Nucleation-dependent propagation of Polycomb modifications emerges during the Drosophila maternal to zygotic transition

Reviewer #1 (Public review):

This well-conceived manuscript investigates the mechanisms that shape the chromatin landscape following fertilization, using the Drosophila embryo as a model system. Importantly, the authors revisit conflicting data using new approaches and analysis to show that the silent H3K27me3 mark deposited by PRC2 is established de novo in the embryo in coordination with the slowing of the nuclear division cycle and activation of zygotic transcription. Unexpectedly, they demonstrate that the transcription factor GAF is not required for the deposition of this mark, but that the well-studied pioneer factor Zelda, which is required for widespread gene expression, is required for H3K27me3 deposition at a subset of regions. The experiments are rigorously performed, and interpretations are clear. Strengths of this manuscript include the rigor of the experimental design, careful analysis, and well-supported conclusions. Some additional citations, analysis, and broadening of the Discussion section to include additional models and data would further strengthen this manuscript.

Reviewer #2 (Public review):

Epigenetic silencing of target genes by the Polycomb pathway is central to maintenance of cell fates during development and depends on repressive chromatin states involving Polycomb complexes and histone modifications. However, the mechanisms by which these chromatin states are built at the earliest stages of development are unclear. Here, Gonzaga-Saavedra and colleagues use the premier experimental system for studying Polycomb gene regulation, Drosophila development, to investigate when Polycomb domains emerge and how they are assembled. Using a combination of CRISPR gene editing, imaging, and genomic profiling, they determine that while H3K27me3 is initially present in the first nuclear cycles, it quickly dissipates and does not re-emerge until mid-nuclear cycle 14, during the major wave of zygotic genome activation (ZGA). This finding helps resolve current discrepancies in the field, informs potential mechanisms of transgenerational inheritance, and indicates that repressive Polycomb domains are built de novo on target genes in embryogenesis. The authors then set out to examine how Polycomb domains are built. Through live imaging and immunofluorescence, they determine that the histone H3K27 methyltransferase, E(z), is present in nuclei at high levels throughout cleavage and blastoderm stages. By contrast, they determine that several Polycomb proteins that bind PREs (cis elements that demarcate Polycomb targets in the genome) are absent from early cleavage nuclei and progressively increase following nuclear cycle 10. These findings suggest that the absence of H3K27me3 in early embryos may be due to failure to assemble functional Polycomb complexes at target genes. Lastly, the authors test the requirement of two transcription factors with important roles in ZGA, GAF, and ZLD. Despite binding to many PREs and regulating chromatin accessibility in early embryos, they find that GAF is largely dispensable for the emergence of H3K27me3 domains. On the other hand, they find that the pioneer factor ZLD is required for proper H3K27me3 emergence; in its absence, some Polycomb domains accumulate greater levels of H3K27me3, whereas other Polycomb domains accumulate less H3K27me3.

Strengths:

The strengths of this study are manifold. It studies an important topic with broad interest to the chromatin and epigenetics fields. It is well-written with detailed method descriptions. In addition, the experimental design and rigor of execution are exceptional despite working with very small amounts of biological material. Example strengths include that the Polycomb proteins studied were tagged with the same epitope, permitting direct quantitative comparisons in imaging and in genomics experiments. Microscopy studies are quantified and performed both via live imaging and via immunofluorescence. The microscopy studies reinforce and extend conclusions made via ChIP. Sophisticated loss-of-function analyses allow for direct mechanistic tests of Polycomb domain emergence.

Weaknesses:

Overall, the study is quite strong already, but it can be further strengthened in several ways. First, several conclusions should be refined based on the data presented. Second, the extent to which ZLD is important for initiating Polycomb domain formation should be made clearer. Third, additional genomic profiling experiments are needed to provide insight into models explaining why H3K27me3 is absent prior to NC14.

Reviewer #3 (Public review):

Gonzaga-Saavedra et al report an analysis on genomic binding of Polycomb group proteins, and of H2Aub1 and H3K27me3 domain formation in the early Drosophila embryo. Using carefully staged embryos during the nuclear cycles (NC) leading up to the cellular blastoderm stage, the authors provide compelling evidence that H3K27me3 domains at PcG target genes are only established during NC14 and do not exist in NC13. In contrast, H2Aub1 domains already start to appear during NC13. The authors show that E(z), the catalytic subunit of the H3K27 histone methyltransferase PRC2, is readily detected in interphase nuclei during the rapid nuclear divisions in pre-blastoderm embryos. In contrast, the DNA-binding proteins Pho, Cg, and GAF that are known (Pho) or have been postulated (Cg, GAF) to anchor PRC2 and PRC1 to Polycomb Response Elements (PREs) in Polycomb target genes only start to show nuclear localization from NC10 onwards with gradually increasing nuclear concentrations, reaching a maximum during NC14. These data strongly corroborate the simple, straightforward view that targeting of PRC2 and PRC1 to PREs by sequence-specific DNA-binding proteins is a prerequisite for the formation of H3K27me3 and H2Aub1 domains at Polycomb target genes.

The authors then explore the potential role of GAF/Trl in this process. They find that in embryos depleted of GAF/Trl, H3K27me3 domain formation is largely unperturbed.

The authors also depleted the pioneer factor Zelda (Zld) and found that removal of Zld results in a more complex outcome. Zelda appears to counteract the accumulation of H3K27me3 at the Polycomb targets eve and zen, but also appears to be required for effective H3K27me3 domain formation at Polycomb targets such as amos or atonal.

This is a very thorough study that reports data of superior technical quality that are highly relevant for the field. The study by Gonzaga-Saavedra et al extends and strengthens previous work from the labs of Eisen (Li et al, eLife 2014) and Zeitlinger (Chen et al, eLife 2013) to convincingly demonstrate that Polycomb domain formation in the early embryo occurs during ZGA but that such domains do not exist prior to ZGA. This should now finally put to rest earlier claims by the Iovino lab (Zenk et al, Science 2017) that H3K27me3 domains present in the zygote nucleus would be propagated and partially maintained during the rapid nuclear cleavage cycles and serve as seeds for H3K27me3 domain formation during ZGA.

The experiments analyzing H3K27me3 domain formation in embryos depleted of GAF/Trl or Zelda will be of great interest to the field.

Author response:

Reviewer 1:

We appreciate the reviewer’s positive assessment and in revision will expand the Discussion to clarify some of the mechanistic insights of this work, as well as to include expanded treatment of related studies in other model systems.

Reviewer 2:

We are grateful for the reviewer’s thorough and supportive comments. We will carefully revise assertions and conclusions for objectivity. Additional analysis of the Zelda experiments will be performed and experimental data tables will be updated to report these results. For the point about providing “insight into models explaining why H3K27me3 is absent prior to NC14,” we have recently submitted a related preprint that addresses this issue directly (Degen, Gonzaga-Saavedra, and Blythe, bioRxiv 2025). In summary, we find evidence that a maternal PcG imprint is indeed maintained through cleavage divisions, albeit through lower-order methylation states (maximally H3K27me2). We chose not to include these additional results in this manuscript to maintain the focus of this study on ZGA. Our revision of this manuscript will include a section in the Discussion that synthesizes the conclusions of the two studies.

Reviewer 3:

We thank the reviewer for recognizing the strength of our data and conclusions, and we agree that our results help settle conflicting claims in the field. We will emphasize Zelda’s context-dependent effects more clearly in the revised manuscript.

References:

Degen EA, Gonzaga-Saavedra N, Blythe SA. Lower-order methylation states underlie the maintenance and re-establishment of Polycomb modifications in Drosophila embryogenesis. bioRxiv [Preprint]. 2025 Jul 29:2025.07.25.666882. doi: 10.1101/2025.07.25.666882. PMID: 40766521; PMCID: PMC12324246.