Figure 6. SUMOylated proteins.

(A) SUMO ChIP-seq profile on rDNA and R1 and R2 consensus sequences. SUMO ChIP-seq signal over the R1 and R2 consensuses and the rDNA unit as in (4A). ChIP-seq data is from Gonzalez et al., 2014. (B) SUMOylation of proteins involved in rDNA transcription and nucleolar function. GFP-tagged proteins were co-expressed with FLAG-SUMO in S2 cells. SUMOylation was detected after immunoprecipitation with anti-GFP antibodies. Several proteins were tagged at either N- or C-terminus. In case of CG3756, only C-terminally tagged protein appears to be SUMOylated. (C) RNAi screen for genes involved in R1 and R2 repression in S2 cells. After knock-down of gene expression using RNAi expression of R1 and R2 transposons was measured by RT-qPCR and normalized to rp49 mRNA. Shown is fold upregulation of R1 and R2 expression upon knock-down compared to control (double-stranded RNA against eGFP gene). Expression levels were measured in three biological replicates. Statistical significance is estimated by two-tailed Student’s t-test; *p<0.05, **p<0.01, ***p<0.001. Information about genes and efficiency of RNAi KD is shown in Supplementary file 4. (D) Tethering of E2 SUMO ligase Ubc9 suppresses rDNA transgene. (Left) The scheme of tethering experiment. The rDNA transgene was modified by insertion of binding site for Csy4 RNA-binding protein into ETS. E2 SUMO ligase Ubc9 was fused with Csy4 protein and co-expressed with rDNA transgene. (Right) The results of RT-qPCR analysis of rDNA transgene expression upon Ubc9 tethering.