Fig. 4.

H3K27me3 demethylation controls transgenerational upregulation of HSFA2. a H3K27me3 levels detected by ChIP-qPCR at the HSFA2 loci. The gene model was shown and the analyzed regions (P1 and P2) were indicated. One intergenic region (NC3) without H3K27me3 mark²⁸ was used as negative background. b EMSA showed HSFA2 probe binding to GST-REF6C containing the C2H2-ZnF cluster. The mutant probe abolished the binding of GST-REF6C. Excess unlabeled probe outcompeted the labeled probe (right panel). The motif is highlighted in red in the probe sequence, with mutated bases shown in lowercase. The region validated by ChIP-qPCR in c was marked by a bar. c ChIP-qPCR validation of REF6 binding at HSFA2 using the REF6-GFP plants. Col was used as the negative control. The TA3 locus was used as the negative-control locus. d Transcript levels of REF6 and BRM, shown as mean ± SD (n = 3). e ChIP-qPCR validation of BRM binding at HSFA2 using the BRM-GFP plants. Col was used as the negative control. f H3K27me3 levels of HSFA2 in the HSFA2Δ and ref6–1 plants. The data were normalized to the corresponding input fraction (a, c, e, f). g HSFA2 transcript levels. Data were shown as means ± SD from three replicates (a, c–g). h Flowering times of indicated lines were assessed (n ≥ 15 for each line). Lowercase letters indicate statistical significance based on one-way (a, c–f) or two-way (g, h) ANOVA with Tukey’s HSD post hoc analysis (P < 0.05)