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. 2002 Jan 22;99(2):1076-81.
doi: 10.1073/pnas.022392999. Epub 2002 Jan 8.

Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements

Byeong-ryool Jeong Br  1 , Dancia Wu-Scharf, Chaomei Zhang, Heriberto Cerutti
Affiliations

Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements

Byeong-ryool Jeong Br et al. Proc Natl Acad Sci U S A. .

Abstract

In the unicellular green alga Chlamydomonas reinhardtii, the epigenetic silencing of transgenes occurs, as in land plants, at both the transcriptional and posttranscriptional levels. In the case of single-copy transgenes, transcriptional silencing takes place without detectable cytosine methylation of the introduced DNA. We have isolated two mutant strains, Mut-9 and Mut-11, that reactivate expression of a transcriptionally silenced single-copy transgene. These suppressors are deficient in the repression of a DNA transposon and a retrotransposon-like element. In addition, the mutants show enhanced sensitivity to DNA-damaging agents, particularly radiomimetic chemicals inducing DNA double-strand breaks. All of these phenotypes are much more prominent in a double mutant strain. These observations suggest that multiple partly redundant epigenetic mechanisms are involved in the repression of transgenes and transposons in eukaryotes, presumably as components of a system that evolved to preserve genomic stability. Our results also raise the possibility of mechanistic connections between epigenetic transcriptional silencing and DNA double-strand break repair.

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Figures

Figure 1
Figure 1
Expression of the RbcS2∷aadA∷RbcS2 transgene is reactivated in the mutant strains. (A) Southern blot analysis of the wild-type untransformed strain (CC-124), the silenced parental strain (11-P[300]), the mutant strains (Mut-9 and Mut-11), and a double mutant strain (Mut-9 Mut-11). Total cell DNA was digested with HindIII and hybridized to the pBluescript vector backbone, which is common to the plasmids containing the chimeric aadA transgene or the tagging rs-3 gene. The fragments corresponding to the transgene (aadA) or the insertional mutagen (rs-3) are indicated. (B) Northern blot analysis of the strains described above. Total cell RNA was isolated from each strain, separated under denaturing conditions, and hybridized to the aadA coding sequence (Upper). The same blot was reprobed with the coding sequence of RbcS2 as a control for equal loading of the lanes (Lower). The faint transcript seen above RbcS2 corresponds to the RbcS1 gene (11). (C) Growth and survival on TAP medium or on TAP medium containing spectinomycin (TAP + SPEC) of the indicated strains. Five-fold serial dilutions of cells, starting with 1 × 105 cells on the left, were spotted on each plate and incubated for 15 days (12).
Figure 2
Figure 2
Reactivation of a retroelement, TOC1, and a DNA transposon, Gulliver, in the mutant strains. Abbreviations are as in the legend to Fig. 1. (A) Northern blot of total RNA probed sequentially for TOC1 (Upper) to examine transcript levels and for RbcS2 (Lower) to test for equal loading of the lanes. (B) Southern blot analysis of TOC1 transposition. Genomic DNA from parallel subcultures (Clones) of the indicated strains was digested with HincII and probed for TOC1. The arrowheads indicate new fragments in the subclones of Mut-9 and Mut-11. (C) Southern blot analysis of Gulliver transposition. Total cell DNA from parallel subcultures (Clones) of the indicated strains was digested with HindIII and probed for Gulliver. The arrowheads indicate missing or new fragments in the subclones of Mut-9 and Mut-9 Mut-11. Although only two subclones are shown for 11-P[300], we did not detect mobilization of either TOC1 or Gulliver in 10 parallel subcultures grown under the same conditions as the mutant strains.
Figure 3
Figure 3
Photoheterotrophic growth of the mutant and wild-type strains. Abbreviations are as in the legend to Fig. 1. Each time point represents the mean (± standard error) of six replicates (three independent experiments). Where the error bars are not visible, they are smaller than the symbols. The exponential phase of the growth curve was used to calculate doubling times. Even though all strains show similar doubling times, Mut-11 and Mut-9 Mut-11 took considerably longer to reach exponential growth (represented by a linear increase in optical density in the semilogarithmic scale).
Figure 4
Figure 4
Effect of DNA-damaging agents on the survival of the mutant and wild-type strains. Each graph point represents the mean (± standard error) of nine replicates (three independent experiments). Where the error bars are not visible, they are smaller than the symbols. The dashed horizontal lines indicate 30% cell survival. Symbols: formula image, wild-type CC-124; ●, Mut-9; Δ, Mut-11; ♦, Mut-9 Mut-11. (A) Survival of the mutants and wild-type C. reinhardtii grown on TAP medium containing increasing concentrations of bleomycin. (B) Survival of the mutants and wild-type C. reinhardtii exposed to increasing doses of UV-C irradiation under nonphotoreactivating conditions. (C) Survival of the mutants and wild-type C. reinhardtii grown on TAP medium containing increasing concentrations of methyl methanesulfonate (MMS).
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