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

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 α2 and a regulatory β subunit. Down-regulation of the MAT II β subunit expression causes a 6–10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the β subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5′-flanking sequences, and elucidated the in vitro and in vivoregulation 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 MAT2Bgene 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.


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
␣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 protein⅐DNA 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 protein⅐DNA 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.
In contrast to MAT ␣ 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).
The expression of the MAT II ␣2, ␣2Ј, and ␤ subunits varies considerably in different tissues. The ␣2, ␣2Ј, 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/␣2Ј subunits but not the ␤ subunits (22,25). By contrast, physiological stimulation of T cells by bacterial superantigens induces an up-regulation of the ␣2/␣2Ј subunits and a down-regulation of the ␤ subunit (25). This results in the formation of ␣2 and ␣2Ј homo-and/or hetero-oligomers (no ␤) with a 3-fold higher K m 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 K m value for AdoMet.
Based on our previous results, we hypothesized that the down-regulation of the MAT II ␤ 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
Isolation and Genomic Organization of the MAT2B Gene-Based on the sequence of the previously reported MAT II ␤ 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 (5Ј-GATTCCTGAGTCCTGTCTTAG) and GSR (5Ј-GCACTTTTGGCTTTCACTCAG), that amplified a 79-bp product that included the 3Ј end of intron 4 and the 5Ј 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 phytohemagglutininstimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2ϫ 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) ϩ 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) ϩ RNA was isolated from 500 ml of human blood by the Poly(A)Tract mRNA isolation system (Promega). Two primers, Bra1 5Ј-GTTCTT-TCTCCCTCCCCACCAT-3Ј (complementary to positions ϩ22-ϩ1 of the open reading frame) and Bra2 5Ј-CAGTTCTTTCTCCCTCCCCACC-3Ј (complementary to positions ϩ24 -ϩ3 of the open reading frame), were synthesized and end-labeled with [␥-32 P]ATP and T4 polynucleotide kinase. Free [␥-32 P]ATP was removed by the QIAquick nucleotide removal kit (Qiagen). Reverse primer extension was carried out using 3 g of mRNA and 5 ϫ 10 5 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 5Ј-flanking DNA of the human MAT2B gene. The promoter was contained within 1.1 kbp. The primers 5Ј-WP (5Ј-GCTC-GAGTAAGATGATCTTGGC) and 3Ј-WP (5Ј-GAAGCTTGCCCGC-CGTCTTCAC) were designed to amplify the region Ϫ998/ϩ204 with respect to the transcription start site. The primers introduced an XhoI site at the 5Ј end and a HindIII site at the 3Ј 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 5Ј-flanking region starting at position ϩ204 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 32 P 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-l final volume by incubating 60 fmol (ϳ50,000 cpm) of 32 P-labeled probe with 5 g of crude nuclear extracts from Jurkat cells in the presence of 2 g of poly(dI-dC), 1 g of salmon sperm DNA in 20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 6 g 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 DNA⅐protein 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 g 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 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(Ϫ998) construct, which was set at 100% activity. Data presented are mean Ϯ 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). 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 ϫ 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 Ͻ2 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-l 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 ϫ g for 1 min. Precleared chromatin solutions were incubated with antibody to Sp1 (10 g), Sp3 (10 g) (Santa Cruz Biotechnology, Inc.) or no antibody (negative control) and rotated at 4°C for 12 h. Immune complexes were collected by adding 80 l 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 l of elution buffer (1% (w/v) SDS and 50 mM NaHCO 3 ). 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 l of 2 M Tris, pH 6.8, 10 l of 0.5 M EDTA, and 2 l of proteinase K (20 mg/ml). The samples were incubated for 2 h at 45°C, and then the DNA was extracted with phenol/CHCl 3 followed by ethanol precipitation and resuspension in 50 l of sterile H 2 O. Five l of DNA solution were used as a template in PCR analysis. Primers were designed to amplify the regions from Ϫ174 or Ϫ4 to ϩ 52. The primer 5Ј-BP8 (5Ј-ACTCGAGAAACTCAAGGC-GATCCACTT) or 5Ј-BP9 (5Ј-CCTCGAGACCGCGCGTACC) was paired with primer 3Ј-BP8 (5Ј-TCTGCCCCCAGCCCACG). The PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide staining.
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 l 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 Ϫ115/Ϫ4 and Ϫ4/ϩ52 were generated by using the mutated pGL3-MAT2B (115)-LUC clones as templates and external primers 3Ј-BP8 (5Ј-TCTGCCCCCAGCCCACG) and 3Ј-BP9 (5Ј-GTTGATTG-GCCACGCTCC). Results from only those mutations that affected promoter activity are described below.

RESULTS
Genomic Organization of the MAT2B Gene-Human genomic clone 22646 was determined to harbor the MAT2B gene and a significant portion of its 5Ј-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 ϳ6.8 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 5Ј-noncoding region and 63 bp of coding sequence. Exon 7 contained 171 bp of coding sequence and 802 bp of 3Ј-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 tended 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 ϳ1.1 kbp of the 5Ј-flanking region of the MAT2B gene is shown in Fig. 3. The sequence from Ϫ15 to ϩ 203 in the 5Ј-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.
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 Ϫ998 to Ϫ115 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 ϩ93 to ϩ204 was included in the construct. The region from ϩ52 to ϩ93 provided minimal promoter activity, and the inclusion of sequences between Ϫ4 and ϩ52 significantly increased activity. This indicated that the 5Ј-noncoding sequences of MAT2B are contributing to promoter function. Another significant enhancement in promoter activity was seen when sequences between positions Ϫ115 and Ϫ4 were also included. Functional studies described below indicated that this enhancement was conferred by the inclusion of a TATA sequence at position Ϫ32/-8. No further enhancement was observed when additional upstream sequences were included. Together the data indicate that the sequences between ϩ52 and ϩ93 provide minimal promoter activity, whereas sequences between Ϫ115 and ϩ93 provide the full promoter activity.
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 Ϫ115 to Ϫ4 were nonspecific (Fig.  5B). By contrast, strong and specific complexes were formed on a probe representing the region from Ϫ4 to ϩ52 (Fig. 5C). Strong complexes were also formed on the region from ϩ52 to ϩ93, which is GC-rich. These complexes were partially competed off with the nonradioactive probe covering the same sequence (Fig. 5D).
The region from Ϫ4 to ϩ93 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.
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 Ϫ174 to Ϫ4 and Ϫ4 to ϩ52 (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 Ϫ115 to ϩ93. Mutation of the Sp1 site at position ϩ9 completely abolished protein⅐DNA complex formation on probes representing sequences from Ϫ115 to ϩ52 or from Ϫ4 to ϩ52 (Fig. 7). By contrast mutation of other Sp1 sites in the region from Ϫ115 to ϩ52 had no effect on complex formation. Similarly, mutation of the three TBF sites located between Ϫ115 and Ϫ4 had no effect on complex formation. Together the data suggest that the Sp1 site at ϩ9 is key for protein-DNA interaction on this region of the promoter. The sequence from ϩ52 to ϩ93 is too heavily GC-rich to mutate in a meaningful way.
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 Ϫ115 and ϩ52 on the in vivo activity of the MAT2B promoter was tested; however, only two mutations affected promoter activity (Fig. 8). The TATA at Ϫ32 and the Sp1 site at ϩ9 were individually or simultaneously mutated on the pGL3-MAT2B(Ϫ115)-Luc or pGL3-MAT2B(Ϫ4)-Luc reporter construct. Mutation of the Sp1 site at position ϩ9 reduced promoter activity by 35-50%, whereas mutation of the other Sp1 sites located in the region from Ϫ115 to ϩ52 had no effect on activity. Mutation of the TATA at Ϫ32 reduced in vivo activity of the MAT2B promoter by only 25%. However, when both the TATA at Ϫ32 and the Sp1 site at ϩ9 were mutated simultaneously, promoter activity was reduced by almost 60%, reach- ing a level that is comparable with that driven by the GC-rich sequence from ϩ52 to ϩ93. Together the data indicate that GC-rich sequences in the region from ϩ52 to ϩ93 can drive MAT2B gene expression up to 25-30% of it full activity, whereas the presence of the Sp1 site at ϩ9/ϩ15 and the TATA sequence at Ϫ32/Ϫ28 are required for 100% activity. DISCUSSION The MAT II isozyme is expressed in all tissues in which it is found as an oligomer that comprises catalytic ␣2 (53 kDa) and ␣2Ј (51 kDa) subunits complexed with the ␤ regulatory subunit (38 kDa) (15,22,31). The ␤ subunit lowers the K m 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.
In leukemic T cells, both the ␣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.
To shed light on the mechanisms underlying the differences in MAT II ␤ subunit expression in different cells, we cloned and characterized the MAT2B gene and its 5Ј-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 ϩ52 and ϩ93, which is rich in GC content. However, full promoter activity was achieved when sequences from Ϫ115 to ϩ52 were included. The Sp1 site located within the 5Ј-noncoding region of the gene (ϩ9/ϩ15) 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 Ϫ32/Ϫ28. When the Sp1 at ϩ9 and the TATA at Ϫ32 are mutated simultaneously, promoter activity is reduced by 60 -70%.
The transcription factor Sp3 seems to bind to the Sp1 site at ϩ9/ϩ15, 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 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.