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| Status |
Public on Nov 28, 2017 |
| Title |
Identification of a conserved maternal-specific repressive domain in Zelda using Cas9-mediated mutagenesis |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome only becomes transcriptionally active hours later. Transcriptional activation is tightly coordinated with the degradation of maternally provided mRNAs during this maternal-to-zygotic transition (MZT). In Drosophila melanogaster, the transcription factor Zelda plays an essential role in widespread activation of the zygotic genome. While Zelda expression is required both maternally and zygotically, the mechanisms by which it functions both during and after the MZT remain unclear. Zelda is a large protein with six C2H2 zinc fingers, but no additional identified domains or predicted enzymatic activities. Using Cas9-mediated genome editing to generate targeted mutations in Zelda, we determined the functional relevance of protein domains conserved amongst Zelda orthologs. Generating these mutations in vivo revealed that neither a conserved N-terminal zinc finger nor an acidic patch were required for activity. Similarly, using a variety of Cas9-enabled approaches we showed that a previously identified splice isoform of zelda is dispensable for viability, and the predicted protein product is not expressed at detectable levels. By contrast, we identified a highly conserved zinc-finger domain that is essential for the maternal, but not zygotic functions of Zelda. A mutation in this zinc-finger domain does not interfere with the capacity of Zelda to activate transcription, but rather leads to a hyperactive form of the protein and enhanced Zelda-dependent gene expression. Embryos inheriting this allele from their mothers die late in embryogenesis. These data define a protein domain critical for controlling Zelda activity and, for the first time, identify a separation between the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are essential for establishing the developmental program during the MZT and that failure to precisely execute this program leads to embryonic lethality.
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| Overall design |
Examination of gene expression profiles from stage 5 wild type embryos or embryos that received maternally deposited zld with a mutation in the second zinc finger (ZnF2). Three replicates of each were analyzed.
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| Contributor(s) |
Hamm DC, Larson ED, Nevil M, Marshall KE, Bondra ER, Harrison MM |
| Citation(s) |
29261646 |
| Submission date |
Sep 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Melissa M Harrison |
| E-mail(s) |
mharrison3@wisc.edu
|
| Organization name |
University of Wisconsin Madison
|
| Department |
Biomolecular Chemistry
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| Lab |
1135 Biochemical Sciences Bldg
|
| Street address |
420 Henry Mall
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platforms (1) |
| GPL13304 |
Illumina HiSeq 2000 (Drosophila melanogaster) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA407471 |
| SRA |
SRP117746 |
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