1. Protection of Germline Gene Expression by the C
Protection of Germline Gene Expression by the C. elegans Argonaute CSR-1 
Christopher J. Wedeles, Monica Z. Wu, Julie M. Claycomb 
Developmental Cell 
Volume 27, Issue 6, Pages (December 2013)
DOI: /j.devcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Developmental Cell 2013 27, 664-671DOI: (10.1016/j.devcel.2013.11.016)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 Characterization of Strains Used in the Tethering Assay
(A) A diagram of the gfp::boxb tethering locus on chromosome (LG) II is shown. The expression of gfp is driven by the germline promoter for pie-1. Five box b hairpin sites are present at the 3′ end of the gfp transcript, followed by the pie-1 3′ UTR. CSR-1::λN recognizes the box b hairpin sequences.
(B) The gfp mRNA (green) and pre-mRNA (blue) levels for the gfp::boxb, csr-1::λN, gfp::boxb, cdk-1::gfp(RNAe) (WM243), and csr-1::λN, cdk-1::gfp (RNAe) strains were measured by qRT-PCR and are expressed relative to the germline expressed gene pie-1. For all parts of this figure (unless noted), error bars represent SEM and the samples are the average of three to five biological replicates (of 50–100 adult hermaphrodites per sample).
(C) The structure of the csr-1::λN transgene is depicted. The scale bar represents 1 kb.
(D) The csr-1::λN mRNA levels for the csr-1::λN, gfp::boxb, csr-1::λN, gfp::boxb (after RNAe), and csr-1::λN, cdk-1::gfp (RNAe) strains were measured by qRT-PCR and are relative to pie-1.
(E) The mRNA levels for three representative CSR-1/22G-RNA germline gene targets (pie-1, hcp-1, and klp-16) were measured in the gfp::boxb (red), csr-1::λN, gfp::boxb (lilac), and cdk-1::gfp(RNAe) (WM243) (purple) strains using qRT-PCR. The values are expressed relative to the nontarget germline gene, sec-23.
(F) CSR-1 chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) demonstrates that CSR-1 associates with the gfp::boxb locus. Endogenous CSR-1 is present in both strains and can associate with gfp::boxb. When CSR-1::λN is present, the levels of CSR-1 at the gfp::boxb transgene locus are consistently higher. This result is consistent with weak licensing of the gfp::boxb transgene in an otherwise wild-type strain background, which enables this transgene to be expressed initially and silenced by the cdk-1::gfp(RNAe) allele. Values at the gfp::boxb tethering locus are relative to the Y47H10A.4 control locus, which is not a target of CSR-1. Input samples and mock ChIPs (performed with beads only) are shown. Error bars are the SD of triplicate experiments. The dashed gray line at 1.33-fold enrichment marks the lower boundary for enrichment by a successful CSR-1 ChIP.
(G) ChIP for histone H3, histone H3 lysine 4 dimethylation (H3K4me2; associated with transcription), histone H3 lysine 9 trimethylation (H3K9me3; associated with transcriptionally silent states) was performed in gfp::boxb (blue), csr-1::λN, gfp::boxb (teal), and cdk-1::gfp(RNAe) (WM243) (green) strains. qPCR was used to examine the gfp locus, cpt-3 (H3K9me3 enriched locus), and hcp-1 (CSR-1 target and H3K4me2 enriched locus) relative to the Y47H10A.4 control locus. Error bars are the SD of triplicate experiments. The dashed gray line at 1.33-fold marks the lower boundary of enrichment. See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
Tethering CSR-1::λN Protects the gfp::boxb Transcript from Silencing by RNAe
(A) An outline of crosses to determine whether the cdk-1::gfp(RNAe) (WM243) allele can silence gfp::boxb is shown. Separate crosses were done to introduce cdk-1::gfp(RNAe) from either the paternal or maternal germline. Both the gfp::boxb and cdk-1::gfp transgenes are inserted in a single copy onto LG II at the same locus, so strains possessing both alleles are maintained as heterozygotes. We segregated gfp::boxb homozygotes in the F2 and verified their genotype by PCR. The strains marked in turquoise and red were analyzed in (B).
(B) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to the germline gene pie-1 are shown. The starting gfp::boxb strain (turquoise) is set to one. For all parts of this figure, each bar on the graph represents the average of three to seven biological replicates of 50–100 adult hermaphrodites per sample, and error bars represent SEM. For the gfp::boxb(RNAe) strains, measurements were taken at the F3 generation and in subsequent generations after the crosses as marked. The parent of origin for the RNAe allele is shown below the generation number. t tests were performed on all samples and all results were significant. Only one set of p values is shown for simplicity (∗p < 0.05, ∗∗p < 0.01 by the Student’s t test.).
(C) An outline of genetic crosses used to determine whether gfp::boxb is silenced via the piRNA pathway. The silenced gfp::boxb(RNAe) strain was generated by crosses described in (A). The wago-9(tm1200) allele was introduced from the male, the gfp::boxb(RNAe) allele was from the hermaphrodite. The gfp::boxb, wago-9(tm1200) homozygous strain was verified by genotyping PCR. The strains marked in turquoise and red were analyzed in (D).
(D) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels in each of strain relative to pie-1 are shown. The starting gfp::boxb(RNAe) strain (turquoise) was derived in (B); this is an independent sample. For the gfp::boxb, wago-9(tm1200) strain, measurements were taken at the F3 generation. Because the gfp levels are greater than the starting strain, expression is activated (∗p < 0.05, ∗∗p < 0.01 by the Student’s t test).
(E) An outline of crosses to determine whether the presence of CSR-1::λN can protect gfp::boxb from silencing by the cdk-1::gfp(RNAe) allele. Separate crosses introduced cdk-1::gfp(RNAe) from either the maternal or paternal germline. Homozygous csr-1::λN, gfp::boxb strains were segregated in the F3 generation and their genotypes verified by PCR. The strains marked in turquoise and red were analyzed in (F).
(F) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to pie-1 are shown. The starting csr-1::λN, gfp::boxb strain (turquoise) is set to one. For the csr-1::λN, gfp::boxb strains after exposure to the cdk-1::gfp(RNAe) allele, measurements were taken at the F3 generation. The small difference in gfp levels for the strain receiving the cdk-1::gfp(RNAe) allele from the maternal germline is not statistically significant by the Student’s t test.
(G) An outline of the genetic crosses to demonstrate that the continued presence of the cdk-1::gfp(RNAe) allele within a csr-1::λN, gfp::boxb strain does not alter the expression of gfp::boxb. The starting strain for these experiments possessed a cdk-1::gfp(RNAe) allele from the maternal germline (Figure 2F, hermaphrodite symbol). The strain was propagated as homozygous for csr-1::λN on LG I and heterozygous for gfp::boxb and cdk-1::gfp(RNAe) on LG II. In each generation (F3….F(n)) csr-1::λN, gfp::boxb homozygotes were segregated and verified by genotyping PCR. The strains marked in turquoise and red were analyzed in (H).
(H) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to pie-1 over several generations are shown. The starting csr-1::λN, gfp::boxb strain (turquoise) is set to one.
(I) An outline of genetic crosses to show that csr-1::λN tethering is important for the protection of gfp::boxb expression. csr-1::3xflag (WM193) was previously shown to rescue the csr-1(tm892) allele (Claycomb et al., 2009). A recombinant chromosome possessing both csr-1::3xflag and gfp::boxb was generated by standard genetic approaches and verified by genotyping PCR. The csr-1::3xflag, gfp::boxb males were crossed to the cdk-1::gfp(RNAe) strain, and then csr-1::3xflag, gfp::boxb alleles were segregated from cdk-1::gfp(RNAe) and maintained as homozygotes. The strains marked in turquoise and red were analyzed in (J).
(J) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to pie-1 are shown. The starting csr-1::3xflag, gfp::boxb strain (turquoise) is set to 1. The F3 generation for the csr-1::3xflag, gfp::boxb (RNAe) strain is shown (∗p < 0.05, ∗∗p < 0.01 by the Student’s t test). See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3
The Introduction of CSR-1::λN into Silent (RNAe) Strains Activates gfp Expression
(A) An outline of crosses to determine whether the introduction of csr-1::λN can activate the expression of a silenced gfp::boxb(RNAe) strain (generated in Figures 2A and 2B). The csr-1::λN allele was introduced from the paternal germline and the silenced gfp::boxb(RNAe) allele was from the hermaphrodite. The strains marked in turquoise and red were analyzed in (B).
(B) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to the germline gene pie-1 are shown. The starting gfp::boxb(RNAe) strain (turquoise) is set to one. For all parts of this figure, each bar on the graph represents the average of three to seven biological replicates from samples of 50–100 adult hermaphrodite worms and error bars represent SEM. After introduction of csr-1::λN into gfp::boxb(RNAe) strains, measurements were taken from each generation indicated. Two gfp::boxb(RNAe) strains were used in these experiments and only differ in which parent introduced the initial cdk-1::gfp(RNAe) silencing signal, as designated. Strains with gfp levels comparable to the starting gfp::boxb(RNAe) strain are silenced, while strains with gfp levels significantly greater than the starting strain are activated (∗p < 0.05, ∗∗p < 0.01 by the Student’s t test).
(C) A diagram of genetic crosses used to show that the continued presence of csr-1::λN, gfp::boxb within the cdk-1::gfp(RNAe) strain activates cdk-1::gfp(RNAe) over several generations is shown. These crosses are the same as those in Figures 2G and 2H, except that we focused on the homozygous csr-1::λN, cdk-1::gfp(RNAe) animals. In each generation (F3….F(n)), csr-1::λN, cdk-1::gfp(RNAe) homozygotes were segregated and verified by genotyping PCR. The strains marked in turquoise and red were analyzed in (D).
(D) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to the pie-1. The starting cdk-1::gfp(RNAe) strain (turquoise) is set to one. Generations are as indicated. Strains with gfp levels comparable to the starting cdk-1::gfp(RNAe) strain are considered to remain silenced, while strains with gfp levels significantly greater than the starting strain are considered to be activated (∗p < 0.05, ∗∗p < 0.01 by the Student’s t test). The parent of origin for the original silencing cdk-1::gfp(RNAe) allele is noted, and had an impact on how quickly activation occurred.
(E) An outline of crosses to determine whether the introduction of csr-1::λN at the same time as cdk-1::gfp(RNAe) can protect gfp::boxb expression is shown. The csr-1::λN, cdk-1::gfp(RNAe) alleles were introduced from the paternal germline and the silenced gfp::boxb(RNAe) allele was from the hermaphrodite. The strains marked in turquoise and red were analyzed in (F).
(F) qRT-PCR measurements of gfp mRNA (green) and pre-mRNA (blue) levels relative to pie-1. The starting gfp::boxb strain (turquoise) is set to one. The F3 generation for the csr-1::λN, gfp::boxb(RNAe) strain is shown (∗p < 0.05, ∗∗p < 0.01 by the Student’s t test).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4
A Model to Summarize How CSR-1 Tethering Can License and Activate Gene Expression
(A) The gfp::boxb transcript possesses pie-1 regulatory elements that can recruit endogenous CSR-1 via 22G-RNAs and allow for the weak licensing of gfp.
(B) When exposed to RNAe, expression of the gfp::boxb transcript is halted by the piRNA pathway. Endogenous CSR-1 in not sufficient to protect the gfp::boxb transcript from the piRNA pathway silencing. (Green small RNAs target gfp, and red small RNAs target box b.)
(C) The tethering of CSR-1::λN to gfp::boxb enables a more stable, or fully licensed, expression state and renders gfp::boxb impervious to silencing.
(D) Over several generations, gfp small RNAs loaded into CSR-1 complexes are capable of acting in trans to license expression of cdk-1::gfp. Tethering CSR-1::λN to gfp::boxb may improve gfp small RNA production and/or loading into CSR-1 complexes, making the activity of the CSR-1 pathway more robust and capable of overcoming silencing by the piRNA pathway.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions