Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltrans ferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal mo tifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. W e used a reverse genetics approach to determine the function of the Zmet2 g ene. Plants homozygous for a Mutator transposable element insertion into mo tif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis o f these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at Cp NpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequence s.