In our previous work, we induced RNA interference (RNAi) against the spectinomycin resistance-conferring aadA transgene by transcribing a long inverted repeat in Chlamydomonas reinhardtii. However, after long-term culture, the level of transcripts of the inverted repeat was markedly decreased. In this study, we performed random insertional mutagenesis of the RNAi strain to identify the genes that contribute to the transcriptional silencing of the silencer construct. We succeeded in isolating several mutants showing derepression of transcription of the inverted repeat. One of these tag mutant strains, 148-10H, had a deletion of the Elongin C gene (ELC), which is a component of some E3 ubiquitin ligase complexes. In the mutant, the level of monomethyl histone H3 on lysine 9 (H3K9me1) was reduced to less than half of the parental strain, and a large portion of deacetylated H3 marks were removed from the promoter region of the silencer construct, while these repressive histone modifications and levels of methyl-CpG levels were retained in the inverted repeat region. The most probable interpretation of the above-mentioned phenomenon is that ELC is essential for stepwise extension of heterochromatin formation that is nucleated in the inverted region over the promoter region.
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