Repression of interrupted and intact rDNA by the SUMO pathway in Drosophila melanogaster
- Division of Biology and Biological Engineering, California Institute of Technology, United States
- Institute of Molecular Genetics, Russian Academy of Sciences, Russian Federation
Figures
Figure 1
Single-copy rDNA transgene allows study of rDNA expression.
(A) Scheme of endogenous rDNA units and the rDNA transgene. Native rDNA units are composed of non-transcribed spacer (NTS) and a transcribed portion that produces the 47S pre-rRNA. The pre-rRNA …
Figure 2
Insertions into 28S rRNA lead to decreased transgene expression.
(A) The scheme of rDNA transgenes. The sequences of R1 and R2 transposons were inserted into their natural integration sites within the 28S rRNA. Transgenes R1’ and R2’ are identical to R1 and R2 …
Figure 3 with 1 supplement
SUMO pathway is required for repression of transposons integrated into rDNA.
(A) Scheme of SUMO pathway. SUMO is activated by E1 enzyme composed of Uba2/Aos1 dimer. Next SUMO is transferred from Aos1 to E2 Ligase Ubc9. Unc9 can directly transfer SUMO to target proteins. …
Figure 3—figure supplement 1
R1 and R2 are strongly up-regulated upon SUMO but not Su(var)2–10 depletion.
Heat maps show RPKM-normalized expression of TE consensuses (RepBase) in SUMO and Su(var)2–10 depleted ovaries (shSmt3 and shSv210) and respective controls (shW) (left) and in SUMO and Su(var)2–10 …
Figure 4
SUMO depletion leads to derepression of both interrupted and intact rDNA transgenes.
(A) SUMO knockdown depresses rDNA transgenes. Expression of rDNA transgenes in ovaries as measured by RT-qPCR upon germline knockdown of SUMO (shSmt3) and in control (shW). Data shows three …
Figure 5 with 1 supplement
SUMO KD does not affect H3K9me3 enrichment over rDNA and R1/R2.
(A) Pol I occupancy increases over rDNA transgenes upon SUMO KD Germline-specific knockdown of SUMO (shSmt3) or control (shW) gene was induced by small hairpin driven by maternal-tubulin-Gal4 …
Figure 5—figure supplement 1
Validation of Rpl135-GFP expression, localization in the nucleolus and binding to rDNA.
(A) Rpl135-GFP localizes to the nucleolus. Rpl135-GFP (green) and nucleolus marker Fibrillarin IF (red) show co-localized in nurse cells of the fly ovary. Scale bar: 10 µm. (B) Rpl135-GFP ChIP-qPCR. …
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)…
Additional files
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Supplementary file 1
Drosophila melanogaster stocks.
- https://cdn.elifesciences.org/articles/52416/elife-52416-supp1-v2.docx
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Supplementary file 2
Primers.
- https://cdn.elifesciences.org/articles/52416/elife-52416-supp2-v2.docx
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Supplementary file 3
Cellular component (CC) GO analysis of SUMOylated proteins identified in S2 cells.
The table shows results from GO enrichment analysis for the cellular component on SUMOylated proteins identified by Handu et al., 2015, performed using the enrichGO function of the clusterProfiler package (Yu et al., 2012).
- https://cdn.elifesciences.org/articles/52416/elife-52416-supp3-v2.xlsx
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Supplementary file 4
RNAi screen for genes involved in R1 and R2 repression in S2 cells For each gene the information column describes it known or predicted function and nucleolus localization.
Association with GO term ‘nucleolus’ (GO:0005730) and its child terms is shown according to Gene Ontology analysis. The presence of SUMOylation motif(s) was indicates the presence of consensus SUMOylation sequence (ψKxE/D) in protein sequence. Expression of R1 and R2 transposons were measured by RT-qPCR in S2 cells after knock-down using double-stranded RNA using primers listed in Supplementary file 2. Shown is fold change in R1 and R2 levels upon knock-down of corresponding gene compared to control KD (double-stranded RNA against eGFP gene). Knockdown efficiency shows the depletion of target gene as measured by RT-qPCR. Expression levels were measured in three biological replicates and normalized to rp49 mRNA. Statistical significance is estimated by two-tailed Student’s t-test; *p<0.05, **p<0.01, ***p<0.001.
- https://cdn.elifesciences.org/articles/52416/elife-52416-supp4-v2.docx
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Supplementary file 5
Numerical data from RNA-seq analysis by sleuth.
- https://cdn.elifesciences.org/articles/52416/elife-52416-supp5-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52416/elife-52416-transrepform-v2.pdf
