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Comparative Study
. 2005 Mar 15;19(6):683-96.
doi: 10.1101/gad.1247705. Epub 2005 Mar 1.

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans

Alla Grishok  1 , Jina L Sinskey, Phillip A Sharp
Affiliations
Comparative Study

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans

Alla Grishok et al. Genes Dev. .

Abstract

The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

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Figures

Figure 1.
Figure 1.
Silencing of an elt-2::gfp/LacZ transgene is induced by L4440 feeding. (A) Structures of the pPD96.04 vector used for constructing the elt-2::gfp/LacZ strain, the L4440 feeding vector, and L4440 with deletion of the LacZ sequence. The backbone sequence (2 kb) and 150-bp LacZ fragment are identical in pPD96.04 and L4440. Drawing is not to scale. (B) elt-2::gfp/LacZ strain not fed with L4440 (top), and elt-2::gfp/LacZ fed with L4440 vector (middle), and with L4440-ΔLacZ vector (bottom). Strains were grown at 20°C.
Figure 2.
Figure 2.
The common vector backbone sequence plays a role in L4440-dependent silencing of elt-2::gfp/LacZ transgene. (A, left) elt-2::gfp/LacZ strain not exposed to L4440 at 16°C; 100% of the observed worms express GFP. (Middle) elt-2::gfp/LacZ worms on L4440 at 16°C; 100% of the observed worms have no GFP expression or very weak expression. (Right) 80%-100% of elt-2::gfp/LacZ worms on L4440-ΔLacZ at 16°C have weak GFP expression. (B-D) RT-PCR-detecting transcript from 2-kb backbone sequence in elt-2::gfp/LacZ strains; 2-kb backbone RNA is not detected in worms silenced by L4440 (C,D, lane 2). ama-1 mRNA is shown as a control for RNA samples. (E) Quantification of the relative levels of gfp/LacZ mRNA in wild-type transgenic worms at 20°C and 16°C, and rde-1(ne300) worms by real-time RT-PCR; ama-1 expression was used for internal reference. Mean values and ranges of the relative LacZ/ama-1 ratios based on three real-time RT-PCR trials are shown.
Figure 3.
Figure 3.
L4440-induced silencing of elt-2::gfp/LacZ is dependent on dcr-1, alg-1/2, and hpl-2. The elt-2::gfp/LacZ strain was fed on L4440 with cloned fragments of genes indicated above each image. Strains were grown at 20°C.
Figure 4.
Figure 4.
RT-PCR analysis of pre-mRNA and mRNA levels during L4440-induced silencing of elt-2::gfp/LacZ. (A) RT-PCR of gfp/LacZ transgene; amplification with exon-specific primers (top) detects products from the mature mRNA (357 bp) and pre-mRNA (458 bp); amplification with intron-specific primers (middle) detects a product of 373-bp; ama-1 mRNA RT-PCR detecting a 236-bp product is shown as control (bottom). (B) Quantification of the relative levels of gfp/LacZ mRNA and gfp/LacZ pre-mRNA shown in A by real-time RT-PCR; ama-1 expression was used for internal reference. Mean values and ranges of the relative LacZ/ama-1 ratios based on two real-time RT-PCR trials are shown.
Figure 5.
Figure 5.
Silencing of elt-2::gfp/LacZ in an rrf-3 background. (A) rrf-3;elt-2::gfp/LacZ at 20°C (top); rrf-3;elt-2::gfp/LacZ at 16°C (middle); rde-1; rrf-3;elt-2::gfp/LacZ at 16°C (bottom). (B) RT-PCR of gfp/LacZ transgene; amplification with exon-specific primers (top), intron-specific primers (middle); ama-1 control (lower middle), and primers specific for the 2-kb backbone sequence (bottom). (C) Real-time RT-PCR quantification of the relative levels of gfp/LacZ mRNA and gfp/LacZ pre-mRNA during silencing induced by feeding the L4440 or L4440 ΔLacZ vectors; ama-1 expression was used for internal reference. Mean values and ranges of the relative LacZ/ama-1 ratios based on two real-time RT-PCR trials are shown.
Figure 6.
Figure 6.
elt-2::gfp/LacZ silencing is associated with decrease in histone H4 acetylation and polymerase II association. (A) ChIP with anti-acetyl-H4 antibodies was performed on extracts from rrf-3; elt-2::gfp strain with (right) and without (left) L4440 feeding. PCR products were amplified with primers specific for the gfp/LacZ transgene (top), 2-kb backbone sequence (bottom) or act-3 gene (middle) as an internal control. (Lane 1) DNA purified from 1/40 of input material was used. (Lane 2) DNA bound to agarose A beads with anti-acetyl-H4 antibodies. (Lane 3) DNA bound to agarose A beads without antibody. (B,C) Real-time PCR quantification of the relative amounts of gfp/LacZ DNA precipitated by anti-acetyl-H4 antibodies or 8WG16 Pol II antibodies at indicated conditions, act-3 was used as internal reference. Mean values and ranges of the relative LacZ/actin ratios from two to three independent experiments are shown.
Figure 7.
Figure 7.
Schematic model showing a possible mechanism of RNAi-TGS at the elt-2::gfp/LacZ repetitive array. Transcription of the gfp/LacZ pre-mRNA (green/blue line) and bidirectional transcription in the repetitive array (black lines) occurs in the transgenic worms. dsRNA produced from the locus would be processed by Dicer, generating siRNAs, which might induce a low level of background silencing of the array. However, silencing of the array is not efficient without additional stimuli. Feeding worms with dsRNA produced from the L4440 vector brings the level of LacZ and backbone-specific siRNA to the threshold level, stimulating chromatin modifications, HP1 localization, and TGS at the locus.
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