Footprinting in vivo reveals changing profiles of multiple factor interactions with the beta-phaseolin promoter...

Whereas in vitro techniques are essentially limited to the analysis of interactions with a single or limited number of cis-elements, in vivo footprinting techniques can be used to assess the total profile of factor interactions with a promoter. By probing with dimethylsulphate and using sensitive ligation-mediated PCR analytical techniques, the in vivo status of the phas promoter was determined in transcriptionally active (embryo) and inactive (leaf) tissues. Changes in factor occupancy were detected during embryogenesis, and the greatest complexity seen (at mid-maturation) was in accordance with the many potential binding sites predicted on the basis of sequence comparison. Evidence was obtained that several cis-elements not previously shown to be used for factor binding in plant promoters are occupied. The great complexity of footprints may represent the need for multiple factor interaction to achieve high levels of transcription. Alternatively, it is possible that the differential levels of expression in individual regions of the embryo evident from histochemical analysis of the GUS reporter result from the interaction of relatively few factors, with the overall footprinting pattern representing a summation of patterns from various tissues.