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| Status |
Public on Mar 17, 2020 |
| Title |
Employing proximity-dependent ligation techniques to estimate EcR/Usp molecular partners in Drosophila |
| Organism |
Drosophila melanogaster |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
The subject of the current study is the finding of possible molecular partners of Drosophila EcR receptor. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. All fused proteins were expressed using the Act5C promoter in Drosophila S2 cells. To ensure functionality of the generated proteins, we verified their ability to bind EcR and Usp sites in the Drosophila genome with the ChIP-Seq. Our results demonstrate that EcR and Usp fusions can be recruited to genomic sites endogenous for the EcR/Usp proteins. Conversely, unfused BioID2 and APEX2 enzymes do not bind to EcR/Usp sites. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. ChIP-Seq experiments revealed an enrichment of CP190 binding on transcription start and end sites of 20E-dependent genes but not at EcR/Usp-bound enhancers.
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| Overall design |
ChIP-Seqs by anti-3xFLAG antibodies were performed after 1 hour treatment with 20-hydroxyecdysone (20E) in S2 Schneider cells stably expressing 3xFLAG-EcR, 3xFLAG-EcR-BioID2, 3xFLAG-EcR-APEX2, 3xFLAG-Usp, 3xFLAG-Usp-APEX2, 3xFLAG-Usp-BioID2, 3xFLAG-BioID2, 3xFLAG-APEX2. ChIP-Seq by anti-CP190 and Rpb3 antibodies were performed after 1 hour treatment with 20E and in sham-treated (DMSO) S2 Schneider cells stably expressing 3xFLAG-EcR. ChIP-Seq by anti-CP190 antibodies were performed on white prepupae (Oregon-Modencode).
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| Contributor(s) |
Vorobyeva NE |
| Citation(s) |
32179799 |
| |
| Submission date |
Oct 24, 2019 |
| Last update date |
Mar 20, 2020 |
| Contact name |
Nadezhda E Vorobyeva |
| E-mail(s) |
nvorobyova@gmail.com
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| Phone |
+79262790219
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| Organization name |
Institute of Gene Biology RAS
|
| Street address |
Vavilova 34/5
|
| City |
Moscow |
| ZIP/Postal code |
119334 |
| Country |
Russia |
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| Platforms (1) |
| GPL25244 |
Illumina NovaSeq 6000 (Drosophila melanogaster) |
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| Samples (17)
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| Relations |
| BioProject |
PRJNA579279 |
| SRA |
SRP226806 |
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