGL3-MAT2B(-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).

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 -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.

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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.


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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 beta  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.

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.


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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.

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 -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.

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 -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).

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 -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.

The region from -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.

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 -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.

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 -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.


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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.

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 -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.


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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.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The MAT II isozyme is expressed in all tissues in which it is found as an oligomer that comprises catalytic alpha 2 (53 kDa) and alpha 2' (51 kDa) subunits complexed with the beta  regulatory subunit (38 kDa) (15, 22, 31). The beta  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 beta  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 beta  protein in mammalian cells represents an intriguing area of study, particularly because beta  is differentially expressed in normal and leukemic T cells and subsequently affects AdoMet levels.

In leukemic T cells, both the alpha 2 and beta  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 alpha 2 subunit only, whereas physiological stimulation via the T cell receptor results in a down-regulation of the beta  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.

To shed light on the mechanisms underlying the differences in MAT II beta  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 beta  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%.

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 alpha 2 and down-regulates the expression of the beta  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 alpha 2 and beta  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.

    FOOTNOTES

* 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.

Dagger Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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