Supplemental Material for Donertas & Sienski Et Al

Supplemental Material for Donertas & Sienski et al.

Supplemental material contains

-Supplementary Tables for used reagents

-Supplementary Figure Legends

-Supplementary Figures S1-S8

Supplementary Table1:Used fly stocks

tj-GAL4 (DGRC # 104055);
tj-Gal4; gypsy-lacZ (Olivieri et al, 2010)
MTD-GAL4 (Ni et al., 2011);
armi RNAi (VDRC # 16205);
mael RNAi (VDRC # 18198 (GD));;
CG3893 RNAi (VDRC # 40480 (GD))
P{GSV6}GS12962 (Kyoto #204406)
UAS-Dcr-2; NGT; nosGAL4, Burdock-lacZ/Tm3,Ser
UAS-Dcr-2; NGT; nosGAL4, HeT-A-lacZ/Tm3,Ser
Short hairpin RNA (shRNA) lines were cloned into the Valium-20 or the Valium-22 vector (Ni et al, 2011) modified with a white selection marker and integrated into the attp2 landing site (Markstein et al., 2008). The hairpin sequence for CG3893 was CTAGCAGTAGCACAGGAGGCCATACTCAATAGTTATATTCAAGCATATTGAGTATGGCCTCCTGTGCTgcg
y,w,hsFlp122;;act<CD2>GAL4, UAS-GFP (Olivieri et al, 2010)
w[1118]; P{ry[+t7.2]=PZ}piwi[1]/CyO (Bloomington #43637)
FLAG-eGFP-CG3893 was cloned by inserting an N-terminal FLAG-eGFP cassette via bacterial recombineering into a genomic rescue construct (chr3L:18891513-18898519)
which was integrated into the attP40 or attP2 landing sites. eGFP-Piwi was cloned by inserting an N-terminal eGFP cassette via bacterial recombineering into a genomic rescue construct which was integrated into the attP2 landing site (Sienski et al, 2012).
Piwi[1] mutant germline clones were generated by w[1118]; FRT40A,piwi[1]/CyO and hsFlp; FRT40A, arm_lacZ/CyO.
GFP-N-Piwi;piwi[1]/piwi[2] (Sienski et al, 2012)
All flies were aged 5 days at 25°C before analysis.

Supplementary Table 2: Primers for QPCR analysis

GENE / Primer / Sequence
rp49 / rp49_fwd / CCGCTTCAAGGGACAGTATCTG
rp49_rev / ATCTCGCCGCAGTAAACGC
ZAM / ZAM_fwd / ACTTGACCTGGATACACTCACAAC
ZAM_rev / GAGTATTACGGCGACTAGGGATAC
HeT-A / HeT-A_fwd / CGCGCGGAACCCATCTTCAGA
HeT-A_rev / CGCCGCAGTCGTTTGGTGAGT
actin5C / act5C_fwd / AAGTTGCTGCTCTGGTTGTCG
act5C_rev / GCCACACGCAGCTCATTGTAG
mdg1 / mdg1_fwd / AAACTCCAACTCCCAATCCG
mdg1_rev / AGTGGTCCCTCGCAGTCGTT
gypsy-spliced / gypsy_fwd / CAACAATCTGAACCCACCAATCT
gypsy_rev / TATGAACATCATGAGGGTGAACG
Blood / blood_fwd / AACAATAGAAAGAAGCCACCGAAC
blood_rev / AGTCATGGACTATTGAGGGTGTTG
CG3893 locus / CG3893_fwd / AAGAAGCTCCAGCAGCACAT
CG3893_rev / GTGTCTGGTGTCTTCGCTGA
piwi locus / piwi_fwd / TTACCCGTACTTCGTCCTGATG
piwi_rev / TTGGGCACCGAAATAACTCA
TSS blood / TSS_blood_fwd / cgcagtcgcagtatttaggtaag
TSS_blood_rev / gtaataaaccaatatatgccaccaattg
TSS gypsy / TSS_gypsy_fwd / attgccgttaaacatcattgt
TSS_ gypsy_rev / gctgaggttcgtcttagacac
TSS mdg1 / TSS_mdg1_fwd / GAATGTAGGCTCGCTTAATAAAAACC
TSS_mdg1_rev / ACACTCTACTCACTCAGATCGC
TSS ZAM / TSS_ZAM_fwd / gacgaagaataaagacgacca
TSS_ZAM_rev / gcggaggtattatcactcaga
light / Lt_fwd / atgcggcggtataactgaac
Lt_rev / tggagatccgaaagtcaagc
TSS actin5c / TSS_act5c_f / CACTTTCAGTCGGTTTATTCCAG
TSS_act5c_r / TTAAGAGCCACGAAACTTTTCAA

Supplementary Table 3: Probes used for Northern Blots

Probe / Sequence
mir-310 / AAAGGCCGGGAAGTGTGCAATA
tj / GGTAATGGGAATGCACTTCTCTTGAA
idefix / AAACTACTGGCAATCGTTTGGGAA

Supplementary Table S4: Used siRNAs for RNAi in OSCs

GENE / siRNA / Sequence
eGFP / eGFP_guide / ACUUCAGGGUCAGCUUGCCTT
eGFP_passenger / GGCAAGCUGACCCUGAAGUTT
armi / armi_guide / UAAACUUAGCUUGACAGCGTT
armi_passenger / CGCUGUCAAGCUAAGUUUATT
mael / mael1_guide / UUCUUCGAACAAGCGAAAGUU
mael1_passenger / GAAGCUUGUUCGCUUUCUAUU
mael2_guide / UUCCAAAUACUACAAGCAUU
mael2_passenger / GGUUUAUGAUGUUCGUAAAUU
piwi / piwi_passenger / CACCUUCACGCCUGGGAGCTT
piwi_guide / GCUCCCAGGCGUGAAGGUGTT
CG3893 / CG3893_1_passenger / GAAACAUUAUAGUACACUAUU
CG3893_1_guide / UAGUGUACUAUAAUGUUUCUU
CG3893_2_passenger / GCUCCAGCAGCACAUCUUAUU
CG3893_2_guide / UAAGAUGUGCUGCUGGAGCUU

Supplementary Figure Legends

Figure S1 (relates to main Figure 1)

(A)Cartoon depicting the CG3893 locus; also shown is the expression level of annotated genes according to RNA-Seq, the position of the GS12962 P-element insertion and sequences that are targeted by VDRC line GD40480 and the cloned shRNA.

(B)Morphology of representative ovaries from CG3893[GS12962] heterozygous and homozygous flies.

Figure S2 (relates to main Figure 2)

(A) Cartoon depicting the setup of the epistasis experiment.

(B) The bar diagram shows fold changes in steady state RNA levels of indicated TEs and genes in piwi-KD, CG3893-KD and double piwi-KD+CG3893-KD OSCsin log10 scale. Values are averages of 3 biological replicates (error bars: StDev) and normalized to control knockdowns.

Figure S3 (relates to main Figure 3)

(A)Confocal images of egg chambers stained for Piwi (magenta) and DNA (DAPI; blue) from CG3893[GS12962]heterozygous (left) or homozygous (right) flies.

(B)Western blots showing protein levels of Piwi orCG3893 in OSC lysates after48h of GFP or CG3893 RNAi.

(C)Northern blots showing levels of indicated piRNAs in total RNA from OSCs transfected with siRNAs (duration given below) against indicated genes. To the left, an RNA size marker is shown. The miR-310 blot serves as loading control.

(D) Normalized piRNA profiles (sense up; antisense down; 200nt windows) from ovarian RNA of CG3893[GS12962]heterozygous (black) or homozygous (red) flies mapping uniquely to the flamenco (left)or the 42AB (right)piRNA clusters.

(E) Scatter plot (log2 scale) showing levels of sense (left) or antisense (right) piRNAs mapping to soma dominant (green), intermediate (yellow) or germline dominant (black) TEs in CG3893[GS12962]heterozygous versus homozygous small RNA libraries.

Figure S4 (relates to main Figure 3)

Normalized piRNA profiles (sense up; antisense down) and the respective small RNA length profiles (siRNAs and piRNAs; red antisense; blue sense) of CG3893[GS12962]heterozygous (black) or homozygous (red) small RNA librariesmapping to gypsy5 (A),HeT-A (B) or the R1 element (C). Mappings up to 3 mismatches were considered and all graphs are normalized to 1 million sequenced miRNAs.

Figure S5 (relates to main Figure 4)

(A) The bar diagrams show quantifications of the average peak area for the three most abundant peptides in control IP and CG3893 IP (two independent mass spec. runs; upper diagram: Piwi-band; lower diagram: CG3893 band). Number of unique peptides and protein coverage in IP and control are indicated.

(B)RPKM values obtained from OSC RNA-seq libraries showing expression levels of indicated piRNA pathway members involved in piRNA biogenesis (grey) or silencing (red) in comparison to Piwi (black).

(C)Confocal image of ovarioles expressing either GFP-Piwi (right ovariole) or GFP-CG3893 (left ovariole). Both transgenes contain the endogenous regulatory regions and were inserted in the identical genomic landing site.

(D) Confocal images of egg chambers stained for Piwi (red), CG3893 (green) and DNA (DAPI, blue) obtained from flies with germline specific knockdowns (GLKD) of Piwi. Several egg chambers with different degrees of CG3893 de-localization are shown to illustrate the variability of the phenotype.

(E) Confocal images of an ovariole where a germline clone for the piwi[1] allele has been induced stained for DNA (DAPI), Piwi (magenta) and CG3893 (green). The mutant egg chamber is marked by the absence of Piwi.

(F) Confocal images of the epithelium of an egg chamber where an RNAi clone for Piwi has been induced. The ovariole was stained for DNA (DAPI), the clonal marker (GFP, green), Piwi (magenta) and CG3893 (red). Clone border indicated by a yellow dashed line.

Figure S6 (relates to main Figure 5)

(A) CLUSTAL-W alignment of Gtsf1 from indicated Drosophila species. Indicated are the N-terminal zinc fingers, the C-terminal peptide 83-115 and the residues mutated for the analyses in Figure 5.

(B) Confocal images of OSCs transfected with the indicated myc-tagged Gtsf1 expression constructs (used in Figure 5) stained for DNA (DAPI) and the Myc-tag (green).

(C) Western blot showing the expression of the indicated Myc-tagged Gtsf1 expression constructs used for the rescue experiment in Figure 5. Piwi levels are shown as control.

Figure S7. Loss of CG3893/Gtsf1 results in increased transcription of transposable elements controlled by the piRNA pathway; relates to main Fig. 6

(A) Displayed are fold changes of RNA Polymerase II occupancy (Pol II ChIP-seq) on TEs in OSCs transfected with indicated siRNAs (normalized to control). Color code for TE groups as in Figure 2 and (Sienski et al. 2012).

(B-C) Density profiles of normalized reads from Pol II ChIP-seq (B) or H3K9me3 ChIP-seq (C) on mdg1, gypsy (group I elements) and Doc (group III element). Grey color indicates signal in control, green in piwi-KD and blue in CG3893/Gtsf1-KD.

(D)Shown is the Pol II ChIP-qPCR enrichment at control genes (light, actin5c; TSS: transcription start site) or TEs (blood, ZAM, HeT-A, gypsy) in ovaries with soma or germline specific CG3893/Gtsf1 knockdowns and the respective controls. Enrichments were calculated over a non-transcribed intergenic region (error bars represent StDev; n=3).

Figure S8 (relates to main Figure 6)

(A)Metaplots of H3K9me3 signals (individual plots for the five bins defined in Figure 6B) in the 50kb window centered on the euchromatic H3K9me3 peak summits in control KD (black), piwi-knockdown (green) or Gtsf1 knockdown OSCs (blue).

(B) Shown are scatter plots of OSC RNA-seq RPKM levels for the 6561 genes that show RPKM of at least 5 in any analyzed library.

(C)Transcriptional activity (Pol II ChIP-seq), H3K9me3 signal (H3K9me3 ChIP-seq) and ChIP input signal at the bancal locus are shown for OSCs with the indicated knockdowns. An OSC-specific gypsy insertion is indicated at the top; the TSS of the shortest bancal isoform is indicated with a dashed line.