Pioneer factors: roles and their regulation in development.

Pioneer factors are a subclass of transcription factors that can bind and initiate opening of silent chromatin regions. Pioneer factors subsequently regulate lineage-specific genes and enhancers and, thus, activate the zygotic genome after fertilization, guide cell fate transitions during development, and promote various forms of human cancers. As such, pioneer factors are useful in directed cell reprogramming. In this review, we define the structural and functional characteristics of pioneer factors, how they bind and initiate opening of closed chromatin regions, and the consequences for chromatin dynamics and gene expression during cell differentiation. We also discuss emerging mechanisms that modulate pioneer factors during development.

The impact of selective HDAC inhibitors on the transcriptome of early mouse embryos.

BACKGROUND: Histone acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays a crucial role in the control of gene expression. HDAC inhibitors (HDACi) have shown potential in cancer therapy; however, the specific roles of HDACs in early embryos remain unclear. Moreover, although some pan-HDACi have been used to maintain cellular undifferentiated states in early embryos, the specific mechanisms underlying their effects remain unknown. Thus, there remains a significant knowledge gap regarding the application of selective HDACi in early embryos. RESULTS: To address this gap, we treated early embryos with two selective HDACi (MGCD0103 and T247). Subsequently, we collected and analyzed their transcriptome data at different developmental stages. Our findings unveiled a significant effect of HDACi treatment during the crucial 2-cell stage of zygotes, leading to a delay in embryonic development after T247 and an arrest at 2-cell stage after MGCD0103 administration. Furthermore, we elucidated the regulatory targets underlying this arrested embryonic development, which pinpointed the G2/M phase as the potential period of embryonic development arrest caused by MGCD0103. Moreover, our investigation provided a comprehensive profile of the biological processes that are affected by HDACi, with their main effects being predominantly localized in four aspects of zygotic gene activation (ZGA): RNA splicing, cell cycle regulation, autophagy, and transcription factor regulation. By exploring the transcriptional regulation and epigenetic features of the genes affected by HDACi, we made inferences regarding the potential main pathways via which HDACs affect gene expression in early embryos. Notably, Hdac7 exhibited a distinct response, highlighting its potential as a key player in early embryonic development. CONCLUSIONS: Our study conducted a comprehensive analysis of the effects of HDACi on early embryonic development at the transcriptional level. The results demonstrated that HDACi significantly affected ZGA in embryos, elucidated the distinct actions of various selective HDACi, and identified specific biological pathways and mechanisms via which these inhibitors modulated early embryonic development.

Zygotic gene activation in mice: profile and regulation.

The zygotic genome is transcriptionally silent immediately after fertilization. In mice, initial activation of the zygotic genome occurs in the middle of the one-cell stage. At the mid-to-late two-cell stage, a burst of gene activation occurs after the second round of DNA replication, and the profile of transcribed genes changes dramatically. These two phases of gene activation are called minor and major zygotic gene activation (ZGA), respectively. As they mark the beginning of the gene expression program, it is important to elucidate gene expression regulation during these stages. This article reviews the outcomes of studies that have clarified the profiles and regulatory mechanisms of ZGA.

Control of Maternal-to-Zygotic Transition in Human Embryos and Other Animal Species (Especially Mouse): Similarities and Differences.

Maternal-to-zygotic transition (MZT) of the control of early post-fertilization development is a key-event conditioning the fate of the future embryo, fetus and newborn. Because of the relative paucity of data concerning human embryos, due to ethical concerns and the poor availability of human embryos donated for research, most data have to be derived from animal models, among which those obtained using mouse embryos are most prevalent. However, data obtained by studies performed in non-mammalian specie can also provide useful information. For this reason, this review focuses on similarities and differences of MZT control mechanisms in humans and other species, with particular attention to the mouse. A number of molecular pathways controlling MZT in mice and humans are compared, pointing out those that could be at the origin of further focused experimental studies and the development of new diagnostic tools based on the translational medicine principles. Data concerning possible candidate molecules to be included in these studies are identified.

Minor zygotic gene activation is essential for mouse preimplantation development.

In mice, transcription initiates at the mid-one-cell stage and transcriptional activity dramatically increases during the two-cell stage, a process called zygotic gene activation (ZGA). Associated with ZGA is a marked change in the pattern of gene expression that occurs after the second round of DNA replication. To distinguish ZGA before and after the second-round DNA replication, the former and latter are called minor and major ZGA, respectively. Although major ZGA are required for development beyond the two-cell stage, the function of minor ZGA is not well understood. Transiently inhibiting minor ZGA with 5, 6-dichloro-1-ß-d-ribofuranosyl-benzimidazole (DRB) resulted in the majority of embryos arresting at the two-cell stage and retention of the H3K4me3 mark that normally decreases. After release from DRB, at which time major ZGA normally occurred, transcription initiated with characteristics of minor ZGA but not major ZGA, although degradation of maternal mRNA normally occurred. Thus, ZGA occurs sequentially starting with minor ZGA that is critical for the maternal-to-zygotic transition.

Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

Dynamics of two key maternal factors that initiate zygotic regulatory programs in ascidian embryos.

In animal embryos, transcription is repressed for a definite period of time after fertilization. In the embryo of the ascidian, Ciona intestinalis (type A; or Ciona robusta), transcription of regulatory genes is repressed before the 8- or 16-cell stages. This initial transcriptional quiescence is important to enable the establishment of initial differential gene expression patterns along the animal-vegetal axis by maternal factors, because the third cell division separates the animal and vegetal hemispheres into distinct blastomeres. Indeed, maternal transcription factors directly activate zygotic gene expression by the 16-cell stage; Tcf7/ß-catenin activates genes in the vegetal hemisphere, and Gata.a activates genes in the animal hemisphere. In the present study, we revealed the dynamics of Gata.a and ß-catenin, and expression profiles of their target genes precisely. ß-catenin began to translocate into the nuclei at the 16-cell stage, and thus expression of ß-catenin targets began at the 16-cell stage. Although Gata.a is abundantly present before the 8-cell stage, transcription of Gata.a targets was repressed at and before the 4-cell stage, and their expression began at the 8-cell stage. Transcription of the ß-catenin targets may be repressed by the same mechanism in early embryos, because ß-catenin targets were not expressed in 4-cell embryos treated with a GSK inhibitor, in which ß-catenin translocated to the nuclei. Thus, these two maternal factors have different dynamics, which establish the pre-pattern for zygotic genetic programs in 16-cell embryos.

CHD1 acts via the Hmgpi pathway to regulate mouse early embryogenesis.

The protein CHD1 is a member of the family of ATPase-dependent chromatin remodeling factors. CHD1, which recognizes trimethylated histone H3 lysine 4, has been implicated in transcriptional activation in organisms ranging from yeast to humans. It is required for pre-mRNA maturation, maintenance of mouse embryonic stem cell pluripotency and rapid growth of the mouse epiblast. However, the function(s) of CHD1 in mouse preimplantation embryos has not yet been examined. Here, we show that loss of CHD1 function led to embryonic lethality after implantation. In mouse embryos in which Chd1 was targeted by siRNA microinjection, the expression of the key regulators of cell fate specification Pou5f1 (also known as Oct4), Nanog and Cdx2 was dramatically decreased, starting at mid-preimplantation gene activation (MGA). Moreover, expression of Hmgpi and Klf5, which regulate Pou5f1, Nanog and Cdx2, was also significantly suppressed at zygotic gene activation (ZGA). Suppression of Hmgpi expression in Chd1-knockdown embryos continued until the blastocyst stage, whereas suppression of Klf5 expression was relieved by the morula stage. Next, we rescued HMGPI expression via Hmgpi mRNA microinjection in Chd1-knockdown embryos. Consequently, Pou5f1, Nanog and Cdx2 expression was restored at MGA and live offspring were recovered. These findings indicate that CHD1 plays important roles in mouse early embryogenesis via activation of Hmgpi at ZGA.

Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development.

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation.

Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

Epigenetic modifications and reprogramming in paternal pronucleus: sperm, preimplantation embryo, and beyond.

Pronuclear/zygotic stage is the very first stage of life. In this period, paternal pronucleus undergoes massive chromatin remodeling called "paternal reprogramming" including protamine-histone replacement and subsequent acquisition of epigenetic modifications. Although these consecutive events are required for the initiation of maternal-zygotic transition, the precise role of paternal reprogramming and its effect on subsequent embryonic development has been largely unknown to date. Recently, various new techniques, especially next-generation sequencing (NGS) and RNAi microinjection contribute to unveil the epigenetic transition from both paternal and maternal to early preimplantation embryos, suggesting not only the simple transcriptional regulation by transcription factors but also dynamic structural alteration of chromatin to initiate the wave of zygotic gene transcription. This review summarizes such recent progress for understanding the epigenetic transition in sperm and preimplantation embryos, and further argue about its transgenerational effect.

Regulation of zygotic gene activation by chromatin structure and epigenetic factors.

After fertilization, the genomes derived from an oocyte and spermatozoon are in a transcriptionally silent state before becoming activated at a species-specific time. In mice, the initiation of transcription occurs at the mid-one-cell stage, which represents the start of the gene expression program. A recent RNA sequencing analysis revealed that the gene expression pattern of one-cell embryos is unique and changes dramatically at the two-cell stage. However, the mechanism regulating this alteration has not yet been elucidated. It has been shown that chromatin structure and epigenetic factors change dynamically between the one- and two-cell stages. In this article, we review the characteristics of transcription, chromatin structure, and epigenetic factors in one- and two-cell mouse embryos and discuss the involvement of chromatin structure and epigenetic factors in the alteration of transcription that occurs between these stages.

Maternal SIN3A regulates reprogramming of gene expression during mouse preimplantation development.

The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A.

DUX4 expression in cancer induces a metastable early embryonic totipotent program.

The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identified cancer cell lines that express DUX4 and showed that DUX4 is transiently expressed in a small subset of the cells. DUX4 expression activates the DUX4-regulated ZGA transcriptional program, the subsequent 8C-like program, and markers of early embryonic lineages, while suppressing steady-state and interferon-induced MHC class I expression. Although DUX4 was expressed in a small number of cells under standard culture conditions, DNA damage or changes in growth conditions increased the fraction of cells expressing DUX4 and its downstream programs. Our demonstration that transient expression of endogenous DUX4 in cancer cells induces a metastable early embryonic stem cell program and suppresses antigen presentation has implications for cancer growth, progression, and immune evasion.

ELF4 is critical to zygotic gene activation and epigenetic reprogramming during early embryonic development in pigs.

Zygotic gene activation (ZGA) and epigenetic reprogramming are critical in early embryonic development in mammals, and transcription factors are involved in regulating these events. However, the effects of ELF4 on porcine embryonic development remain unclear. In this study, the expression of ELF4 was detected in early porcine embryos and different tissues. By knocking down ELF4, the changes of H3K9me3 modification, DNA methylation and ZGA-related genes were analyzed. Our results showed that ELF4 was expressed at all stages of early porcine embryos fertilized in vitro (IVF), with the highest expression level at the 8-cell stage. The embryonic developmental competency and blastocyst quality decreased after ELF4 knockdown (20.70% control vs. 17.49% si-scramble vs. 2.40% si-ELF4; p < 0.001). Knockdown of ELF4 induced DNA damage at the 4-cell stage. Interfering with ELF4 resulted in abnormal increases in H3K9me3 and DNA methylation levels at the 4-cell stage and inhibited the expression of genes related to ZGA. These results suggest that ELF4 affects ZGA and embryonic development competency in porcine embryos by maintaining genome integrity and regulating dynamic changes of H3K9me3 and DNA methylation, and correctly activating ZGA-related genes to promote epigenetic reprogramming. These results provide a theoretical basis for further studies on the regulatory mechanisms of ELF4 in porcine embryos.

nudt7 gene depletion causes transcriptomic change in early development of zebrafish.

The Nudt family has been identified as enzymes performing Coenzyme A to 3'5'-ADP + 4'-phospho pantetheine catalysis. The members of this family have been shown to be particularly involved in lipid metabolism, while their involvement in gene regulation through regulating transcription or mRNA metabolism has also been suggested. Here, we focused on peroxisomal NUDT7, possessing enzymatic activity similar to that of its paralog, peroxisomal NUDT19, which is involved in mRNA degradation. No reports have been published about the Nudt family in zebrafish. Our transcriptomic data showed that the Nudt family members are highly expressed around zygotic gene activation (ZGA) in developing zebrafish embryos. Therefore, we confirmed the computational prediction that the products of the nudt7 gene in zebrafish were localized in the peroxisome and highly expressed in early embryogenesis. The depletion of nudt7 genes by the CRISPR/Cas9 system did not affect development; however, it decreased the rate of transcription in ZGA. In addition, H3K27ac ChIP-seq analysis demonstrated that this decrease in transcription was correlated with the genome-wide decrease of H3K27ac level. This study suggests that peroxisomal Nudt7 functions in regulating transcription in ZGA via formation of the H3K27ac domain in active chromatin.

Comparative Embryogenesis in Angiosperms: Activation and Patterning of Embryonic Cell Lineages.

Following fertilization in flowering plants (angiosperms), egg and sperm cells unite to form the zygote, which generates an entire new organism through a process called embryogenesis. In this review, we provide a comparative perspective on early zygotic embryogenesis in flowering plants by using the Poaceae maize and rice as monocot grass and crop models as well as Arabidopsis as a eudicot model of the Brassicaceae family. Beginning with the activation of the egg cell, we summarize and discuss the process of maternal-to-zygotic transition in plants, also taking recent work on parthenogenesis and haploid induction into consideration. Aspects like imprinting, which is mainly associated with endosperm development and somatic embryogenesis, are not considered. Controversial findings about the timing of zygotic genome activation as well as maternal versus paternal contribution to zygote and early embryo development are highlighted. The establishment of zygotic polarity, asymmetric division, and apical and basal cell lineages represents another chapter in which we also examine and compare the role of major signaling pathways, cell fate genes, and hormones in early embryogenesis. Except for the model Arabidopsis, little is known about embryopatterning and the establishment of the basic body plan in angiosperms. Using available in situ hybridization, RNA-sequencing, and marker data, we try to compare how and when stem cell niches are established. Finally, evolutionary aspects of plant embryo development are discussed.

DPPA2, DPPA4, and other DPPA factor epigenomic functions in cell fate and cancer.

Many gene networks are shared between pluripotent stem cells and cancer; a concept exemplified by several DPPA factors such as DPPA2 and DPPA4, which are highly and selectively expressed in stem cells but also found to be reactivated in cancer. Despite their striking expression pattern, for many years the function of DPPA2 and DPPA4 remained a mystery; knockout of Dppa2 and Dppa4 did not affect pluripotency, but caused lung and skeletal defects late in development, long after Dppa2 and Dppa4 expression had been turned off. A number of recent papers have further clarified and defined the roles of these important factors, identifying roles in priming the chromatin and maintaining developmental competency through regulating both H3K4me3 and H3K27me3 at bivalent chromatin domains, and acting to remodel chromatin and facilitate reprogramming of somatic cells to induced pluripotency. These findings highlight an important regulatory role for DPPA2 and DPPA4 at the transitional boundary between pluripotency and differentiation and may have relevance to the functions of DPPA2 and 4 in the context of cancer cells as well.

How to improve mouse cloning.

The mouse is the most extensively used mammalian laboratory species in biology and medicine because of the ready availability of a wide variety of defined genetic and gene-modified strains and abundant genetic information. Its small size and rapid generation turnover are also advantages compared with other experimental animals. Using these advantages, somatic cell nuclear transfer (SCNT) in mice has provided invaluable information on epigenetics related to SCNT technology and cloning, playing a leading role in relevant technical improvements. These improvements include treatment with histone deacetylase inhibitors, correction of Xist gene expression (controlling X chromosome inactivation), and removal of methylated histones from SCNT-generated embryos, which have proven to be effective for SCNT cloning of other species. However, even with the best combination of these treatments, the birth rate in cloned offspring is still lower than intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). One remaining issue associated with SCNT is placental enlargement (hyperplasia) found in late pregnancy, but this abnormality might not be a major cause for the low efficiency of SCNT because many SCNT-derived embryos die before their placentas start to enlarge at midgestation (early postimplantation stage). It is known that, at this stage, undifferentiated trophoblast cells in the extraembryonic tissue of SCNT-derived embryos fail to proliferate. Understanding the molecular mechanisms is essential for further technical improvements of mouse SCNT, which might also provide clues for technical breakthroughs in mammalian SCNT and cloning in general.

Dynamic Alternative Splicing During Mouse Preimplantation Embryo Development.

The mechanism of alternative pre-mRNA splicing (AS) during preimplantation development is largely unknown. In order to capture the dynamic changes of AS occurring during embryogenesis, we carried out bioinformatics analysis based on scRNA-seq data over the time-course preimplantation development in mouse. We detected numerous previously-unreported differentially expressed genes at specific developmental stages and investigated the nature of AS at both minor and major zygotic genome activation (ZGA). The AS and differential AS atlas over preimplantation development were established. The differentially alternatively spliced genes (DASGs) are likely to be key splicing factors (SFs) during preimplantation development. We also demonstrated that there is a regulatory cascade of AS events in which some key SFs are regulated by differentially AS of their own gene transcripts. Moreover, 212 isoform switches (ISs) during preimplantation development were detected, which may be critical for decoding the mechanism of early embryogenesis. Importantly, we uncovered that zygotic AS activation (ZASA) is in conformity with ZGA and revealed that AS is coupled with transcription during preimplantation development. Our results may provide a deeper insight into the regulation of early embryogenesis.