Analysis of the methylation pattern of the maize opaque-2 (O2) promoter and in vitro binding studies indicate that the O2 b-Zip protein and other endosperm factors can bind to methylated target sequences

The maize opaque-2 locus (o2) has an endosperm-specific expression and is positively autoregulated by its gene product, a b-Zip protein, to a TGACGTTG motif. The genomic sequencing method was used here to describe, in leaf and endosperm, the methylation pattern of a 390-base pair region of the o2 promoter. In leaf, 96% of the C residues are methylated, whereas in endosperm the 5-methylcytosine content is 84%. Comparison of these methylation patterns indicates that the o2 tissue-specific expression does not result from the demethylation of any specific C residue and that, in vivo, O2 interacts with a methylated target sequence. Consistently, gel-shift experiments using a CpG-methylated, partially methylated, and hemimethylated o2 promoter fragments showed that, in vitro, the O2 protein binds to the major groove of a methylated target sequence, although its binding activity decreases at increasing levels of C-methylation and is more effectively reduced by methylation of the lower strand than of the upper strand of the DNA. Using partially purified endosperm cell extracts, we also show that, besides the O2 protein, other proteins specifically bind to a partially methylated o2 promoter fragment, therefore indicating that in plants a subset of different proteins may mediate the expression of a naturally occurring methylated o2 promoter.