Figure 7. Drosophila SetDB1 is required for Su(var)2–10 mediated reporter silencing and physically interacts with Su(var)2–10.

(A) Drosophila SetDB1 and Wde contain predicted SIMs. Diagrams show computationally predicted SIMs (red marks) of SetDB1 and Wde homologs in three representative Drosophila species and in human. Asterisks mark the previously described functional SIM of human SetDB1 and Wde (Ivanov et al., 2007; Uchimura et al., 2006). Grey boxes show position of conserved domains.

(B) SetDB1 and Wde interact with SUMOylated proteins. Total protein lysates from S2 cells co-expressing FLAG-HA-SUMO and GFP-SetDB1 or GFP-Wde were immunopurified using anti-GFP nanotrap beads. Cells not expressing FLAG-HA-SUMO were used as negative control.

(C) Wde interacts with SUMO through its SIM motifs. Total protein lysates from S2 cells expressing GFP-Wde fragments including SIM 3–6 and SIM 7 were incubated with recombinant GST-SUMO and immunopurified using anti-GFP nanotrap beads. SIM interaction deficient SUMO mutant and Wde SIM 7 mutant were used to probe specificity of interactions.

(D) SetDB1 is required for Su(var)2–10-induced reporter repression and H3K9me3 deposition at reporter locus. Plots show expression (RT-qPCR) and H3K9me3 enrichment (ChIP-qPCR) at the reporter locus upon tethering of λN-GFP, or λN-GFP-Su(var)2–10 in control (shW) and SetDB1-depleted ovaries. λN-GFP, shW and λN-GFP-Su(var)2–10, shW data is the same as in Fig 5B, D. Dots represent independent biological replicates; bars show mean and SD.

(E) Wde is required for Su(var)2–10 induced reporter repression. Plot shows reporter expression upon tethering of λN-GFP, or λN-GFP-Su(var)2–10 in Wde GLKD or control (shW) ovaries. Dots correspond to three independent biological replicates; bars indicate the mean and SD.

(F) Su(var)2–10 and SUMO are not required for Wde-induced repression. Plot shows reporter expression upon tethering of λN-GFP, or λN-GFP-Wde in ovaries depleted of Su(var)2–10, SUMO or SetDB1 by shRNAs. *λN-GFP-Wde mediated repression is significantly reverted only upon SetDB1 GLKD (p<0.01, single factor ANOVA followed by Tukey post-hoc test). Dots correspond to three independent biological replicates; bars indicate the mean and SD.

(G) Su(var)2–10 interacts with SetDB1. Protein lysates from ovaries expressing FLAG-Su(var)2–10 and GFP-SetDB1 were immunoprecipitated with either anti-FLAG or anti-GFP nanotrap beads. In each experiment, lysate from ovaries not expressing the bait protein was used as negative control.

See also Figure S6.