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

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Summary

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.

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