Kick-starting the zygotic genome: licensors, specifiers, and beyond

Abstract

Zygotic genome activation (ZGA), the first transcription event following fertilization, kickstarts the embryonic program that takes over the control of early development from the maternal products. How ZGA occurs, especially in mammals, is poorly understood due to the limited amount of research materials. With the rapid development of single-cell and low-input technologies, remarkable progress made in the past decade has unveiled dramatic transitions of the epigenomes, transcriptomes, proteomes, and metabolomes associated with ZGA. Moreover, functional investigations are yielding insights into the key regulators of ZGA, among which two major classes of players are emerging: licensors and specifiers. Licensors would control the permission of transcription and its timing during ZGA. Accumulating evidence suggests that such licensors of ZGA include regulators of the transcription apparatus and nuclear gatekeepers. Specifiers would instruct the activation of specific genes during ZGA. These specifiers include key transcription factors present at this stage, often facilitated by epigenetic regulators. Based on data primarily from mammals but also results from other species, we discuss in this review how recent research sheds light on the molecular regulation of ZGA and its executors, including the licensors and specifiers.

Keywords: Early Embryo; Licensor; Specifier; Transcription Factor; ZGA.

Figure 1. ZGA “licensor” and “specifier” model.

The electric lever, switches, and lamps serve as a model for illustrating the process of ZGA. In this analogy, ZGA “licensors” act as the electric lever, governing the flow of electricity (permission of transcription). ZGA “specifiers” function as distinct switches, regulating the activation of specific lamps (genes).

Figure 2. Epigenetic reprogramming around mouse ZGA.

In mouse, ZGA is executed by RNA Pol II and facilitated by transcription factors (TFs) and chromatin regulators (CRs), amid dramatic epigenetic reprogramming. After fertilization, genome-wide loss of DNA methylation starts from the zygote stage and reaches the lowest level in blastocysts. On the paternal genome, H3K36me3, H3K4me3, H3K27me3, H3K27ac, and H2AK119ub are quickly reset after fertilization based on the globally distinct patterns between sperm and late 1-cell embryo, consistent with the protamine-histone exchange that occurs shortly following fertilization. From the maternal genome, H3K36me3 is briefly inherited by 1-cell embryos and is then reset after ZGA to mark newly transcribed genes. H3K27ac is absent from chromatin of MII eggs and is re-established around pronuclear stage 3-4 (PN3-4) after fertilization. H3K4me3 is briefly inherited from oocytes to early 2-cell embryos. H3K27ac and H3K4me3 then transit from non-canonical, broad domains to canonical sharp peaks during ZGA. H3K27me3 and H2AK119ub exhibit broad, non-canonical patterns in oocytes and preimplantation embryos, with H3K27me3 domains away from promoters being inherited to blastocysts and H2AK119ub1 exhibiting more dynamic changes. Both marks switch to canonical patterns with sharp peaks at promoters after implantation (not shown here). The global patterns of H3K9me3 on both alleles show distinct distributions in 1-cell embryos compared to those in gametes, indicating epigenetic resetting on both genomes. H3K9me3 at early stages is considered non-repressive and is thus marked as non-canonical. 3D chromatin structures including loops and topologically associating domains (TADs) are substantially reduced in MII eggs, 1-cell and 2-cell embryos but are gradually re-established after ZGA. The presence of loops, TADs, as well as the patterning of histone modifications in mouse sperm are currently under debate (Yin et al, 2023).

Figure 3. Models of ZGA timing control.

(A) “Titration/removal of repressors” model. The dilution of transcriptional repressors (red triangles) by DNA replication and cell division initiates the activation of ZGA genes. (B) “Accumulation of activators” model. Active factors (in green) are gradually translated (by ribosomes in blue) after fertilization and act as a molecular timer to trigger ZGA. (C) “Nucleus gating” model. The maturation of nuclear import pathways facilitates the entry of key regulators (in green) into the nucleus, which activates ZGA.

Figure 4. TPRXs and OBOXs function in human and mouse ZGA, respectively.

Human TPRX1/2 genes and mouse Croxs/Obox genes originated from the duplication of the Otx family gene Crx and both belong to the Eutherian Totipotent Cell Homeobox (ETCHbox) genes (Royall et al, 2018). Mouse Croxs/Obox are highly divergent orthologues of TPRX1 and TPRX2, respectively. TPRXL was likely generated by reverse transcription of TPRX1 mRNA followed by genome integration (Booth and Holland, 2007). TPRX1/2/L triple knockdown in human 3 pronuclei (3PN) embryos and Obox1/2/3/4/5/7 maternal-zygotic KO in mouse downregulated about 31% and 48% of human and mouse ZGA genes (Ji et al, ; Zou et al, 2022), respectively, indicating their conserved roles in ZGA.