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Identification of Ku70 Domain-Specific Interactors Using BioID2.

ABSTRACT: Since its inception, proximity-dependent biotin identification (BioID), an in vivo biochemical screening method to identify proximal protein interactors, has seen extensive developments. Improvements and variants of the original BioID technique are being reported regularly, each expanding upon the existing potential of the original technique. While this is advancing our capabilities to study protein interactions under different contexts, we have yet to explore the full potential of the existing BioID variants already at our disposal. Here, we used BioID2 in an innovative manner to identify and map domain-specific protein interactions for the human Ku70 protein. Four HEK293 cell lines were created, each stably expressing various BioID2-tagged Ku70 segments designed to collectively identify factors that interact with different regions of Ku70. Historically, although many interactions have been mapped to the C-terminus of the Ku70 protein, few have been mapped to the N-terminal von Willebrand A-like domain, a canonical protein-binding domain ideally situated as a site for protein interaction. Using this segmented approach, we were able to identify domain-specific interactors as well as evaluate advantages and drawbacks of the BioID2 technique. Our study identifies several potential new Ku70 interactors and validates RNF113A and Spindly as proteins that contact or co-localize with Ku in a Ku70 vWA domain-specific manner.

SUBMITTER: Abbasi S

PROVIDER: S-EPMC8001828 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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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Identification of Ku70 Domain-Specific Interactors Using BioID2.

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