METHIONINE ADENOSYLTRANSFERASE4 Mediates DNA and Histone Methylation.

Plant physiology (2018-03-25)
Jingjing Meng, Lishuan Wang, Jingyi Wang, Xiaowen Zhao, Jinkui Cheng, Wenxiang Yu, Dan Jin, Qing Li, Zhizhong Gong
ABSTRACT

DNA and histone methylation coregulate heterochromatin formation and gene silencing in animals and plants. To identify factors involved in maintaining gene silencing, we conducted a forward genetic screen for mutants that release the silenced transgene Pro35S::NEOMYCIN PHOSPHOTRANSFERASE II in the transgenic Arabidopsis (Arabidopsis thaliana) line L119 We identified MAT4/SAMS3/MTO3/AT3G17390, which encodes methionine (Met) adenosyltransferase 4 (MAT4)/S-adenosyl-Met synthetase 3 that catalyzes the synthesis of S-adenosyl-Met (SAM) in the one-carbon metabolism cycle. mat4 mostly decreases CHG and CHH DNA methylation and histone H3K9me2 and reactivates certain silenced transposons. The exogenous addition of SAM partially rescues the epigenetic defects of mat4 SAM content and DNA methylation were reduced more in mat4 than in three other mat mutants. MAT4 knockout mutations generated by CRISPR/Cas9 were lethal, indicating that MAT4 is an essential gene in Arabidopsis. MAT1, 2, and 4 proteins exhibited nearly equal activity in an in vitro assay, whereas MAT3 exhibited higher activity. The native MAT4 promoter driving MAT1, 2, and 3 cDNA complemented the mat4 mutant. However, most mat4 transgenic lines carrying native MAT1, 2, and 3 promoters driving MAT4 cDNA did not complement the mat4 mutant because of their lower expression in seedlings. Genetic analyses indicated that the mat1mat4 double mutant is dwarfed and the mat2mat4 double mutant was nonviable, while mat1mat2 showed normal growth and fertility. These results indicate that MAT4 plays a predominant role in SAM production, plant growth, and development. Our findings provide direct evidence of the cooperative actions between metabolism and epigenetic regulation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Millipore
ANTI-FLAG® M1 Agarose Affinity Gel
Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-Histone H3 Antibody, CT, pan, serum, Upstate®
Sigma-Aldrich
Anti-monomethyl-Histone H3 (Lys27) Antibody, Upstate®, from rabbit
Sigma-Aldrich
ChIPAb+ Monomethyl-Histone H3 (Lys9) - ChIP Validated Antibody and Primer Set, clone CMA306, from mouse, purified by using protein G