Effects of DNA methylation on DNA binding and transcriptional activity of the Opaque2 protein
--Rossi, V; Solinas, G; Motto, M; Pellegrini, L

In animals, DNA methylation appears to influence the tissue-specific expression of developmentally regulated genes by affecting the interactions of both chromatin proteins and transcription factors with DNA. Similarly, in plants methylation of the C residue embedded within the consensus sequence for mammalian CREB factors, TGACGTCA, was found to inhibit in vitro DNA binding activity of CREB-like factors present in nuclear extracts of pea, wheat, soybean and cauliflower (Inamdar et al., Plant Mol. Biol. 17:111-123, 1991). However, binding of plant nuclear proteins to hemimethylated target sequences has been reported for the tobacco CG-1 nuclear factor(s) (Staiger et al., PNAS 86:6930-6934, 1989) and the maize Ac-encoded transposase (Kunze and Starlinger, EMBO J. 8:3177-3185, 1989).

The maize Sn and O2 promoters have been shown to be highly methylated at the time and place of their expression (Ronchi et al., EMBO J. 14:5318-5328, 1995; Rossi et.al., unpublished). Particularly, the methylation pattern of the O2 promoter disclosed in immature endosperms indicates the occurrence of m5C within both DNA strands of the O2-protein binding site (TGACGTTG), thus suggesting the capability of this bZIP transcription factor to bind to a methylated consensus sequence and to activate the expression of a highly methylated promoter. To address these questions, gel-shift experiments were performed to investigate the DNA binding activity of a recombinant O2-protein to O2 promoter fragments (spanning from -399 to -211) whose methylation state mimics the O2 methylation patterns disclosed by genomic sequencing. We used O2 promoter fragments methylated at CpG sites, hemimethylated on either the upper or the lower strand, fully methylated, and containing different percentages of m5C. Results showed that O2 efficiently binds to a CpG-methylated O2 promoter. Band shifts were also observed with a 30%- and 50%-methylated promoter fragment. However, higher percentages of m5C were found to impair O2 in vitro binding activity. Interestingly, hemimethylated O2 promoter fragments can still be retarded, although a DNA fragment hemimethylated on the lower strand was bound with reduced efficiency compared to a promoter fragment hemimethylated on the upper strand.

An in vitro transcription initiation system consisting in a partially methylated O2 promoter fragment (spanning from -762 to +60), HeLa cells nuclear extracts, and E. coli extracts expressing a recombinant O2-protein was developed to investigate the capability of the O2-protein to bind and activate transcription of a highly methylated promoter. Results showed that a 50%-methylated O2 promoter allows formation of active preinitiation complexes and the synthesis of specific O2 transcripts.

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