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

Leighton Legros,Abdel-Baset Halim,Margaret E Chamberlin,Arthur Geller,Malak Kotb

doi:10.1074/jbc.m102816200

Leighton Legros, Abdel-Baset Halim + Show 3 more

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https://doi.org/10.1074/jbc.m102816200

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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 alpha2 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.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY033822
The Methionine adenosyltransferase (MAT) II isozyme is expressed in all tissues including the liver (9, 14 –21)
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]

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY033822. The MAT I/III isozymes are, respectively, a tetramer and dimer of a catalytic ␣1 subunit that is expressed only in liver [7, 11,12,13]. 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 Km 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. 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].

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