2.7 BES1 directly targets APX2 and HSFA3 in the memory phase
Since BES1 is critical for the sustained activation of HS-associated memory genes, we further examine whether BES1 directly target these genes for activation. Yeast one-hybrid (Y1H) assays indicate that BES1 directly activates the expression of APX2 and HSFA3 (Fig. 6A). We next conducted an electrophoretic mobility shift assay (EMSA) towards the fragment of APX2 and HSFA3 promoter containing an E-box, the potential BES1 protein binding site. As expected, the EMSA assay showed that MBP-BES1 proteins could bind to APX2 and HSFA3 probes, but MBP did not (Fig. 6B, C). We further confirmed that BES1 directly binds to the promoter of APX2 andHSFA3 in vivo using a chromatin immunoprecipitation (ChIP)-qPCR assay. Obvious enrichment was found in the promoter of APX2 andHSFA3 (Fig. 6D). These results suggest that APX2 andHSFA3 are transcriptional targets of BES1 proteins during the thermomemory process.