6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda

A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. We have previously shown that N6-methyladenine (6mA) DNA modification is highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. Here we report that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to-zygotic transition. We find that zelda encoding the pioneer factor Zelda is marked by 6mA. Our genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, our findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.


Responses to the reviewer3's comments
Remarks to the Author: The authors have partially addressed my concerns but there remains a lack of a clear mechanistic link between Jumu, Zelda and the observed changes in gene expression. While the manuscript claims to have identified a "Jumu-Zelda-ZGA regulatory axis" the data cannot distinguish direct from indirect effects of Zelda, neither do the data permit one to distinguish regulation by Zelda versus by "other factors".
Response：First, we would like to thank the reviewer for her/his comments. In this study, we provided compelling genetic evidence showing that Jumu functions as a maternal factor to regulate early embryonic development. Since loss of maternal Jumu leads to up-regulation of Zelda in early embryos, we tried to link the Jumu and Zelda. Our genetic analysis showed that 1) maternal overexpression of Zelda caused embryonic lethal phenotype, which mimics that observed in Jumu maternal mutant embryos; 2) partial loss of Zelda could antagonize the embryonic lethal phonotypes induced by loss of maternal Jumu, suggesting that Zelda is one of critical target genes of Jumu. Moreover, RNA-seq analysis suggested that Jumu-target genes were highly overlapped with Zelda-target genes in early embryos. Because zygotic gene regulation by Zelda is quite complex, we agree with the reviewer's point, we cannot exclude the possibility that Jumu regulates early embryonic development through additional factors. In the final version, we have toned down our conclusion, and avoided to use the "Jumu-Zelda-ZGA regulatory axis".
For example, the authors have now separately analyzed Group 1 and Group 2 GSRJ genes relative to Zelda-bound genes. Both Groups are significantly affected: Group 1 is upregulated upon Zelda overexpression while Group 2 is downregulated (response Fig. 5). They claim that the results are "expected". I find this result to be unexpected: since Zelda is a positive regulator of gene expression, why would the effect on Group 2 be down rather than up (or no change)? These data highlight the absence of a mechanistic explanation.
Response：Previous studies have suggested that loss of maternal Zelda leads to either down-regulation or up-regulation of target genes in Drosophila embryos, suggesting that Zelda regulates gene expression in either direct or indirect manner, although Zelda is a positive regulator of gene expression 1, 2 . For the case of indirect targets of Zelda, Zelda could activate a set of miRNAs, which inhibit sets of downstream target genes. Regarding the relationship between Zelda binding and its target gene expression, the situation is also very complicated. It has been shown that 1) not all binding genes were activated by Zelda, only a subset of binding gene was activated, Zelda binding may poise genes for later activation; 2) Zelda's function to activated genes may be mediated, in part, by local histone acetylation and other histone modifications; 3) Zelda-mediated transcriptional activation may be potentiated by the subsequent binding of additional transcription factors 3 .
Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.