Isolation of Heat Shock Factor HsfA1a-Binding Sites in vivo Revealed Variations of Heat Shock Elements in Arabidopsis thaliana

Abstract

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.

Analysis of the Regulation of Target Genes by anArabidopsisHeat Shock Transcription Factor, HsfA2

“…22) In fact, Hsf-dependent expression of HSresponsive genes is required for cooperation between HSE and CCAAT-box. 23) Recently, Guo et al 24) reported that HsfA1a bound the stress responsive element (STRE: AGGGG). However, the present experiments showed that only HSE modules of target genes were essential for transcriptional activation by HsfA2, suggesting that other cis elements are not required.…”

Overexpression of Arabidopsis HsfA1a enhances diverse stress tolerance by promoting stress-induced Hsp expression

“…Expression of Hsps as chaperones, including Hsp18.2 and Hsp70, protects cells against injuries associated with various stressors (Simões-Araújo et al, 2003;de Azevedo et al, 2012;Magdalena et al 2012). On the other hand, HsfA1a can bind various promoter targets that are not limited to classical Hsp genes and also include other genes (Guo et al, 2008), implying a broad physiological basis for the stress tolerance role of HsfA1a. However, determining the exact genome-wide target genes of individual HsfA1 members, and whether target genes regulated by HsfA1a in response to distinct stressors are identical or only partially overlap require further investigation.…”

“…For the sorbitol treatment, sorbitol was added into the SIB buffer to a final concentration of 1 M, followed by incubation at room temperature for 20 min under vacuum. The stressed plants were subjected to formaldehyde crosslinking/chromatin immunoprecipitation (X-Chip) using HsfA1a antibodies, and the immunoprecipitated DNA was analyzed by PCR using primer pairs specific to HSP18.2 and HSP70 gene promoters, according to Guo et al (2008). A primer pair amplifying a far upstream sequence of the HSP18.2 promoter was used as a negative control.…”

HEAT-INDUCED TAS1 TARGET1Mediates Thermotolerance via HEAT STRESS TRANSCRIPTION FACTOR A1a–Directed Pathways inArabidopsis

“…Class B and C Hsfs lack the AHA motif and have no activator function on their own (Döring et al, 2000;Nover et al, 2001;Kotak et al, 2004). It has been demonstrated that HsfA1a and other plant Hsf members bind the heat shock element (HSE) consisting of tandem inverted repeats of the short consensus sequence nGAAn (nTTCnnGAAnnTTCn, called the perfect HSE) and the AGGGG motif (von Guo et al, 2008). The roles of several Hsfs of class A (15 members) in Arabidopsis that show high functional diversification and genetic redundancy are still unknown, and the key elements mediating HsfA1 genes in the thermotolerance remain obscure.…”