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. 2005 Apr 1;19(7):782-7.
doi: 10.1101/gad.332305. Epub 2005 Mar 17.

Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences

Valérie J P Robert  1 , Titia Sijen, Josien van Wolfswinkel, Ronald H A Plasterk
Affiliations

Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences

Valérie J P Robert et al. Genes Dev. .

Abstract

Protection of genomes against invasion by repetitive sequences, such as transposons, viruses, and repetitive transgenes, involves strong and selective silencing of these sequences. During silencing of repetitive transgenes, a trans effect ("cosuppression") occurs that results in silencing of cognate endogenous genes. Here we report RNA interference (RNAi) screens performed to catalog genes required for cosuppression in the Caenorhabditis elegans germline. We find factors with a putative role in chromatin remodeling and factors involved in RNAi. Together with molecular data also presented in this study, these results suggest that in C. elegans repetitive sequences trigger transcriptional gene silencing using RNAi and chromatin factors.

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Figures

Figure 1.
Figure 1.
Germline cosuppression of pie-1::gfp::H2B. (A) Germline GFP expression in AZ212, a strain homozygous for pie-1::gfp::H2B. A lineup of oocytes expressing GFP localized in their nuclei is shown. (B) Cosuppression is induced in lines NL3847 and NL3864 that respectively contain dpy-30::gfpΔ::unc-54 and him-14::gfpΔ::unc-54 integrated arrays; lineups of oocytes that no longer express the GFP are shown. Cosuppression induced by the dpy-30::gfpΔ::unc-54 array (line NL3848) is released in an rde-2 (pk716) background (strain NL3858). (C) In cosuppressed lines gfp mRNA levels are strongly reduced as apparent from RPAs using a probe specific for the full-length gfp mRNA. For control on RNA loading and developmental stage, a pie-1-specific probe was added in each RPA and the ratio of gfp to pie-1 mRNA was determined. The reference strain N2 and a yeast tRNA (–) were used as negative controls. (D) The pie-1::gfp::H2B locus is not silenced by an array of promoterless gfp copies. (E) RPAs detect dsRNA molecules in cosuppressed lines. Samples were pretreated to contain total RNA (tot), only dsRNA (ds), or no RNA (–). The RPA probe (of anti-sense polarity) discriminates unspliced and spliced gfpΔ or gfp full sequences. Protected fragments in the dsRNA lane correspond mainly to the unspliced form of the gfpΔ transcripts. Similar results were obtained with NL3847 and NL3848 (data not shown).
Figure 3.
Figure 3.
Analysis of release of cosuppression. (A) GFP re-expression was studied in a line in which pie-1::gfp::H2B is cosuppressed by an extrachromosomal array of repetitive dpy-30::gfp::unc-54 sequences (including a rol6 marker) after loss of the extrachromosomal array. The nonroller progeny (F1, F2, and F3) of a roller parent (F0) exhibit a progressive re-expression of pie-1::gfp::H2B. The number of positive animals out of 20 examined is indicated for each generation. (B) Release of cosuppression involves release of transcriptional gene silencing. The ratio of spliced to unspliced gfp transcripts expressed from pie-1::gfp::H2B was compared in silenced and re-expressed states either after crossing the cosuppressed line NL3848 in an rde-2(pk716) background (NL3858) or after feeding cosuppressed line NL3847 (results not shown for NL3848 and NL3864) dsRNA-food targeting the mut/cde/rde gene mut-16. The spliced/unspliced ratio remains the same in cosuppressed and reexpressed states but increases upon re-expression of pk1660 (NL3700) that is post-transcriptionally silenced by natural Tc1 siRNA (Sijen and Plasterk, 2003). Similar results were obtained after feeding NL3848 and NL3864 with dsRNA food targeting mut-16.
Figure 2.
Figure 2.
Identification of cde genes and their involvement in other silencing processes. (A) Re-expression of GFP upon feeding strain NL3847 or NL3864 with food expressing dsRNA targeting cde gene pgl-1 or K10D2.3, respectively. (B) pk1673, a deletion allele of ppw-2, removes most of the coding sequences (dashed line; see Supplemental Material)—including the conserved domains PAZ and PIWI (plain lines)—and exhibits a cde phenotype when genetically combined to pie-1::gfp::H2B in the presence of a him-14::gfpΔ::unc-54 array. (C) The cde genes pgl-1, rha-1, egl-13, and K10D2.3 are involved in RNAi. Deletion alleles for pgl-1, egl-13, rha-1, ppw-2, K10D2.3, and hpl-2 (see Supplemental Material) were tested for RNAi competence on food targeting the germline-expressed genes pos-1 (gray bars) and par-1 (white bars). Wild-type OP50 bacteria were used as a negative control (black bars). For pgl-1, rha-1, K10D2.3, hpl-2, and ppw-2, the percentage of dead embryos was estimated (left panel); for egl-13, which is egg-laying deficient, the brood size of single animals was estimated (right panel).
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