Investigation of gene regulatory functions of U-shaped by targeted protein depletion
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Abstract
Compensatory mechanisms can occur when RNAi is used to knock down target genes. Additionally, it is challenging to distinguish between the immediate, intermediate, and long-term consequences of gene knock down with high temporal resolution. Targeted protein depletion systems enable the rapid degradation of a protein of interest (POI), circumventing the problems described above. Here, two such systems were established, the GFP-Degron system and the Auxin inducible degron 2 (AID2) system. The GFP-Degron system can deplete a GFP-tagged as well as the AID 2 system can degrade mAID-tagged proteins within a few hours.
To deplete U-shaped (Ush), a master regulator of Drosophila hematopoiesis, the GFP-Degron system was established in stably transfected S2 cells. Depletion of Ush was detectable as soon as 6 h after induction of the GFP-Degron system. Following RNAi mediated ush knockdown the cell growth of S2 cells was impaired after 48 h, with the GFP-Degron system this effect was already visible after 24 h. RNA-Seq analysis revealed the depletion of Ush with the GFP-Degron system resulting in deregulation of the whole S2 cell transcriptome. It was found that a proportion of plasmatocyte marker genes was profoundly downregulated following Ush depletion whereas a proportion of lamellocyte marker genes was significantly upregulated. Furthermore, the morphology of S2 cells was altered after Ush depletion. The S2 cells became less round, spread out forming spiky protrusions and gained adhesiveness. Immunofluorescence analyses revealed structural alterations of cytoskeletal F-actin filaments following Ush depletion. Integrins are important mediators between the extracellular matrix and the actin cytoskeleton. Indeed, four out of seven Drosophila integrin genes were upregulated following Ush depletion. In addition, the localisation and the intensity of the integrin signals of inflated and ItgaPS4 changed upon Ush degradation. Focal adhesions appeared as punctae at the leading edge of the spiky protrusions. All in all, these changes are reminiscent of the transdifferentiation from plasmatocytes to lamellocytes. Upon loss of Ush, the S2 cells lose their plasmatocyte-like identity and gain a lamellocyte-like identity. This thesis provides a novel model system to study transdifferentiation in vitro.
The GFP-Degron system was also established in S2 cells with GFP-tagged dMi-2 which is an ATP-dependent chromatin remodeler. However, the depletion rate was insufficient for dMi-2. Therefore, the AID2 system was established for the depletion of dMi-2. The establishment was successful and future experiments will show how fast the AID2 system can deplete dMi-2.
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Issued: 2026-01-22
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FB20:Medizin
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en
Keywords
Differentiationtransdifferentiationgene regulationU-shapedTargeted protein depletionS2 cellsGFP-DegronAID2
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2.11-05 - Allgemeine Genetik und funktionelle Genomforschung2.11-03 - Zellbiologie
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Trummel, Deborah: Investigation of gene regulatory functions of U-shaped by targeted protein depletion. : 2026-01-22.
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Except where otherwised noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International