(A) Diakinetic oocyte chromosomes in wild type and drh-3 or ego-1 RNAi-depleted animals. Six discrete DAPI figures are observed in wild type, while greater than six figures are present in mutant oocytes.
(B) Incidence of males in wild type (N2) and 3× Flag csr-1 rescue.
(C) Viable progeny per brood in wild type (N2), 3× Flag csr-1 rescue, and csr-1(tm892).
(D) DAPI-stained wild type (N2) and RNAi-depleted embryos undergoing the first mitotic division. Anaphase bridging is evident (white arrowhead). An aberrant piece of DNA is visible in ego-1 (yellow arrowhead).
(E) Fluorescence in situ hybridization with probes for chromosome V 5S rDNA in wild type and csr-1 RNAi-depleted embryos (DNA, blue; FISH signal, green). Left panels in each set show FISH signal alone. White dotted lines indicate embryo (large oval) and nuclei (circles). Yellow dotted lines indicate polar bodies. Images are projections of Z-stacks through the entire embryo after deconvolution.

(A) Single confocal sections showing kinetochore organization in the first cell division of wild type (N2) and csr-1 RNAi-depleted embryos (HCP-3, red; tubulin, green; DNA, blue).
(B) HCP-3/inner kinetochore disorganization frequency in wild type (N2), vs. ego-1 and csr-1 RNAi-depleted embryos (example metaphase images, HCP-3, red; DNA, green).
(C) BUB-1/outer kinetochore disorganization frequency in wild type (N2), vs. ego-1 and csr-1 RNAi-depleted embryos (example metaphase images, BUB-1, red; DNA, green).
(D) KLE-2/condensin disorganization frequencty in wild type (N2), vs. ego-1 and csr-1 RNAi-depleted embryos (example metaphase images, KLE-2, red; DNA, green).

(A) Western blots of developmentally staged protein lysates (left) or various germline mutant lysates (right) probed for EGO-1, DRH-3, EKL-1, CSR-1 (multiple isoforms), and tubulin (as a loading control). L1, L2, L3 and L4 are larval stages; YA, Young Adults; GA, Gravid Adults; Embryos, mixed stage embryos. GA 25°C, Gravid Adults grown at 25°C; fem-1(hc17), no sperm at 25°C; fog-2(q71) enriched to 95% males by filtration (20°C); and glp-4(bn2), no germline at 25°C.
(B) Wild type perinuclear germline localization of DRH-3, CSR-1, and EGO-1 (left, yellow) (DNA, center, blue).
(C) DRH-3 (left, green) colocalizes with the P Granule component, PGL-1 (center, red; DNA, blue).
(D) DRH-3 and CSR-1 (left, yellow) remain localized to P Granules in the embryonic P cell lineage (dashed circles; DNA, center, blue).
(E) Single confocal sections of PGL-1 (red) in wild type and csr-1(tm892) mutant germlines through the germline surface and core. P Granules become detached from the nuclear periphery in csr-1(tm892) (DNA, green; distal is to the left).

(A) Single confocal sections of CSR-1 (left, red) in wild type oocytes. CSR-1 is enriched on diakinetic chromosomes as oocytes mature (yellow arrowhead), and remains in some P Granules (blue arrow) (DNA, center, green; distal is to the left).
(B) to (E) Single confocal sections of CSR-1 (B), DRH-3 (C), EGO-1 (D), EKL-1 (E) (red) in wild type embryo prophase/prometaphase (tubulin, green; DNA, blue).
(F) to (I) Single confocal sections of CSR-1 (F), DRH-3 (G), EGO-1 (H), EKL-1 (I) (red) in wild type embryo metaphase (tubulin, green; DNA, blue).
(J) to (K) Single confocal sections of EKL-1 in wild type embryo early (J) and late (K) anaphase (tubulin, green; DNA, blue).

(A) Line plot comparing the relative proportions of small RNA classes between wild type (N2) Input (left) and CSR-1 IP (right) samples. (AS=antisense, S=sense)
(B) Box and whisker plot of the relative proportion of small RNA reads for each locus targeted within each small RNA class, in the CSR-1 IP relative to Input. Loci with values closer to 1 indicate enrichment of small RNA reads in the IP, a value of 0.5 indicates equal proportions of reads in the IP and input, and values closer to 0 indicate loci depleted of small RNA reads in the IP. Boxes contain 50% of siRNA loci (between the 25^th and 75^th percentile), with the line inside each box representing the median value. Lines extending to the right of the box represent the most enriched value, and lines extending to the left of the box represent the most depleted value in the IP. X axis is relative proportion of reads (measured as IP value divided by Input plus IP values for any given locus). Dotted lines indicate the values corresponding to two-fold enrichment (a value of 0.66) or depletion (a value of 0.33). Calculations were made with small RNA cutoffs as described in Supplemental Experimental Procedures.
(C) Venn diagram depicting the proportion of loci that possess a two-fold or greater depletion of 22G-RNAs in the glp-4(bn2) mutant that are also enriched two-fold or more in the CSR-1 IP. Only loci present in both datasets with 25 reads per million or more are represented.
(D) Box and whisker plot of the relative proportion of small RNA reads for each locus in the csr-1(tm892) and ego-1(om97) relative to a congenic wild type strain (DA1316). Protein coding genes (red) and repeat elements (blue) are represented. drh-3 and ekl-1 small RNA analyses are described in (Gu et al., cosubmitted).

(A) Box and whisker plot of mRNA expression from microarray experiments in wild type vs. csr-1(tm892) mutants. The analysis was done for all genes measured by the array (left), and the subset of only CSR-1 22G-RNA target genes (right).
(B) Western blot analysis of wild type and csr-1(tm892) protein lysates, probed for CSR-1 22G-RNA target proteins. EKL-1 is a loading control.
(C) ChIP/Quantitative real-time PCR analysis of CSR-1 enrichment at CSR-1 22G-RNA or WAGO-1 22G-RNA target loci. Fold enrichment is calculated relative to the Y47H10A.3 locus, which, like clp-3, Y47H10A.4, and M01G12.9, is not targeted by small RNAs. Data from a single, representative set of experiments is presented, error bars are standard deviation. (IP with CSR-1, blue; IP with beads only/no antibody, red)
(D) Density of CSR-1 22G-RNA target genes on each chromosome. Each bar represents the numbers of genes in a 100 kb bin. (Watson strand, blue; Crick strand, red). Chromosome number is as indicated. Scale bar represents one gene.

(A) 22G-RNA synthesis: In the germline, DRH-3, EGO-1 and CSR-1 localize to perinuclear P Granules, where DRH-3 and EGO-1 initiate the synthesis of 22G-RNAs from transcripts that are important for germline development and early embryogenesis. These 22G-RNAs are loaded onto CSR-1 and can guide the complex to its targets.
(B) Initial targeting of genomic loci: In oocytes, CSR-1 22G-RNA complexes move into the nucleus where they target nascent transcripts, possibly by cleaving them. Chromatin modifying factors may associate with CSR-1 complexes to promote local modification of histones at and near CSR-1 target loci, establishing pericentromeric chromatin domains (green nucleosomes). A complex containing EGO-1, DRH-3, and possibly EKL-1 is proposed to amplify the signal in a positive feedback loop, by generating more 22G-RNAs in the nucleus with the CSR-1 22G-RNA-targeted nascent transcripts as the template.
(C) Establishment and maintenance of chromatin domains: The CSR-1 22G-RNA dependent chromatin domains containing modified histones (green nucleosomes) may promote the proper binding and organization of other components such as condensins and cohesins in embryo mitotic divisions. Furthermore, these chromatin domains could both help to recruit and restrict the incorporation of the centromeric Histone H3 variant, HPC-3/CENP-A (red nucleosomes) in chromatin domains adjacent to those targeted by CSR-1 22G-RNA complexes. Regions of the chromatin loop out and self-associate, permitting the assembly of a proper planar, rigid kinetochore on the poleward faces of condensed chromosomes. As cell divisions continue, chromatin domains could be maintained epigenetically, possibly even by EKL-1, thus becoming less reliant on CSR-1 22G-RNA activity throughout development.