Methylation of the Opaque2 box in zein genes is parent-dependent and affects O2 DNA binding activity in vitro

In the maize endosperm, the expression of the subfamily 4 (SF4) of the zein genes is under the transcriptional control of the Opaque2 (O2) basic leuci ne zipper transcriptional activator, which binds to the O2-box 5'-TCCACGTAG A-3'. Southern experiments showed that the O2-box core sequence ACGT is hea vily methylated in sporophytic tissues but becomes hypomethylated in the en dosperm. DNA analyses of two inbred lines and their reciprocal crosses indi cate that the hypomethylation state is established on the maternal compleme nts of the endosperm. Electrophoretic mobility shift assay (EMSA) and south western experiments with endosperm protein extracts and recombinant O2 usin g oligonucleotides with methylated and unmethylated cytosines in the O2-box indicate an inhibitory effect of modified sequences on O2-binding activity . These results suggest that DNA methylation modulates O2 activity in vivo and shed light on molecular mechanisms involved in the parent-dependent zei n gene expression in maize endosperm.