The DNA‐binding activity of Gal4 is inhibited by methylation of the Gal4 binding site in plant chromatin

Summary Derivatives of the Saccharomyces cerevisiae transcription factor Gal4 which act as effective transcription activators in yeast, Drosophila, mammalian cells and plant protoplasts are shown to direct expression from a GUS reporter construct when expressed in transgenic tobacco. However, in comparison to 35S‐GUS controls, Gal4‐mediated expression of the reporter gene was relatively weak and extremely variable. GUS expression was lost as plants matured and it was almost undetectable in most of their progeny. Gal4‐mediated gene expression could be restored by treating tissues with 5‐aza‐cytidine, implicating cytosine methylation in the loss of Gal4‐mediated expression. Restoration of reporter expression was not accompanied by an increase in steady‐state levels of the activator transcript. We propose that the DNA‐binding activity of Gal4 is sensitive to methylation of its binding site in plant chromatin. The Gal4‐DNA co‐crystal predicts that 5‐methylcytosine at either of the outer two positions of the binding site will effectively prevent Gal4 binding. We show that these positions become extensively methylated in transgenic plants and that methylation of Gal4‐binding sites interferes with Gal4 binding in vitro. These observations suggest that the Gal4 DNA‐binding domain is intrinsically sensitive to cytosine methylation and that, despite the success of Gal4‐based expression systems in yeast and Drosophila, Gal4 is not ideal for use in plant gene expression technology.