Stress-induced activation of heterochromatic transcription

Abstract

Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved.

Figure 1. A temperature shift can release transcriptional silencing of a transgenic locus.

Representative images of histochemical staining for GUS activity (left) performed on seedlings grown under the conditions defined on the right. Plants grown for 3 days at 21°C were transferred to 4°C for 3–9 weeks (a–c) and then shifted to either 21°C (d–f) or 37°C (g–i) for 1 day. Seedlings at 3, 7, and 9 days post-sowing were transferred at 4°C for 1 week and shifted to 37°C for 1 day (j–l), or directly shifted to 37°C for 1 day omitting the cold treatment (m–o).

Figure 2. The ITS-induced release of transcriptional silencing is transient.

(a) Experimental scheme of the control and stress treatments. (b–i) Representative images of histochemical staining for GUS activity performed on seedlings grown under the indicated conditions. (j) Reverse-transcription-PCR detection of GUS transcripts from total RNA of the indicated samples. Amplification of 18S rRNA was used to normalize the amounts of RNA template. Negative controls lacked reverse transcriptase (RT -).

Figure 3. Temperature shift induces transient transcriptional activation of endogenous silent loci.

RNA was purified from plants of the Zurich (Zh) and Col-0 accessions after the indicated treatments. Detection of MULE-F19G14 transcripts was performed by Northern blot. Hybridization with an 18S rRNA-specific probe is shown as a loading control. Transcripts corresponding to 106B, 5S and 180-bp repeats were detected by reverse transcription-PCR (RT-PCR). Amplification of 18S rRNA was used to normalize the amounts of RNA template. Negative controls lacked reverse transcriptase (RT -).

Figure 4. ITS-induced transcriptional activation occurs without detectable changes in the levels of DNA methylation at endogenous loci.

(A) Southern blot analysis of DNA methylation at 106B, 5S and 180-bp repeats using the indicated methylation-sensitive restriction endonucleases. (B) Southern blot analysis of DNA methylation at MULE F19G14 was performed by digesting genomic DNAs with SspI (methylation insensitive), followed by digestion with the indicated methylation-sensitive restriction endonucleases.

Figure 5. Impact of mutations in epigenetic regulators on ITS-induced transcriptional switches.

(A) RNA was extracted from ddm1, mom1 and rts1 mutant plants and the corresponding wild types (WT) after the indicated treatments. Detection of MULE-F19G14 transcripts was performed by Northern blot. Hybridization with an 18S rRNA-specific probe is shown as a loading control. Transcripts corresponding to 106B, 5S and 180-bp repeats were detected by reverse transcription-PCR (RT-PCR). Amplification of 18S rRNA was used to normalize the amounts of RNA template. Negative controls lacked reverse transcriptase (RT-). (B) RT-PCR analysis of transcripts from 106B repeats in the indicated mutant backgrounds and corresponding WT. Amplification of ACTIN2 (ACT2) RNA was used to normalize the amounts of RNA template. Negative controls lacked reverse transcriptase (RT -).

Figure 6. Genome-wide analysis of ITS-induced transcriptional changes.

The relative densities of repeats and 5-methylcytosines (mC) along the 5 chromosomes of Arabidopsis are shown at the top. (A) Top graphs show chromosome-wide changes in transcript abundance in ITS versus CTS plants in a sliding 100-kb window. Middle and lower graphs represent distribution and variation in transcript accumulation from gypsy- and copia-type LTR retrotransposons, respectively, in ITS plants compared with CTS plants. (B) Upper graphs represent chromosome-wide changes in transcript accumulation in ITS+2d versus CTS+2d plants in a sliding 100-kb window. Lower graphs indicate distribution and enrichment in gypsy- and copia-type LTR retroelement transcript in ITS+2d plants compared with CTS+2d plants.