Analysis of heat-shock transcription factor-DNA binding in Arabidopsis suspension cultures by UV laser crosslinking.

Abstract

Crosslinking techniques are important in examining protein-DNA interactions in living cells. Formaldehyde and UV light emitted by conventional lamps are the most commonly used crosslinking agents. The crosslinking step is followed by immunoprecipitation of specific protein-DNA adducts, and by analysis and quantification of the co-immunoprecipitated DNA. There are a few reported cases of fruitful in vivo protein-DNA crosslinking experiments, but not in plants. In this report, we analyse the binding of heat-shock transcription factor (HSF) to heat-shock promoters in intact Arabidopsis cells. Efficient protein-DNA crosslinking by irradiation of Arabidopsis suspension culture tissue was achieved using UV laser pulses. In addition, methods for immunoprecipitation and detection of the co-immunoprecipitated DNA are reported. Our results suggest that Arabidopsis HSFs immunoreactive for HSF1 antibodies bind constitutively to the HSP18.2 gene.