Regulation of the human MAT2B gene encoding the regulatory beta subunit ofmethionine adenosyltransferase, MAT II

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenos ylmethionine (AdoMet), a key molecule in transmethylation reactions and pol yamine biosynthesis, The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit. Down-regulation of the MAT II beta subunit expre ssion causes a 6-10-fold increase in intracellular AdoMet levels. To unders tand the mechanism by which the beta subunit expression is regulated, we cl oned the MAT2B gene, determined its organization, characterized its 5 ' -fl anking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position -203 relat ive to the translation start site, Promoter deletion analysis defined a min imal promoter between positions +52 and +93 base pairs, a GC-rich region, I nclusion of the sequences between -4 and +52 enhanced promoter activity; th is was primarily because of an Sp1 recognition site at +9/+15, The inclusio n of sequences up to position -115 provided full activity; this was attribu ted 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 s howed no effect of the anti-Spl antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein DNA complex formation, sug gesting that Sp3 is one of the main factors binding to this Sp1 site. Chrom atin immunoprecipitation assays supported the involvement of both Sp1 and S p3 in complexes formed on the MAT2B promoter. The data show that the 5 ' -u ntranslated sequences play an important role in regulating the MAT2B gene a nd 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.