Germline expression of Bonus is required for oogenesis.

A) Bon is expressed throughout oogenesis. Stacked confocal image of wild-type Oregon-R flies stained for Bon. B) Bar graph shows the relative expression of Bon (normalized to rp49 level) in control and Bon-depleted ovaries (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.). C) Confocal images of egg chambers from wild-type Oregon-R flies (control) and flies expressing MT-Gal4-driven shRNA against Bon stained for Bon (scale bar: 20 μm). D) Bon depletion leads to rudimentary ovaries. Phase contrast images of dissected ovaries from flies of indicated genotypes. Wild-type Oregon-R flies were used as control. E) Top: Phase contrast image of dissected ovaries with different phenotypes from flies with Bon GLKD driven by nos-Gal4. Bottom: graph showing the percentage of normal, hypomorphic and rudimentary ovary phenotypes of indicated genotypes (n= 85, 175, 52, 71, 64, 58, respectively). F) Confocal images of whole ovaries from wild-type Oregon-R flies (control) and flies with Bon GLKD driven by bam+nos double driver stained for Vasa (red) and DAPI (blue) (scale bar: 30 μm).

Bonus functions as a repressor of tissue-specific genes in ovary.

A) Bon GLKD leads to misexpression of tissue-specific genes in the ovary. Volcano plot shows fold changes in genes expression upon Bon GLKD driven by nos-Gal4 in the ovary as determined by RNA-seq (n =3). Siblings that lack shRNA against Bon produced in the same cross were used as a control. Genes that change significantly (log2FC>1, qval< 0.05, LRT test, sleuth (Pimentel et al., 2017)) are highlighted. Genes bon, pst, Rbp6, ple are labeled. Genes with infinite fold change values (zero counts in control ovaries) are not shown. B) Bon represses genes with diverse functions. Bubble plot shows the analysis of GO enrichment at the level of biological processes (BP) for genes that are de-repressed upon Bon GLKD driven by nos-Gal4 (log2FC>1, qval< 0.05, LRT test, sleuth (Pimentel et al., 2017)). Only GO terms above the established cut-off criteria (p-value<0.01 and >3 genes per group) are shown. BP are ranked by fold enrichment values. The most significant processes are highlighted in purple, and the less significant in yellow according to log10(FDR) values. The bubbles size reflects the number of genes, assigned to the GO BP terms. C) Normal expression level of de-regulated genes upon Bon GLKD in the tissues where they are normally expressed indicates Bon-mediated silencing of genes normally expressed in the head and digestive system. The graph shows the percentage of de-repressed genes upon Bon GLKD driven by nos-Gal4 (log2FC>1, qval< 0.05, LRT test, sleuth (Pimentel et al., 2017)) with given expression level in the indicated enriched tissues. Expression levels according RPKM values from modENCODE anatomy RNA-seq dataset are no expression (0-0), very low (1-3), low (4-10), moderate (11-25), moderate high (26-50), high (51-100), very high (101-1000), extremely high (>1000). D) GLKD of Bon leads to ple expression in follicular cells. Confocal images of egg chambers show RNA in situ HCR detecting ple and bonus mRNAs in flies with MT-Gal4>Bon GLKD and control siblings from the same cross that lack Bon shRNA (scale bar: 20 μm). E) Bon represses rbp6 in the germline. Confocal images of egg chambers show RNA in situ HCR detecting rbp6 and bonus mRNAs in flies with MT-Gal4>Bon GLKD and control siblings from the same cross that lack Bon shRNA (scale bar: 20 μm). F) Bar graph shows the relative expression of ple and rbp6 (normalized to rp49 level) in control and Bon-depleted ovaries (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.).

Bonus induces transcriptional silencing.

A) Schematics of the reporter construct in flies that allows Bon recruitment to nascent reporter transcript in flies. λN-GFP-Bonus and the mKate reporter encoding 4BoxB hairpins are co-expressed in germline cells of the ovary (driven by MT-Gal4). B) Bon tethering leads to transcriptional silencing of the reporter. Bar plot shows reporter expression (normalized to rp49 level) upon tethering of λN-GFP-Bonus or λN-GFP control ovaries (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.). C) Bon tethering leads to H3K9me3 accumulation. Bar plot shows H3K9me3 enrichment upon tethering of λN-GFP-Bonus or λN-GFP control ovaries (ChIP-qPCR, dots correspond to 2 independent biological replicates; error bars indicate st. dev.). D) Heatmap shows H3K9me3 distribution across Bon targets in control and nos-Gal4>Bon GLKD ovaries (input-normalized log2 values). E) RNA-seq and ChIP-seq tracks show counts per million (CPM)-normalized coverage for CG1572 in control and nos-Gal4>Bon GLKD ovaries. The gene structure is depicted at the top; arrow indicates the direction of transcription. The ChIP (blue) and input (gray) signals are overlaid. Numbers show the CPM values of the exonic regions (RNA-seq) or the normalized ChIP/input signal (ChIP-seq) in a manually selected genomic location. F) Bon depletion results in a slight decrease in H3K9me3 over some Bon target genes. Bar graph shows H3K9me3 levels at the genes CG3191 and Spn88Eb in control and Bon-depleted ovaries (ChIP-qPCR, dots correspond to 2 independent biological replicates; error bars indicate st. dev.).

Bonus interacts with Mi-2, Rpd3 and SetDB1.

A) Reporter silencing by Bon depends on Mi-2 and SetDB1. Bar plot showing the reporter expression (normalized to rp49 level) upon tethering of control λN-GFP or λN-GFP-Bonus in ovaries with Rpd3, Mi-2, SetDB1 GLKD, and control white GLKD (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.). B-C) Bon interacts with SetDB1 and Rpd3. Western blot analysis of immunoprecipitation experiment using GFP nanotrap beads from S2 cells co-expressing GFP-Bonus and Flag-tagged SetDB1 (B) and Flag-tagged Rpd3 (C). Lysates not expressing GFP-Bonus were used as negative control. D) Bon interacts with the C-terminus of Mi-2. Top: schematic illustration of full-length Drosophila Mi-2 and its truncated versions as defined by the amino acids: C-terminal truncated Mi-2 (1-1680) and N-terminal truncated Mi-2 (1681-1982). Bottom: Western blot analysis of immunoprecipitation experiment using GFP nanotrap beads from S2 cells co-expressing GFP-Bonus and Flag-tagged Mi2 fragments. Lysate not expressing GFP-Bonus was used as negative control.

Bonus is SUMOylated.

A) Schematic representation of putative SUMOylation sites within Bon. SUMOylation consensus sites are shown and boxed. Canonical consensus sites are in bold. Putative SUMOylated lysines were mutated to arginines individually (K9R, K20R, K763R) or in combination (3KR). B) Bon is SUMOylated at specific residues. Western blot analysis shows the SUMOylation levels of GFP-tagged Bon and SUMO-deficient triple mutant 3KR expressed in fly ovaries. SUMOylated form of Bon was detected only in wild-type GFP-Bonus (WT). Total protein lysates from flies co-expressing Flag-SUMO and λN-GFP-Bonus or λN-GFP-Bonus[3KR] were immunopurified using anti-GFP nanotrap beads. Flies not expressing λN-GFP-tagged protein were used as a negative control. C) Bon is predominantly SUMOylated at K20. Western blot analysis shows the associated SUMOylation levels of GFP-tagged Bon and SUMO-deficient triple mutant 3KR and single mutated K9R, K20R expressed in S2 cells. Single mutation K9R reduced, while the K20R mutation and triple 3KR mutation completely abolished Bon SUMOylation. Total protein lysates from S2 cells co-expressing HA-SUMO and GFP-Bonus or GFP-Bonus[3KR], GFP-Bonus[K9R], GFP-Bonus[K20R] were immunopurified using anti-GFP nanotrap beads. Lysate not expressing GFP-tagged protein was used as a negative control. D) SUMOylation site of Bon is conserved in insects. Sequence alignment of the Bon protein sequence from 12 Drosophila species and other insects shows conserve action of canonical SUMOylation consensus at K20 (boxed and indicated by the arrowhead). E) SUMO-deficient Bon mislocalizes into nuclear foci. Confocal images of egg chambers show the localization of MT-Gal4-driven λN-GFP-tagged Bonus and SUMO-deficient triple mutant λN-GFP-Bonus[3KR] flies. Images on the right panel show isolated nurse cell nuclei (scale bar: 20μm). F) Chromatin association of Bon depends on its SUMOylation. Western blot analysis shows the fractionation of cytoplasmic (cyto), nuclear (nuclei) and chromatin compartments of MT-Gal4-driven λN-GFP-tagged Bonus (WT) and SUMO-deficient triple mutant λN-GFP-Bonus (3KR) fly ovaries. Lamin and Histone H3 were used as markers for nuclear and chromatin fractions. G) Bon-mediated reporter repression depends on Bon SUMOylation. Bar plot shows the reporter expression (normalized to rp49 level) upon tethering of λN-GFP-Bonus, SUMO-deficient triple mutant λN-GFP-Bonus[3KR] or λN-GFP control ovaries (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.). H) Bar plot shows the reporter expression (normalized to rp49 level) upon tethering of control λN-GFP or λN-GFP-Bonus in ovaries with SUMO GLKD, and control white GLKD (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.). I) Western blot analysis shows the SUMO-dependent interaction between Bon and SetDB1. Total protein lysates from S2 cells co-expressing Flag-SetDB1 and GFP-Bonus (WT) or triple mutant GFP-Bonus[3KR] (3KR) were immunopurified using anti-GFP nanotrap beads. Lysate from cells not expressing GFP-tagged protein was used as a negative control.

SUMO E3-ligase Su(var)2-10 interacts with Bonus and regulates its SUMOylation.

A) Western blot analysis shows the interaction between Bon and SUMO E2-conjugating enzyme Ubc9. Total protein lysates from S2 cells co-expressing Flag-Ubc9 and GFP-Bonus (WT), SUMO-deficient triple mutant 3KR or single mutated K9R, K20R were immunopurified using anti-GFP nanotrap beads. Lysate from cells not expressing GFP-tagged proteins was used as a negative control. B) Bon interacts with Su(var)2-10. Western blot analysis of immunoprecipitation experiment using GFP nanotrap beads from S2 cells co-expressing GFP-Bonus and Flag-tagged Su(var)2-10. Lysate expressing only Flag-Su(var)2-10 was used as a negative control. C) Western blot analysis shows the loss of SUMOylated Bon in fly ovaries upon Su(var)2-10 depletion. Total protein lysates from flies co-expressing MT-Gal4-driven Flag-SUMO and λN-GFP-Bonus and shRNAs against Su(var)2-10 (Sv210) or control white (ctrl) were immunopurified using anti-GFP nanotrap beads. Ovarian lysates from flies expressing only Flag-SUMO, only expressing λN-GFP-Bonus, or lacking Su(var)2-10 shRNA were used as controls. D) Reporter repression by Bon depends on Su(var)2-10. Bar plot shows reporter expression (normalized to rp49 level) upon tethering of control λN-GFP or λN-GFP-Bonus in ovaries with Su(var)2-10 GLKD (shSv210), and control white GLKD (RT-qPCR, dots correspond to 3 independent biological replicates; error bars indicate st. dev.). E) Bon H3K9me3 depositing requires Su(var)2-10. Bar plot shows H3K9me3 enrichment upon tethering of control λN-GFP or λN-GFP-Bonus in ovaries with Su(var)2-10 GLKD (shSv210), and control white GLKD (ChIP-qPCR, dots correspond to 2 independent biological replicates; error bars indicate st. dev.).