Benjamin Czech, Jonathan B. Preall, Jon McGinn, and Gregory J. Hannon

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1 Molecular Cell, Volume 50 Supplemental Information A Transcriptome-wide RNAi Screen in the Drosophila Ovary Reveals Factors of the Germline pirna Pathway Benjamin Czech, Jonathan B. Preall, Jon McGinn, and Gregory J. Hannon 1

2 Figure S1. Enrichment Analysis of RNA-Seq from Ovaries and Other Tissues, Related to Figure 1 (A) Scatter plots comparing the relative expression levels of protein-coding genes in Drosophila ovaries to carcasses, embryos or OSS cells (log 10 FPKM). (B) VENN diagram showing genes with shared and specific expression. Only genes with FPKM > 1 in either tissue were included to the analysis. (C) Scatter plot displaying relative expression ratio of all genes in ovary versus carcass RNAseq data as a function absolute expression level in ovary (log 10 FPKM). Statistically significant (P < 0.01) or sub-significant (P > 0.01) levels of differential expression are denoted with black dots and grey dots, respectively. Genes with ovary FPKM > 1, which is the range screened in our study, are highlighted by yellow background. 2

3 Figure S2. Candidate Hit Calling, Related to Figure 2 Box plots summarizing z-scores of all screened genes, positive armi controls, and negative white controls, respectively. Data for each of the tested transposons (HeTA, TAHRE, blood, burdock) is shown separately. 3

4 Figure S3. Localization of GASZ and Del, Related to Figure 4 (A) Shown is the schematic domain structure of murine and Drosophila GASZ. The ankyrin repeat domain (green), sterile alpha motif (SAM) (red), and putative basic leucine zipper domain (yellow) are indicated. E-values represent homology with consensus sequence for the indicated domains. Domains were predicted using the NCBI conserved domain prediction algorithm ( Note that using our stringency (Evalue threshold = 0.1), we did not detect the basic leucine zipper motif in Drosophila (Dm) or mouse (Mm) GASZ. (B) Expression of a N-terminally tagged GFP-Del transgene from the ubiquitin promoter in the ovary. Shown are germarium and stage 2-6 egg chambers (top), and a stage 9 egg chamber (bottom). GFP-Del is evident in nuclear foci and restricted to the germ cell lineage despite being driven by a ubiquitous promoter. (C) N-terminally tagged UASp-GFP-GASZ transgene driven in the female germline by nos- GAL4. Shown are stage 4-6 egg chambers (top), germarium (gm) and stage 2 egg chambers (bottom). The GFP-GASZ transgene can be seen in diffuse perinuclear structures extending well into the syncytial cytoplasm. (D) OSS cells expressing N- terminal GFP-GASZ (green in merge) show extensive co-localization with Mitotracker CMXRos (red in merge), suggesting association with mitochondria. 4

5 Figure S4. Effect of gasz Knockdown on pirna Levels, Related to Figure 5 (A) Shown are scatter plots (log 10 scale) comparing the levels of pirnas mapping to germline dominant (orange), intermediate (grey), or soma dominant (green) transposons in the indicated germline-knockdown libraries. (B) GASZ depletion in somatic cells compromises pirna production. Size distribution of 18- to 29-nt small RNAs derived from each strand of the flam and 42AB clusters are shown as histogram (blue sense; red antisense). The fraction of mirnas (green) for the indicated libraries is highlighted in the cake diagrams. (C) Histograms show relative pirna levels for a series of soma- and germline-dominant clusters. Total reads were normalized across libraries to 42ABunique pirnas, which are unaffected by tj-gal4-driven knockdowns. Changes in pirna levels are shown with reference to white knockdowns and set to 100%. 5

6 Figure S5. Silencing Requirements Differ for Distinct Transposons, Related to Figure 6 (A) Scatter plot comparing transposon de-repression (as z-scores) for HeTA and burdock (top 500 candidates from the primary screen are shown in red, median 500 candidates are indicated in blue). Known pirna pathway components are highlighted in green. (B) Similar as in (A), but comparing blood to burdock. 6

7 Supplemental Experimental Procedures Fly Stocks and Husbandry All fly strains for the primary screen were purchased from the VDRC. RNAi lines for re-screening were obtained from the VDRC or TRiP (Harvard). Strains carrying the X-chromosome linked UAS-Dcr2 (P{UAS-Dcr-2.D}1) transgene and female germline driver nos-gal4 (P{GAL4-nos.NGT}40) (#25751), as well as the stock containing a hs-hid transgene on the Y-chromosome (P{w[+mC]=hs-hid}Y) (#8846) were obtained from Bloomington. For facilitated virgin collection, we generated a conditional virginator stock that contains the Dcr2 transgene and nos-gal4 driver, as well as the hs-hid transgene (males: Dcr2/hs-hid(Y); nos- GAL4 and females: Dcr2; nos-gal4). Mutant strains del 3 /CyO and del KG10262 /CyO were obtained from Bloomington (#4979 and #15249, respectively). Flies were kept on standard media at 25 C. Virgin females were obtained by two consecutive 2 hr heat shocks at 37 C (spaced by 24 hrs) of bottles containing 0-24 hr old embryos. For germline-knockdown experiments, five males expressing RNAi constructs were crossed to five virgin females (3-5 day old) expressing nos-gal4 and UAS-Dcr2. After 7 days of mating, parental flies were discarded. F1 offspring flies were transferred to fresh food media for 2.5 days before collection of six females into 96-well collection tubes (seeded with one 5 mm stainless steel bead per well, Qiagen) and stored at -80 C until further processing. For immunofluorescence experiments and small RNA libraries, F1 offspring flies were additionally supplemented with yeast paste and ovaries were dissected in cold 1x PBS. RNA Isolation, Reverse Transcription, and qpcr For screening experiments, total RNAs were isolated using the RNeasy 96 Universal Tissue kit (Qiagen) according to the manufacturer s instructions. In brief, 750 l QIAzol Lysis Reagent were added to each collection tube and tissue was ground using a Mixer Mill MM400 (Retsch) twice for 5 min at 25 Hz. 150 l chloroform were added to each sample, mixed and centrifuged. The aqueous phase containing the RNA was transferred to square blocks and mixed with 400 l of 70% EtOH. The mixture was transferred to RNeasy 96-well plates placed on a vacuum manifold (Qiagen). DNase treatment was performed on column following the manufacturer s instructions. Following DNase digest, RNAs were washed once with 800 l Buffer RW1, and twice with 800 l Buffer RPE. RNAs were eluted in 100 l RNase-free water by centrifugation. Complementary DNA was obtained by reverse transcription using 2.5 l RNA as input, oligo(dt) 20 primers, and the SuperScript III Reverse Transcriptase system (Invitrogen). Multiplexed qpcrs were carried out using TaqMan Universal Master Mix II, no UNG (Applied Biosystems) and primers listed in Table S2. Experiments were performed on a Chromo4 Real-Time PCR Detector (BioRad), and data was quantified using the Opticon Monitor software. Z-scores 7

8 [transposon] [rp49 for transposon expression were calculated on C T values (C T C T control] ). For dissected ovaries, TRIzol reagent (Invitrogen) was used to extract total RNAs. 1 g RNA was treated with DNase I Amplification Grade (Invitrogen) according to the manufacturer s instructions. Reverse transcription was carried out with oligo(dt)20 primers using the SuperScript III Reverse Transcriptase system (Invitrogen). QPCRs were either carried out using SYBR Green PCR Master Mix or TaqMan Universal Master Mix II, no UNG (Applied Biosystems) with primers listed in Table S2. Transposon levels were quantified using the C T method (Livak and Schmittgen, 2001), normalized to rp49 and fold changes were calculated relative to knockdown of white. All experiments were carried out in triplicates, with the average and standard deviation shown. Immunofluorescence The immunofluorescence experiments were carried out as described (Preall et al., 2012). In brief, ovaries were fixed in freshly prepared 4% paraformaldehyde for 20 min at room temperature. Blocking and permeabilization were carried out simultaneously in wash buffer (50 mm Tris ph6.8, 150 mm NaCl, 0.5% NP-40) supplemented with bovine serum albumin (BSA) (5 mg/ml). Primary antibodies were diluted 1:1000 and incubated overnight at 4 C in wash buffer supplemented with 1 mg/ml BSA. Anti-Piwi and anti-ago3 were generated in the Hannon laboratory (Brennecke et al., 2007). Anti-Aub and anti- Armi were gifts from Mikiko Siomi (Nishida et al., 2007; Saito et al., 2010). Secondary antibodies (AlexaFluor-488 and -568) were purchased from Invitrogen and used at 1:1000 dilutions. Images were acquired on a Perkin Elmer UltraVIEW spinning disk confocal microscope. Small RNA Libraries and Bioinformatic Analysis Small RNA libraries were constructed similar as described (Brennecke et al., 2007), with slightly modified adapters that enabled multiplexed sequencing. The below small RNA libraries were prepared for this study: 19- to 28-nt from nos-gal4-expressed dsrna-white 19- to 28-nt from nos-gal4-expressed dsrna-yb 19- to 28-nt from nos-gal4-expressed dsrna-armi 19- to 28-nt from nos-gal4-expressed dsrna-gasz 19- to 28-nt from nos-gal4-expressed dsrna-spn-e 19- to 28-nt from nos-gal4-expressed dsrna-aub 19- to 28-nt from nos-gal4-expressed dsrna-del 19- to 28-nt from tj-gal4-expressed dsrna-white 19- to 28-nt from tj-gal4-expressed dsrna-yb 19- to 28-nt from tj-gal4-expressed dsrna-armi 19- to 28-nt from tj-gal4-expressed dsrna-gasz 8

9 19- to 28-nt from tj-gal4-expressed dsrna-spn-e 19- to 28-nt from tj-gal4-expressed dsrna-zuc Libraries were sequenced in-house using the Illumina HiSeq platform. The analysis of small RNA libraries was performed similar as described (Czech et al., 2008; Preall et al., 2012). Illumina reads were stripped of the 3 linker and collapsed. The resulting small RNA sequences were matched to release 5 of the Drosophila genome (version dm3). Only reads that met these conditions were used for further analyses. Sequences were annotated using a combination of mirbase (micrornas), Flybase (protein coding genes; noncoding RNAs), and UCSC (transposons; non-coding RNAs) tracks, as well as custom annotations for synthetic cloning markers, endo-sirnas from structured loci and individual mir and mir* strands. Following removal of reads corresponding to structural RNAs and synthetic markers, total small RNA counts (18- to 29-nt) were set to one million reads in the dsrna-white library. For comparison between samples, all libraries were normalized based on unique pirna-sized (23- to 29-nt) mappers to the flam (nos-gal4 knockdowns) or 42AB (tj-gal4 knockdowns) clusters. Size profiles for flam and 42AB pirna clusters were obtained by extracting the abundance and read length of sequences uniquely matching to these loci. For ping-pong analysis of 42AB-derived pirnas, we recorded the abundance of all neighboring pirnas on the opposite genomic strand within a certain window (20-nt upstream and 10-nt downstream) as well as their relative 5 end distance, with each sequence only counted once per offset even if multiple ping-pong pairs were detected. We calculated z-scores to display ping-pong signatures within the probed window. A peak at position 9 is indicative of more pirna partners at the opposite strand that overlap by precisely 10-nt and serves as signature for ping-pong amplified pirnas. To compare pirna levels matching to individual transposons across libraries, we extracted all mapping reads based on annotations and calculated the ratio relative to white control knockdowns (log 2 transformation was used for improved display). Density plots were generated by matching all pirna reads against the batumi consensus sequence allowing zero mismatches. RNA-Seq Experiments and Transcriptome Analysis For transcriptome libraries, 6 g of total RNA were extracted from ovaries of 2-3 day old females, carcasses of the same ovariectomized females, 0-1 hr dechoronized embryos, or OSS cells using TRIzol (Invitrogen). All flies were of the genotype (UAS-Dcr2; nos-gal4) used as the driver line for our screen. Two independent biological replicates were analyzed for each tissue type. The samples were depleted of rrna using Ribo-Zero Gold (Epicentre) and used as input for the ScriptSeq v2 kit (Epicentre). Libraries were multiplexed using TruSeq barcoding PCR primers and sequenced in-house using the Illumina HiSeq platform. Raw sequencing reads were mapped iteratively to release 5 of the Drosophila genome (total of 13,795 unique protein-coding genes; version dm3, 9

10 refseq release 4.48 from 9/13/2011) using Bowtie (Langmead et al., 2009) tolerating up to two mismatches. The remaining reads were mapped to the RefGene-annotated exon junctions using TopHat (Trapnell et al., 2009). Transcripts were quantified using Cufflinks (Trapnell et al., 2010) and converted in fragments per kilobase of exon per million reads (FPKM), which was used to select candidates for initial screening. For differential expression analysis, Bowtie-mapped reads aligning to Drosophila RefSeq genes were counted using HTSeq and processed using the DESeq R package (Anders and Huber, 2010). For the estimation of ovary-expressed genes, we only considered protein-coding mrnas, and removed all non-coding transcripts (i.e., snornas, snrnas, trnas). For analysis of transposon expression in knockdown ovaries, RNAseq libraries were prepared from dissected ovary RNA and mapped as described above to obtain the total number of genomic mappers as a normalization factor. All reads were then mapped using Bowtie to the canonical Drosophila transposon sequence set from FlyBase (version 9.4.2). Reads per million were calculated for each transposon, and baseline expression was established as the average of five negative controls: two independent biological replicates each of the UAS-Dcr2; nos-gal4 driver strain, two of nos-gal4 driving dsrna-white, and one of nos- GAL4 driving dsrna-yb. Log-transformed fold-changes of each knockdown were then calculated relative to this baseline. VDRC strain identifiers used in this experiment are available upon request. 10

11 Supplemental References Anders, S., and Huber, W. (2010). Differential expression analysis for sequence count data. Genome Biol 11, R106. Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R., and Hannon, G.J. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, Czech, B., Malone, C.D., Zhou, R., Stark, A., Schlingeheyde, C., Dus, M., Perrimon, N., Kellis, M., Wohlschlegel, J.A., Sachidanandam, R., et al. (2008). An endogenous small interfering RNA pathway in Drosophila. Nature 453, Langmead, B., Trapnell, C., Pop, M., and Salzberg, S.L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, Nishida, K.M., Saito, K., Mori, T., Kawamura, Y., Nagami-Okada, T., Inagaki, S., Siomi, H., and Siomi, M.C. (2007). Gene silencing mechanisms mediated by Aubergine pirna complexes in Drosophila male gonad. Rna 13, Preall, J.B., Czech, B., Guzzardo, P.M., Muerdter, F., and Hannon, G.J. (2012). shutdown is a component of the Drosophila pirna biogenesis machinery. Rna 18, Saito, K., Ishizu, H., Komai, M., Kotani, H., Kawamura, Y., Nishida, K.M., Siomi, H., and Siomi, M.C. (2010). Roles for the Yb body components Armitage and Yb in primary pirna biogenesis in Drosophila. Genes Dev 24, Trapnell, C., Pachter, L., and Salzberg, S.L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, Trapnell, C., Williams, B.A., Pertea, G., Mortazavi, A., Kwan, G., van Baren, M.J., Salzberg, S.L., Wold, B.J., and Pachter, L. (2010). Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28,