Isolation of heat shock factor HsfA1a-binding sites in vivo revealed variations of heat shock elements in Arabidopsis thaliana.

The information about DNA-binding sites of regulatory protein is important to understanding the regulatory network of DNA-protein interactions in the genome. In this report we integrated chromatin immunoprecipitation with DNA cloning to isolate genomic sites bound in vivo by heat shock factor HsfA1a in Arabidopsis thaliana. Plantlets were subjected to formaldehyde crosslinking, followed by immunoprecipitation of chromatin. The immunoprecipitated DNA was amplified by PCR and cloned. From a library enriched in putative HsfA1a-binding sites, 21 different genomic fragments were identified (65-332 bp). Six fragments contained known HsfA1a-binding motif (perfect heat shock element). Six fragments contained novel HsfA1a-binding motifs: (1) gap-type, (2) TTC-rich-type, (3) stress responsive element (STRE). Representatives of each were verified by in vitro electrophoretic mobility shift assay. About 81% of the isolated fragments contained the HsfA1a-binding motifs, and/or could be bound by HsfA1a, demonstrating that the method is efficient in the isolation of genomic binding sites of a regulatory protein. The nearest downstream genes to the HsfA1a-binding fragments, which were considered as potential HsfA1a target genes, include a set of classical heat shock protein genes: Hsp17.4, Hsp18.2, Hsp21, Hsp81-1, Hsp101, and several novel genes encoding a non-race specific disease resistance protein and a transmembrane CLPTM1 family protein.