A conserved maternal-specific repressive domain in Zelda revealed by Cas9-mediated mutagenesis in Drosophila melanogaster

Abstract

In nearly all metazoans, the earliest stages of development are controlled by maternally deposited mRNAs and proteins. The zygotic genome becomes transcriptionally active hours after fertilization. Transcriptional activation during this maternal-to-zygotic transition (MZT) is tightly coordinated with the degradation of maternally provided mRNAs. 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 to remodel the embryonic genome and prepare the embryo for development remain unclear. Using Cas9-mediated genome editing to generate targeted mutations in the endogenous zelda locus, we determined the functional relevance of protein domains conserved amongst Zelda orthologs. We showed that neither a conserved N-terminal zinc finger nor an acidic patch were required for activity. Similarly, a previously identified splice isoform of zelda is dispensable for viability. By contrast, we identified a highly conserved zinc-finger domain that is essential for the maternal, but not zygotic functions of Zelda. Animals homozygous for mutations in this domain survived to adulthood, but embryos inheriting these loss-of-function alleles from their mothers died late in embryogenesis. These mutations did not interfere with the capacity of Zelda to activate transcription in cell culture. Unexpectedly, these mutations generated a hyperactive form of the protein and enhanced Zelda-dependent gene expression. These data have defined a protein domain critical for controlling Zelda activity during the MZT, but dispensable for its roles later in development, for the first time separating the maternal and zygotic requirements for Zelda. This demonstrates that highly regulated levels of Zelda activity are required for establishing the developmental program during the MZT. We propose that tightly regulated gene expression is essential to navigate the MZT and that failure to precisely execute this developmental program leads to embryonic lethality.

Dynamic multifactor hubs interact transiently with sites of active transcription inDrosophilaembryos

“…The highly anomalous nature of the movement of Zld that differs from that of chromatin associated His2B and Bcd suggests that its motion depends at least in part on interactions off of DNA. Most of the amino acid sequence of Zld consists of intrinsically disordered domains, some of which are required for its function (Hamm et al 2017) . We and others have shown that intrinsically disordered domains mediate weak, multivalent proteinprotein interactions between regulatory factors (Chong et al 2018; Boehning et al 2018; Kovar 2011; Burke et al 2015; Altmeyer et al 2015; Xiang et al 2015; Friedman et al 2007) that lead to the selective enrichment of factors in hubs (Chong et al 2018; Boehning et al 2018 .…”

“…However, how it accomplishes this remains unclear. Zelda has a cluster of four C2H2 Znfingers near the Cterminus that mediate its DNA binding activity and 2 additional ZFs near the Nterminus which have been implicated in controlling its activation potential (Hamm et al 2017) , but most of the rest of the protein consists of varying types of lowcomplexity sequence.…”

Temporal Control of Transcription by Zelda in living Drosophila embryos

“…To better understand this surprising result, we examined Zld dynamics in living embryos and particularly during mitosis ( Figure 4). We took advantage of an endogenously tagged fluorescent version of Zld 24 . The GFP-zld flies are homozygous viable, thereby showing that the GFP-tagged version of Zld retains wild-type physiological properties.…”

“…RNAi were expressed following crosses between this recombinant and UASp-shRNA-w (BL35573) or UASp-shRNA-zld 21 , virgin females expressing RNAi, MCP-GFP-His2Av-mRFP were crossed with MS2 containing transgenic constructs. The GFP-zld strain was obtained by CRISPR 24 . GFP-ash1 26 and His2Av-mRFP (BL23651).…”

Zelda and the maternal‐to‐zygotic transition in cockroaches

“…The functional study of Zelda domains was approached using a Cas9 engineering system to obtain D. melanogaster insects with point mutations in the Zinc finger 1, in the acidic patch, or in the Zinc finger 2 (JAZ) [16]. Mutations in the Zinc finger 1 or in the acidic patch did not affect the viability of the insects, whereas mutations in the Zinc finger 2 (JAZ) led to a maternal-effect lethal phenotype [16]. A transcriptomic analysis of mutated flies showed that the Zinc finger 2 (JAZ) acts as a maternal repressive domain during embryo development, being associated with the transcriptional regulation of the zygotic Zelda targets and with the clearance of maternal transcripts [16].…”