Analysis of heat-shock transcription factor-DNA binding in Arabidopsis suspension cultures by UV laser crosslinking.
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.
Zhang
L
L
ZMBP-Allgemeine Genetik, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
Eggers-Schumacher
G
G
Schöffl
F
F
Prändl
R
R
D017360:Arabidopsis / Q000502:physiology* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D017931:DNA Primers; D018744:DNA, Plant / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005557:Formaldehyde / Q000494:pharmacology; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D007834:Lasers; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays
D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D018744:DNA, Plant; D004268:DNA-Binding Proteins; C000620151:HSF1 protein, Arabidopsis; C446287:HSP18.2 protein, Arabidopsis; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D010940:Plant Proteins; D014157:Transcription Factors; D005557:Formaldehyde