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

Abstract

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. Methionine adenosyltransferase (MAT)1 (ATP⅐L-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 ␣1 subunit that is expressed only in liver (7, 11-13). The MAT II isozyme is expressed in all tissues including the liver (9, 14 -21). Previous studies from our group (15, 22) have determined that MAT II from human leukemic T and B cells, as well from activated human lymphocytes, is a hetero-oligomer that consists of ␣2 (53 kDa), ␣2Ј (51 kDa), and ␤ (38 kDa) subunits. The ␣2/␤ hetero-oligomeric composition of MAT II was also determined in bovine brain, Ehrlich ascites tumor, and calf thymus (15,19). The ␣2 subunit is responsible for the enzyme catalytic activity and is post-translationally modifie...

Expression Pattern, Regulation, and Functions of Methionine Adenosyltransferase 2β Splicing Variants in Hepatoma Cells

“…12 A critical Sp1 site at +9 was important for basal promoter activity in Cos-1 and Jurkat cells. Our results on V1 basal promoter activity in HepG2 cells agree in general with the work of LeGros et al 12 While the gene that encodes for the catalytic subunit of MAT is essential to life, MAT2β appears dispensable. Indeed, some tissues express very low to absent levels of either V1 or V2.…”

Transcriptional regulation of the promoter of the rat frizzled related protein gene by CREB

“…R6 13-8 cells were formaldehyde crosslinked and immunoprecipitated as described (Legros et al, 2001) with slight modifications. R6 13-8 cultures were fixed by the addition of formaldehyde at a concentration of 0.4% for 15 min.…”

Methionine Adenosyltransferase 2A/2B and Methylation: Gene Sequence Variation and Functional Genomics

“…Although MAT1A and MAT2A share a high degree of amino acid sequence identity (84% in humans) (Chamberlin et al, 2000;Mato et al, 2001), multimers of these enzymes differ in their physical and substrate kinetic properties (Okada et al, 1981;Kotb and Geller, 1993;Kotb et al, 1997;Halim et al, 1999;Mato et al, 2001). A third gene, MAT2B, unrelated in amino acid sequence to MAT1A or MAT2A, encodes a regulatory subunit that physically associates with the MAT2A dimer, forming a heterotetramer (LeGros et al, 2000(LeGros et al, , 2001Martínez-Chantar et al, 2003). When MAT2B is bound to the MAT2A dimer, the apparent K m of the complex for methionine is greatly reduced-from 100 to 20 M (Halim et al, 1999;LeGros et al, 2000LeGros et al, , 2001Mato et al, 2001;Martínez-Chantar et al, 2003).…”

“…A third gene, MAT2B, unrelated in amino acid sequence to MAT1A or MAT2A, encodes a regulatory subunit that physically associates with the MAT2A dimer, forming a heterotetramer (LeGros et al, 2000(LeGros et al, , 2001Martínez-Chantar et al, 2003). When MAT2B is bound to the MAT2A dimer, the apparent K m of the complex for methionine is greatly reduced-from 100 to 20 M (Halim et al, 1999;LeGros et al, 2000LeGros et al, , 2001Mato et al, 2001;Martínez-Chantar et al, 2003). The human MAT2A and MAT1A genes map to chromosomes 2p11.2 and 10q22, respectively, span 6.1 (MAT2A) and 17.8 kilobases (kb) (MAT1A), and their structures both include nine exons that encode 395 amino acids (Mato et al, 2001).…”