Figure 1. Heat stress releases heterochromatin silencing without altering DNA methylation.

(A) Scheme representing the method used to submit rosette leaves to a control stress (23°C) or a heat stress (37°C). The L5-GUS transgene is reactivated in leaves subjected to heat stress. GUS: β-glucuronidase. (B) RT-qPCR analysis of transcripts from MULE-AT2G15810 and the L5-GUS transgene in L5 transgenic plants at 23 or 37°C, normalized to the reference gene AT5G12240 and further normalized to the mean of L5 samples at 23°C. Error bars represent standard error of the mean across three biological replicates. (C) RT-qPCR analysis of transcripts from endogenous repeats in L5 transgenic plants at 23 or 37°C (Transcriptionally Silent Information [TSI]). Amplification of 18S rRNA was used as a loading control. PCR in the absence of reverse transcription (RT-) was performed to control for genomic DNA contamination. (D) (top) Transcriptional changes in WT plants subjected to heat stress represented along chromosomes by log2 ratios (37/23°C) of mean reads per kilobase per million mapped reads (RPKM) values calculated in 100 kb windows. (bottom) Density of TEs detected as significantly up-regulated in WT plants subjected to heat stress is plotted in red (left y axis) with total TE density in grey (right y axis), both calculated by 100-kb windows. Windows containing up-regulated ONSEN elements (AtCOPIA78) are marked with an asterisk. (E) Average cytosine methylation levels by 500-kb windows calculated in CG, CHG, and CHH contexts in a WT subjected to a control stress (23°C) or to heat stress (37°C). (F) PCGs or TEs up-regulated in heat-stressed WT plants were aligned at their 5′-end or 3′-end and average cytosine methylation levels in the indicated nucleotide contexts were calculated from 3 kb upstream to 3 kb downstream in a WT subjected to a control stress (23°C) or to heat stress (37°C). Upstream and downstream regions were divided in 100 bp bins, whereas annotations were divided in 40 bins of equal length.