
==== Front
9111375
2199
Curr Opin Genet Dev
Curr Opin Genet Dev
Current opinion in genetics & development
0959-437X
1879-0380

37339553
10.1016/j.gde.2023.102062
nihpa2020689
Article
Principles of genome activation in the early embryo
Zhou Coral Y *
Heald Rebecca *
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
* Twitter account: @CoralZhou, @rebeccaheald

Corresponding authors: Zhou, Coral Y (coral.zhou@berkeley.edu), Heald, Rebecca (bheald@berkeley.edu)
12 9 2024
8 2023
18 6 2023
23 9 2024
81 102062102062
https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A major hurdle in an embryo’s life is the initiation of its own transcriptional program, a process termed Zygotic Genome Activation (ZGA). In many species, ZGA is intricately timed, with bulk transcription initiating at the end of a series of reductive cell divisions when cell cycle duration increases. At the same time, major changes in genome architecture give rise to chromatin states that are permissive to RNA polymerase II activity. Yet, we still do not understand the series of events that trigger gene expression at the right time and in the correct sequence. Here we discuss new discoveries that deepen our understanding of how zygotic genes are primed for transcription, and how these events are regulated by the cell cycle and nuclear import. Finally, we speculate on the evolutionary basis of ZGA timing as an exciting future direction for the field.
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pmcIntroduction

For sexually reproducing organisms, embryogenesis begins following the fertilization of an egg by a sperm. However, the embryo does not acquire its own identity until it starts transcribing its own genome in a process called Zygotic Genome Activation (ZGA) [1,2]. ZGA begins as a specialized set of transcription factors called pioneer factors bind to nucleosomes at promoters and enhancers, allowing downstream transcription activators to access the DNA and leading to an increase in RNA polymerase II (Pol II) activity [3]. The misregulation of ZGA timing or activity leads to developmental delay, arrest, or even embryo death [1].

Classic studies in Xenopus demonstrated that the largest burst of transcription occurs after a series of rapid and synchronous cell division cycles that partition the large maternal egg cytoplasm into thousands of smaller cells [4,5]. These experiments laid the groundwork for current investigations of how and to what degree ZGA timing is regulated by changes in the nuclear-to-cytoplasmic (N/C) ratio, developmental time, cell cycle duration, and cell size across diverse species [1]. A more recent area of great interest in the field is dissecting how changes in the 3D genome architecture during ZGA relate to changes in transcriptional output (reviewed in [6–8]). However, it is still unclear how all of these events work together to trigger ZGA at a precise developmental stage, and how these mechanisms differ across metazoans.

Here we summarize recent developments in the ZGA field in the context of two fundamental questions: 1) How is the genome primed for ZGA? and 2) What determines the timing of ZGA within and across species?

Pioneer factors and histones prime the chromatin landscape for zygotic genome activation

Pioneer factors create accessibility by peeling open the genome

Early models of ZGA proposed a role for maternally loaded transcriptional repressors that are progressively titrated away from the genome due to an increasing N/C ratio [4,5]. We now recognize that an additional barrier to activating a naïve genome is the ability of transcription factors to bind and create accessibility at promoters and enhancers (Figure 1c–d). There is emerging evidence that accessibility is initiated by pioneer factors, which bind to specific DNA sequences on nucleosomes and expose nearby regions of DNA that can then recruit additional activating factors such as chromatin remodeling enzymes [3,9]. The classic ZGA-specific pioneer factors include Pou5f3, Nanog and Sox19b in zebrafish [10,11]; Zelda in Drosophila [12]; and DUX [13–15], NFY [16], and more recently Nr5a2 [17••] in mouse. In all cases, depletion, inhibition, or knockout of one or more pioneer factors results in decreased transcription of zygotic genes accompanied by decreased chromatin accessibility at key regulatory regions.

Since the discovery of Zelda as a major regulator of ZGA in Drosophila 15 years ago [12], it has been a mystery how approximately half of Zelda-regulated zygotic genes remained accessible even when maternal Zelda was depleted. Also, the premature expression of Zelda was not sufficient to precociously activate the genome [18]. Recent studies have solved this conundrum by demonstrating bona fide pioneer activities for CLAMP [19•] and GAF (GAGA Factor) [20•], both of which bind and create accessibility at GA repeats near Zelda-regulated genes. The discovery of GAF as a pioneer factor is of particular interest as it was recently demonstrated that in Drosophila hemocytes, single molecules of GAF spend an unusually long time bound to chromatin (~140 s), 20–30-fold longer than other studied transcription factors [21••]. This ‘kinetic dominance’ allows GAF to occupy ~100% of its target sites at any given time [21••]. Unlike Zelda, GAF binds to developmental patterning genes during mitosis as well as in interphase [22], actively bookmarking these genes for expression through the cell cycle [23••]. Together these data suggest that the unique ability of GAF to persist on chromatin for long periods of time, and perhaps especially during mitosis, is important for establishing transcriptional memory during the many nuclear divisions of early embryogenesis.

The observation that multiple pioneer factors can co-exist at the same locus raises the question of if and how pioneer factors cooperate with one another to create accessibility across the genome. Two recent studies directly addressed this question using zebrafish by creating maternal knockouts of the three ZGA factors Nanog (n), Pou5f3 (p), and Sox19b (s) in various combinations [24••]. One group created a triple knockout mutant of all three factors (MZnps) and asked if the loss of accessibility at zygotic genes could be rescued by injecting mRNAs encoding one or more of them. They found that while each factor alone had its own pioneering activity, Nanog was required to open more regions than the other two factors. At regions bound by all three factors, about a third required the presence of one factor for the others to bind. Another study comparing double (MZps) versus single knockout (MZs) mutants suggested that Sox19b and Pou5f3 mostly act independently to open chromatin [25]. These results, similar to findings in Drosophila [19,20•], suggest that pioneer factors can act independently or cooperatively depending on the genomic context, with Nanog being the most crucial. Consistent with these observations, live imaging experiments in zebrafish showed that Nanog was the first of the three factors to co-localize with Pol II-enriched transcription hubs prior to ZGA, and embryos containing mutant Nanog no longer formed these hubs [26•]. In the future, it will be interesting to dissect the molecular mechanism behind this privileged role for Nanog during ZGA.

The histone code is reshuffled during zygotic genome activation to establish an open chromatin landscape

As pioneer factors find their target sequences on nucleosomes, histones themselves are also being marked, exchanged and redistributed across the genome during early embryogenesis [27]. Activating histone marks and variants can either cooperate with pioneer factors to maintain open promoters and enhancers, or act independently to regulate ZGA (Figure 1). For example, the acetylation of histones loosens inter-nucleosomal contacts in vitro [28] and correlates with active transcription in vivo [29]. In mouse embryos, the inhibition of Brd4 and p300, the reader and writer for H3K27ac, respectively, resulted in aberrant ZGA, loss of chromatin accessibility, and developmental arrest [30••]. Injection of Brd4 and p300 into zebrafish embryos was sufficient to induce premature ZGA of some genes [31]. However, treatment of wild-type embryos with inhibitors of Brd4 and p300 was not sufficient to block chromatin opening at the sites that were most transcriptionally down-regulated in the MZnps mutant [24••]. Together these results suggest that histone acetylation is necessary but not always sufficient to activate the genome and sometimes acts downstream of pioneer factor activity.

Histone variants also play a role in ZGA but potentially through a different pathway. In Drosophila, the histone variant H2A.Z was shown to mark 65% of zygotic genes before they are activated, and knockdown of the H2A.Z-specific chaperone Domino diminished ZGA [32••]. Importantly, genes regulated by H2A.Z were largely unoccupied by Zelda, and the knockdown of Domino did not affect Zelda binding, suggesting the H2A.Z pathway functions independently of Zelda to activate zygotic transcription [32••]. In mouse embryos, redistribution of histone variant H3.3 by the histone chaperone CAF1 is required for normal ZGA though underlying mechanisms are unclear [33•]. Examining the physical interplay between histones and pioneer factors at zygotic genes in the early embryo is an exciting area for future work.

Events related to the cell cycle converge to trigger zygotic genome activation

Multiple mechanisms have been proposed to trigger ZGA including the titration of maternally loaded factors due to increasing N/C ratio [4,5,34–37], a threshold cell size [38] or nuclear size [39], developmental time [31] or cell cycle duration [40]. Here we summarize the most recent work characterizing the contributions of N/C ratio, cell cycle length and nuclear import on ZGA timing.

Nuclear-to-cytoplasmic ratio and cell cycle duration independently contribute to zygotic genome activation

The exponential increase in genome copies within a finite volume of maternal cytoplasm results in a dramatic increase in N/C ratio as development proceeds (Figure 1a, Figure 2a), ultimately reaching a threshold N/C ratio that has been proposed to trigger ZGA [4,5]. Indeed, decreasing the total genome content three-fold using Xenopus cybrids (Figure 2a) [41] or two-fold using haploid Drosophila (Figure 2b) [43] delayed ZGA. However, the interpretation of this result is confounded by the long-standing observation that decreasing genome content causes embryos to undergo an additional cell division just before lengthening their cell cycle [4,42]. To distinguish how ZGA is influenced by N/C ratio versus cell cycle duration, one group used live imaging to track the accumulation of specific zygotic transcripts in Drosophila haploids, diploids, and a chk1 mutant that shortens the nuclear cycle around the time of ZGA (Figure 2b) [43••]. For roughly half of the zygotic genes tested, the chk1 mutant phenocopied the delay in ZGA observed in haploids, suggesting that the activation of these genes is regulated by cell cycle duration and not N/C ratio [43••], consistent with earlier work in Drosophila [40]. For another set of transcripts, the chk1 mutant activated transcription at the same time as wild-type diploids, suggesting that N/C ratio was instead the major regulator of ZGA timing [43••]. In a separate study, prolonged treatment of Xenopus embryos with cycloheximide to induce interphase arrest triggered ZGA several hours earlier than in control embryos (Figure 2c) [44••], consistent with previous work in zebrafish [31]. Remarkably, the cycloheximide-treated embryos contained ~10-fold fewer cells than normal embryos, with a N/C ratio far lower than that of wild-type embryos during ZGA onset, yet still produced transcription profiles almost identical to untreated embryos [44••]. Taken together, these results demonstrate how N/C ratio and cell cycle duration can independently activate transcription. Future work will be required to identify underlying molecular mechanisms, which could be both gene-specific and species-specific.

Nuclear import gates access of pioneer factors to the genome

An important missing piece of the puzzle is how ZGA timing is linked to chromatin remodeling activities that prime the genome for transcription. Two new studies introduce a potential role for nuclear import as a rate-limiting step for ZGA (Figure 1). One study in zebrafish used proteomics to quantify changes in nuclear proteins during the early cleavage stages [45••]. The authors discovered that among the proteins that increased most dramatically in nuclear abundance were key pioneer factors that trigger ZGA including Nanog, Sox19b and Pou5f3 [45••]. Using electron microscopy (EM) and immunofluorescence, they further showed that the nuclear pore complexes (NPCs) gradually increased in structural complexity leading up to ZGA [45••]. Together, these results suggest that the NPC assembly state can serve as a barrier to the entry of pioneer factors during early cleavage stages, thereby acting as a timer for ZGA. In a complementary story using similar approaches in Xenopus, the authors found that NPC proteins were among the first to be imported into the nucleus and that different transcription factors enter the nucleus at different times throughout the cleavage divisions [46••]. Using an extract-based biochemical assay, the authors went further to demonstrate that the timing of transcription factor entry correlates with their affinity for importin α/β, the major nuclear import machinery [46••]. This result is particularly exciting because it posits a simple biochemical model in which nuclear import gates the access of pioneer factors and other chromatin remodeling machinery to the genome, creating a temporally regulated sequence of events that ultimately trigger ZGA (Figure 1). Such a mechanism could also explain why increasing interphase duration can trigger ZGA as more time is allowed for importing key factors.

Zygotic genome activation across evolution

ZGA timing differs dramatically across evolution. While flies, frogs and fish all initiate their major ZGA after 10–13 nuclear cycles [1,2], mice begin ZGA after only one cell division [47,48] and humans begin transcribing their genome at the one-cell stage [49•]. Despite these differences, we speculate that there are a few unifying principles for how ZGA is timed. First, since mammalian eggs are several orders of magnitude smaller than that of amphibian species that possess similar genome sizes, the N/C ratio at which ZGA initiates across species is not vastly different. We speculate that there is a universal N/C ratio threshold above which transcription will be active, and which could be tuned to different egg sizes among closely related species. For mammals with small eggs, this threshold is reached almost immediately upon fertilization, while for amphibians this threshold is only reached after multiple rounds of reductive cell divisions. There could also be a lower threshold for the N/C ratio below which transcription is silenced. Recent work comparing developmental timing across Xenopus species with a six-fold variation in genome size and 3.5-fold variation in egg size showed that early cleavage divisions and gastrulation occur with the same kinetics, suggesting that ZGA timing is conserved despite differences in the N/C ratio [50•]. A second unifying theme is that actively transcribing embryos, regardless of the species, have slow cell cycles on the timescale of hours or days, while rapidly cleaving embryos are transcriptionally silent [51]. One proposed explanation is that the machinery that silences or activates transcription, which is generally well-conserved across species, needs time to remodel its genomic targets [52–55]. In the future, it will be fascinating to test these ideas across a wide range of species, including close animal relatives [56•] to dissect the evolutionary history of ZGA.

Summary and conclusions

The idea that a genome must be ‘awakened’ in an early embryo has puzzled researchers for more than 40 years [4,5]. Now with modern tools, we have a more detailed understanding of ZGA timing and mechanisms at the level of molecular players and specific genes. Armed with this knowledge, we envision that the next phase of ZGA research will integrate these findings into both universal and species-specific principles for transcriptional activation across molecular, cellular, and organismal scales.

Acknowledgements

We would like to thank all of the authors who contributed to this field, including those who we were not able to include in this short review due to space limitations. This work was supported by the Jane Coffin Childs Memorial Fund Fellowship to C.Y.Z. and the National Institutes of Health (NIH) MIRA grant R35GM118183 and the Flora Lamson Hewlett Chair in Biochemistry to R.H.

Data Availability

No data was used for the research described in the article.

Figure 1 A series of chromatin remodeling events triggers ZGA. (a) ZGA occurs toward the end of a series of reductive cleavage divisions. (b) As cleavage divisions progress, NPCs increase in size and complexity [45••]. Due to differences in the affinity of pioneer factors for nuclear import machinery, the green pioneer factor is imported before the brown pioneer factor [46••]. (c) At the nucleosome level, pioneer factors peel open nucleosomal DNA at their target site, creating accessibility for downstream factors such as histone chaperones that exchange canonical histones with variants [32,33•], and histone acetyltransferases [18,30••]. Ultimately these events create an open genome structure that allows RNA Pol II to begin transcribing. (d) Legend for different molecules displayed.

Figure 2 N/C ratio and cell cycle duration can independently trigger ZGA. (a) Left: As embryos progress through cleavage divisions, the increase in genome copies within a finite volume of maternal cytoplasm results in a dramatic increase in N/C ratio. Right: In Xenopus, decreasing genome content delays the onset of bulk ZGA [41•]. (b) In Drosophila, wild-type embryos initiate transcription at nuclear cycle 13 (NC13) while haploid embryos initiate transcription after an additional cell cycle, at nuclear cycle 14 (NC14). A diploid chk1 mutant that has a slow NC13 phenocopies wild-type embryos for the activation of the blue transcript suggesting regulation by cell cycle length. However, the activation of the pink transcript is delayed, similar to the haploid embryos, suggesting regulation mainly by N/C ratio [43••]. (c) In Xenopus, the accumulation of total zygotic transcripts can be measured by the incorporation of fluorescent 5-ethynyluridine into nascent RNA. While control embryos initiate transcription once a threshold cell size is reached, treatment of pre-ZGA embryos with cycloheximide results in early onset ZGA in large cells [44••].

Declaration of Competing Interest

The authors Coral Y. Zhou and Rebecca Heald declare no conflicts.
==== Refs
References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

1. Schulz KN , Harrison MM : Mechanisms regulating zygotic genome activation. Nat Rev Genet 2019, 20 :221–234.30573849
2. Jukam D , Shariati SAM , Skotheim JM : Zygotic genome activation in vertebrates. Dev Cell 2017, 42 :316–332.28829942
3. Zaret KS : Pioneer transcription factors initiating gene network changes. Annu Rev Genet 2020, 54 :367–385.32886547
4. Newport J , Kirschner M : A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell 1982, 30 :675–686.6183003
5. Newport J , Kirschner M : A major developmental transition in early Xenopus embryos: II. control of the onset of transcription. Cell 1982, 30 :687–696.7139712
6. Zhang Y , Xie W : Building the genome architecture during the maternal to zygotic transition. Curr Opin Genet Dev 2022, 72 :91–100.34896808
7. Ing-Simmons E , Rigau M , Vaquerizas JM : Emerging mechanisms and dynamics of three-dimensional genome organisation at zygotic genome activation. Curr Opin Cell Biol 2022, 74 :37–46.35065445
8. Vallot A , Tachibana K : The emergence of genome architecture and zygotic genome activation. Curr Opin Cell Biol 2020, 64 :50–57.32220807
9. Kobayashi W , Tachibana K : Awakening of the zygotic genome by pioneer transcription factors. Curr Opin Struct Biol 2021, 71 :94–100.34256217
10. Lee MT , Bonneau AR , Takacs CM , Bazzini AA , DiVito KR , Fleming ES , Giraldez AJ : Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature 2013, 503 :360–364.24056933
11. Leichsenring M , Maes J , Mössner R , Driever W , Onichtchouk D : Pou5f1 transcription factor controls zygotic gene activation in vertebrates. Science 2013, 341 :1005–1009.23950494
12. Liang H-L , Nien C-Y , Liu H-Y , Metzstein MM , Kirov N , Rushlow C : The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila. Nature 2008, 456 :400–403.18931655
13. Hendrickson PG , Doráis JA , Grow EJ , Whiddon JL , Lim J-W , Wike CL , Weaver BD , Pflueger C , Emery BR , Wilcox AL , : Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nat Genet 2017, 49 :925–934.28459457
14. Whiddon JL , Langford AT , Wong C-J , Zhong JW , Tapscott SJ : Conservation and innovation in the DUX4-family gene network. Nat Genet 2017, 49 :935–940.28459454
15. Iaco AD , Planet E , Coluccio A , Verp S , Duc J , Trono D : DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nat Genet 2017, 49 :941–945.28459456
16. Lu F , Liu Y , Inoue A , Suzuki T , Zhao K , Zhang Y : Establishing chromatin regulatory landscape during mouse preimplantation development. Cell 2016, 165 :1375–1388.27259149
17.•• Gassler J , Kobayashi W , Gáspár I , Ruangroengkulrith S , Mohanan A , Hernández LG , Kravchenko P , Kümmecke M , Lalic A , Rifel N , : Zygotic genome activation by the totipotency pioneer factor Nr5a2. Science 2022, 378 :1305–1315, 10.1126/science.abn7478.36423263
This paper demonstrates bona fide pioneer activity for the orphan nuclear receptor Nr5a2 in human embryos. Nr5a2 binds to nucleosomes in vitro and chemical inhibition of Nr5a2 results in decreased transcription and lower accessibility at 72% of zygotic genes.

18. Larson ED , Komori H , Fitzpatrick ZA , Krabbenhoft SD , Lee C-Y , Harrison M : Premature translation of the Drosophila zygotic genome activator Zelda is not sufficient to precociously activate gene expression. G3 Genes Genomes Genet 2022, 12 :jkac159.
19.• Duan J , Rieder L , Colonnetta MM , Huang A , Mckenney M , Watters S , Deshpande G , Jordan W , Fawzi N , Larschan E : CLAMP and Zelda function together to promote Drosophila zygotic genome activation. Elife 2021, 10 :e69937.34342574
This work demonstrates that CLAMP is a key pioneer factor for ZGA in Drosophila. CLAMP binds nucleosomes in vitro and collaborates with the classic ZGA factor Zelda to create accessibility at zygotic genes.

20.• Gaskill MM , Gibson TJ , Larson ED , Harrison MM : GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo. Elife 2021, 10 :e66668.33720012
By degrading the maternal supply of GAF in Drosophila, these authors demonstrate that GAF activates a subset of zygotic genes largely independent of Zelda. Additionally, GAF is retained on mitotic chromosomes during cleavage divisions and appears to have a role in maintaining chromosome stability, particularly during mitosis.

21.•• Tang X , Li T , Liu S , Wisniewski J , Zheng Q , Rong Y , Lavis LD , Wu C : Kinetic principles underlying pioneer function of GAGA transcription factor in live cells. Nat Struct Mol Biol 2022, 29 :665–676.35835866
The authors tracked the dynamics of single particles of GAF in Drosophila hemocytes demonstrating that GAF has an unusually long residence time on chromatin, independent of chromatin remodeling enzymes. Combining these data with genomics studies, the authors propose that GAF has a unique ‘kinetic dominance’ to allow it to occupy essentially every target site in the genome at any given time.

22. Blythe SA , Wieschaus EF : Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis. Elife 2016, 5 :e20148.27879204
23.•• Bellec M , Dufourt J , Hunt G , Lenden-Hasse H , Trullo A , Aabidine AZE , Lamarque M , Gaskill MM , Faure-Gautron H , Mannervik M , : The control of transcriptional memory by stable mitotic bookmarking. Nat Commun 2022, 13 :1176.35246556
This paper combines genomics and live imaging in Drosophila to show that GAF binds tightly to interphase and mitotic chromatin, is required to increase accessibility at zygotic genes, and plays a role in transcriptional memory by bookmarking active genes through mitosis.

24.•• Miao L , Tang Y , Bonneau AR , Chan SH , Kojima ML , Pownall ME , Vejnar CE , Gao F , Krishnaswamy S , Hendry CE , : The landscape of pioneer factor activity reveals the mechanisms of chromatin reprogramming and genome activation. Mol Cell 2022, 82 :986–1002e9.35182480
This paper uses genomics to analyze how each of the three major ZGA factors in zebrafish (Pou5f3, Sox19b and Nanog) contributes to creating an open chromatin landscape to initiate transcription. They discover that depending on the gene of interest, the factors can act independently or collaboratively.

25.• Gao M , Veil M , Rosenblatt M , Riesle AJ , Gebhard A , Hass H , Buryanova L , Yampolsky LY , Grüning B , Ulianov SV , : Pluripotency factors determine gene expression repertoire at zygotic genome activation. Nat Commun 2022, 13 :788.35145080
This paper combines genomics and classic embryology to demonstrate how Sox19b and Pou5f3 pattern operate mostly independently to turn on genes that specify the ventral, but not the dorsal side of the zebrafish embryo.

26.• Kuznetsova K , Chabot NM , Ugolini M , Wu E , Lalit M , Oda H , Sato Y , Kimura H , Jug F , Vastenhouw NL : Nanog organizes transcription bodies. Curr Biol 2022, 33 :164–173e5, 10.1016/j.cub.2022.11.015.36476751
The authors use live imaging in zebrafish to demonstrate that the three major ZGA factors (Pou5f3, Sox19b and Nanog) co-localize with Pol II with different dynamics. Nanog appears first, before transcription begins, and is required for the formation of transcription bodies.

27. Shindo Y , Brown MG , Amodeo AA : Versatile roles for histones in early development. Curr Opin Cell Biol 2022, 75 :102069.35279563
28. Allahverdi A , Yang R , Korolev N , Fan Y , Davey CA , Liu C-F , Nordenskiöld L : The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association. Nucleic Acids Res 2011, 39 :1680–1691.21047799
29. Shvedunova M , Akhtar A : Modulation of cellular processes by histone and non-histone protein acetylation. Nat Rev Mol Cell Bio 2022, 23 :329–349.35042977
30.•• Wang M , Chen Z , Zhang Y : CBP/p300 and HDAC activities regulate H3K27 acetylation dynamics and zygotic genome activation in mouse preimplantation embryos. Embo J 2022, 41 :e112012.36215692
This group uses genomics to characterize the dynamics of H3K27ac, an activating mark associated with enhancers and promoters, during early mouse development. They show that inhibiting the writer and reader of the mark (CBP/p300) results in aberrant ZGA, loss of chromatin accessibility and developmental arrest.

31. Chan SH , Tang Y , Miao L , Darwich-Codore H , Vejnar CE , Beaudoin J-D , Musaev D , Fernandez JP , Benitez MDJ , Bazzini AA , : Brd4 and P300 confer transcriptional competency during zygotic genome activation. Dev Cell 2019, 49 :867–881e8.31211993
32.•• Ibarra-Morales D , Rauer M , Quarato P , Rabbani L , Zenk F , Schulte-Sasse M , Cardamone F , Gomez-Auli A , Cecere G , Iovino N : Histone variant H2A.Z regulates zygotic genome activation. Nat Commun 2021, 12 :7002.34853314
These authors introduce the role of the histone variant H2A.Z as an additional ZGA factor in Drosophila, acting at genes not regulated by Zelda. Knockdown of the H2A.Z-specific chaperone Domino results in decreased activation of zygotic genes and lower Pol II occupancy.

33.• Ishiuchi T , Abe S , Inoue K , Yeung WKA , Miki Y , Ogura A , Sasaki H : Reprogramming of the histone H3.3 landscape in the early mouse embryo. Nat Struct Mol Biol 2021, 28 :38–49.33169018
This study uses genomics to track the dynamics of H3.3 in mouse oocytes and early embryos. The authors found that H3.3 is redistributed from heterochromatin to euchromatin around the same time as ZGA. Inhibition of the histone chaperone chromatin assembly factor-1 (CAF-1) results in decreased incorporation of H3.3, developmental arrest and aberrant ZGA.

34. Shindo Y , Amodeo AA : Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo. Curr Biol 2021, 31 :2633–2642e6.33848457
35. Amodeo AA , Jukam D , Straight AF , Skotheim JM : Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. Proc Natl Acad Sci 2015, 112 :E1086–E1095.25713373
36. Joseph SR , Pálfy M , Hilbert L , Kumar M , Karschau J , Zaburdaev V , Shevchenko A , Vastenhouw NL : Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos. Elife 2017, 6 :e23326.28425915
37. Collart C , Allen GE , Bradshaw CR , Smith JC , Zegerman P : Titration of four replication factors is essential for the Xenopus laevis midblastula transition. Science 2013, 341 :893–896.23907533
38. Chen H , Einstein LC , Little SC , Good MC : Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo. Dev Cell 2019, 49 :852–866e7.31211992
39. Jevtić P , Levy DL : Nuclear size scaling during Xenopus early development contributes to midblastula transition timing. Curr Biol 2015, 25 :45–52.25484296
40. Strong IJT , Lei X , Chen F , Yuan K , O’Farrell PH : Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate mid-blastula transition events at a low nuclear-cytoplasmic ratio. PLOS Biol 2020, 18 :e3000891.33090988
41.• Jukam D , Kapoor RR , Straight AF , Skotheim JM : The DNA-to-cytoplasm ratio broadly activates zygotic gene expression in Xenopus. Curr Biol 2021, 31 :4269–4281e8.34388374
These authors alter genome size by three-fold by manipulating fertilization conditions in Xenopus embryos to show that decreasing ploidy also delays activation of 90% of zygotic genes. This work supports the hypothesis that the N/C ratio is a major driver in ZGA timing.

42. Edgar BA , Kiehle CP , Schubiger G : Cell cycle control by the nucleo-cytoplasmic ratio in early Drosophila development. Cell 1986, 44 :365–372.3080248
43.•• Syed S , Wilky H , Raimundo J , Lim B , Amodeo AA : The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities. Proc Natl Acad Sci 2021, 118 :e2010210118.33790005
To distinguish between contributions of cell cycle duration and N/C ratio ZGA timing, these authors use live imaging to track the dynamics of transcription activation in various mutants in Drosophila. They discover that the importance of each of these cues on ZGA timing is dependent on the identity of the transcript.

44.•• Chen H , Good MC : Nascent transcriptome reveals orchestration of zygotic genome activation in early embryogenesis. Curr Biol 2022, 32 :4314–4324e7, 10.1016/j.cub.2022.07.078.36007528
These authors demonstrate that lengthening the cell cycle by treating Xenopus embryos with cycloheximide is sufficient to trigger an early ZGA. These results support the hypothesis that cell cycle duration, not the N/C ratio, is the major determinant of ZGA timing.

45.•• Shen W , Gong B , Xing C , Zhang L , Sun J , Chen Y , Yang C , Yan L , Chen L , Yao L , : Comprehensive maturity of nuclear pore complexes regulates zygotic genome activation. Cell 2022, 185 :4954–4970e20, 10.1016/j.cell.2022.11.011.36493774
This group shows that during pre-ZGA cleavage divisions in zebrafish, NPCs gradually increase in size and complexity, concomitant with the increase in the nuclear entry of key pioneer factors. These results suggest that the capacity for nuclear import could act as a timer for ZGA.

46.•• Nguyen T , Costa EJ , Deibert T , Reyes J , Keber FC , Tomschik M , Stadlmeier M , Gupta M , Kumar CK , Cruz ER , : Differential nuclear import sets the timing of protein access to the embryonic genome. Nat Commun 2022, 13 :5887.36202846
These authors use proteomics to demonstrate that different transcription factors enter the nucleus at different times during cleavage divisions in Xenopus, which correlates with differences in the affinity of each factor with nuclear import machinery.

47. Nothias JY , Miranda M , DePamphilis ML : Uncoupling of transcription and translation during zygotic gene activation in the mouse. Embo J 1996, 15 :5715–5725.8896464
48. Aoki F , Worrad DM , Schultz RM : Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev Biol 1997, 181 :296–307.9013938
49.• Asami M , Lam BYH , Ma MK , Rainbow K , Braun S , VerMilyea MD , Yeo GSH , Perry ACF : Human embryonic genome activation initiates at the one-cell stage. Cell Stem Cell 2021,29 :209–216e4.34936886
Using high-resolution, single-cell RNA-seq, the authors demonstrate that the first zygotic transcription begins shortly after fertilization, at the one-cell stage in human embryos.

50.• Miller KE , Cadart C , Heald R : Dodecaploid Xenopus longipes provides insight into the emergence of size scaling relationships during development. Curr Biol 2023, 33 :1327–1336e4.36889317
These authors compare the onset of the mid-blastula transition (MBT) among Xenopus species that differ in genome size by six-fold. They show that MBT onset occurs at different N/C ratios across species, supporting the idea that different species have evolved different mechanisms to set ZGA timing.

51. O’Farrell PH : Growing an embryo from a single cell: a hurdle in animal life. Cold Spring Harb Perspect Biol 2015, 7 :a019042.26254311
52. Seller CA , Cho C-Y , O’Farrell PH : Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila. Gene Dev 2019, 33 :403–417.30808658
53. Shermoen AW , O’Farrell PH : Progression of the cell cycle through mitosis leads to abortion of nascent transcripts. Cell 1991, 67 :303–310.1680567
54. Kwasnieski JC , Orr-Weaver TL , Bartel DP : Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns. Genome Res 2019, 29 :1188–1197.31235656
55. Rothe M , Pehl M , Taubert H , Jäckle H : Loss of gene function through rapid mitotic cycles in the Drosophila embryo. Nature 1992, 359 :156–159.1522901
56.• Olivetta M , Dudin O : The nuclear-to-cytoplasmic ratio drives cellularization in the close animal relative Sphaeroforma arctica. Curr Biol 2023, 33 :1597–1605e3, 10.1016/j.cub.2023.03.019.36996815
Using a close animal relative Sphaeroforma arctica, these authors show that the N/C ratio regulates the timing of multi-cellularization through interactions at the cell cortex. This study suggests that the N/C ratio as a major regulator of major developmental transitions predates animals.
