The lysine deacetylase activity of histone deacetylases 1 and 2 is required to safeguard zygotic genome activation in mice and cattle.

The genome is transcriptionally inert at fertilization and must be activated through a remarkable developmental process called zygotic genome activation (ZGA). Epigenetic reprogramming contributes significantly to the dynamic gene expression during ZGA; however, the mechanism has yet to be resolved. Here, we find histone deacetylases 1 and 2 (HDAC1/2) can regulate ZGA through lysine deacetylase activity. Notably, in mouse embryos, overexpression of a HDAC1/2 dominant-negative mutant leads to developmental arrest at the two-cell stage. RNA-seq reveals that 64% of downregulated genes are ZGA genes and 49% of upregulated genes are developmental genes. Inhibition of the deacetylase activity of HDAC1/2 causes a failure of histone deacetylation at multiple sites, including H4K5, H4K16, H3K14, H3K18 and H3K27. ChIP-seq analysis exhibits an increase and decrease of H3K27ac enrichment at promoters of up- and downregulated genes, respectively. Moreover, HDAC1 mutants prohibit the removal of H3K4me3 by impeding expression of Kdm5 genes. Importantly, the developmental block can be greatly rescued by Kdm5b injection and by partially correcting the expression of the majority of dysregulated genes. Similar functional significance of HDAC1/2 is conserved in bovine embryos. Overall, we propose that HDAC1/2 are indispensable for ZGA by creating correct transcriptional repressive and active states in mouse and bovine embryos.

Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency.

The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice.

KDM5-mediated redistribution of H3K4me3 is required for oocyte-to-embryonic transition in cattle†.

Reprogramming of histone modifications is critical to safeguard correct gene expression profile during preimplantation development. Of interest, trimethylation of lysine 4 on histone 3 (H3K4me3) exhibits a unique and dynamic landscape with a potential species-specific feature. Here, we address how it is reprogrammed and its functional significance during oocyte maturation and early embryonic development in cows. Notably, the overall signal of H3K4me3 decreased sharply during embryonic genome activation (EGA). By using low input ChIP-seq, we find widespread broad H3K4me3 domains in oocytes and early cleaved embryos. The broad domains are gradually removed after fertilization, which is obviously seen during EGA. Meanwhile, H3K4me3 becomes enriched at promoter regions after the removal of broad H3K4me3. Interestingly, the gene expression level displays a positive correlation with the relative H3K4me3 signal of their promoters when embryos reach 16-cell stage. Importantly, disruption of KDM5 (H3K4me3 demethylases) increases H3K4me3 level, decreases the embryonic developmental rate, and results in dysregulation of over a thousand genes. Meanwhile, KDM5 deficiency causes a redistribution of H3K4me3 across genome. In particular, H3K4me3 in gene body or intergenic regions cannot be removed, and H3K4me3 in promoter regions is aberrantly reduced. Besides, the positive correlation between promoter H3K4me3 enrichment and gene expression level disappears. Overall, we describe the genomic reprogramming of H3K4me3 with a greater resolution during bovine preimplantation development and propose that KDM5-mediated redistribution of H3K4me3 plays an important role in modulating oocyte-to-embryonic transition.

Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and chromatin structure†.

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during preimplantation embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and preimplantation embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in preimplantation embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to a compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in the expression of genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine preimplantation development.

NOTCH signaling pathway is required for bovine early embryonic development†.

The NOTCH signaling pathway plays an important role in regulating various biological processes, including lineage specification and apoptosis. Multiple components of the NOTCH pathway have been identified in mammalian preimplantation embryos. However, the precise role of the NOTCH pathway in early embryonic development is poorly understood, especially in large animals. Here, we show that the expression of genes encoding key transcripts of the NOTCH pathway is dynamic throughout early embryonic development. We also confirm the presence of active NOTCH1 and RBPJ. By using pharmacological and RNA interference tools, we demonstrate that the NOTCH pathway is required for the proper development of bovine early embryos. This functional consequence could be partly attributed to the major transcriptional mediator, Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ), whose deficiency also compromised the embryo quality. Indeed, both NOTCH1 and RBPJ knockdown cause a significant increase of histone H3 serine 10 phosphorylation (pH3S10, a mitosis marker) positive blastomeres, suggesting a cell cycle arrest at mitosis. Importantly, RNA sequencing analyses reveal that either NOTCH1 or RBPJ depletion triggers a reduction in H1FOO that encodes the oocyte-specific linker histone H1 variant. Interestingly, depleting H1FOO results in detrimental effects on the developmental competence of early embryos, similar with NOTCH1 inhibition. Overall, our results reveal a crucial role for NOTCH pathway in regulating bovine preimplantation development, likely by controlling cell proliferation and maintaining H1FOO expression.

Functional roles of the chromatin remodeler SMARCA5 in mouse and bovine preimplantation embryos†.

Upon fertilization, extensive chromatin reprogramming occurs during preimplantation development. Growing evidence reveals species-dependent regulations of this process in mammals. ATP-dependent chromatin remodeling factor SMARCA5 (also known as SNF2H) is required for peri-implantation development in mice. However, the specific functional role of SMARCA5 in preimplantation development and if it is conserved among species remain unclear. Herein, comparative analysis of public RNA-seq datasets reveals that SMARCA5 is universally expressed during oocyte maturation and preimplantation development in mice, cattle, humans, and pigs with species-specific patterns. Immunostaining analysis further describes the temporal and spatial changes of SMARCA5 in both mouse and bovine models. siRNA-mediated SMARCA5 depletion reduces the developmental capability and compromises the specification and differentiation of inner cell mass in mouse preimplantation embryos. Indeed, OCT4 is not restricted into the inner cell mass and the formation of epiblast and primitive endoderm disturbed with reduced NANOG and SOX17 in SMARCA5-deficient blastocysts. RNA-seq analysis shows SMARCA5 depletion causes limited effects on the transcriptomics at the morula stage, however, dysregulates 402 genes, including genes involved in transcription regulation and cell proliferation at the blastocyst stage in mice. By comparison, SMARCA5 depletion does not affect the development through the blastocyst stage but significantly compromises the blastocyst quality in cattle. Primitive endoderm formation is greatly disrupted with reduced GATA6 in bovine blastocysts. Overall, our studies demonstrate the importance of SMARCA5 in fostering the preimplantation development in mice and cattle while there are species-specific effects.

The nucleolar protein NOP2 is required for nucleolar maturation and ribosome biogenesis during preimplantation development in mammals.

The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by Transmission Electron Microscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and Fluorescence In Situ Hybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis.

Sin3a regulates the developmental progression through morula-to-blastocyst transition via Hdac1.

Suppressor interacting 3a (Sin3a) is a scaffold component of the chromatin repressive complex Sin3/histone deacetylase (Hdac). Sin3a has been shown as a hub gene driving preimplantation development in both mice and humans. However, its precise functions during preimplantation development remain unclear. Here, we show that the embryos arrested at morula stage upon specific depletion of Sin3a in mouse early embryos. Given the reduced cell number in Sin3a-depleted embryos, blocked cell proliferation is observed, likely because of the increased level of Trp53 acetylation at lysine 379. Moreover, we found that Sin3a depletion reduces Cdx2 and Tir Na Nog (Nanog), suggesting a failure of the first cell fate decision. In addition, we noted a striking increase of genome-wide DNA methylation, likely attributed to the increased nuclear DNA methyltransferase 1 observed in Sin3a-depleted embryos. Notably, RNA sequencing analyses showed 717 genes are differentially expressed, and Gene Ontology analysis of down-regulated genes (e.g., Hdac1) revealed top enriched terms involving protein deacetylation. Consistently, we confirmed a significant decrease of Hdac1 mRNA and protein abundance. Importantly, the development and Trp53 acetylation in Sin3a-depleted embryos could be rescued by expression of Hdac1 but not Hdac2. In summary, our results indicate a vital role of Sin3a in safeguarding the developmental progression through the morula-to-blastocyst transition via Hdac1.-Zhao, P., Li, S., Wang, H., Dang, Y., Wang, L., Liu, T., Wang, S., Li, X., Zhang, K. Sin3a regulates the developmental progression through morula-to-blastocyst transition via Hdac1.

Overlapping functions of RBBP4 and RBBP7 in regulating cell proliferation and histone H3.3 deposition during mouse preimplantation development.

Preimplantation development is critical for reproductive successes in mammals. Thus, it is important to understand how preimplantation embryogenesis is regulated. As a key event of preimplantation development, epigenetic reprogramming has been widely studied, yet how epigenetic complexes regulate preimplantation development remains largely unknown. Retinoblastoma binding protein 4 (RBBP4) and 7 (RBBP7) are integral components of epigenetic complexes including SIN3A, NuRD, and CoREST. Here, we demonstrate that double knockdown of Rbbp4 and 7, but not individually, causes embryonic lethality during the morula-to-blastocyst transition. Mechanistically, depletion of RBBP4 and 7 results in dysregulation of genes related to cell cycle, lineage development, and regulation of transcription, which is accompanied by cell cycle block, disrupted lineage specification and chromatin structure. Interestingly, RBBP4/7 depletion leads to a dramatic increase in H3.3 and H3K27ac abundance during morula-to-blastocyst transition. ChIP-seq analysis in early embryos and embryonic stem cells reveals enrichment of H3.3 at the promoter regions of RBBP4/7 target genes. In summary, our studies demonstrate the compensatory role of RBBP4/7 and reveal its potential mechanisms in preimplantation development.Summary sentence:RBBP4 and RBBP7 play a compensatory role in regulating cell proliferation, apoptosis, and histone H3.3 deposition during preimplantation development.

SIN3A Regulates Porcine Early Embryonic Development by Modulating CCNB1 Expression.

SIN3A is the central scaffold protein of the SIN3/histone deacetylase (HDAC) transcriptional repressor complex. SIN3A participates in the mouse preimplantation development by fine-tuning HDAC1 expression. However, it remains unresolved if this functional significance of SIN3A was conserved in other mammals. Herein, RNA-seq results show a large amount of SIN3A mRNA is present in oocytes and early embryos prior to embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in pigs, cattle, mice, and humans. Interestingly, immunofluorescence data show that SIN3A protein level peaks at four-cell stage in pigs compared with morula stage in cattle. SIN3A depletion in early embryos causes a developmental arrest at two-cell stage in pigs but does not affect bovine early embryonic development. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause two-cell block but leads to a reduced blastocyst rate. By using unbiased RNA-seq approach, we found that Cyclin B1 (CCNB1) transcript level is dramatically reduced. Moreover, CCNB1 knockdown results in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development to pass two-cell stage. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, which probably involves the regulation of CCNB1 expression.

Transcriptomic Prediction of Pig Liver-Enriched Gene 1 Functions in a Liver Cell Line.

The newly identified liver-enriched gene 1 (LEG1) encodes a protein with a characteristic domain of unknown function 781 (DUF781/LEG1), constituting a protein family with only one member in mammals. A functional study in zebrafish suggested that LEG1 genes are involved in liver development, while the platypus LEG1 homolog, Monotreme Lactation Protein (MLP), which is enriched in the mammary gland and milk, acts as an antibacterial substance. However, no functional studies on eutherian LEG1s have been published to date. Thus, we here report the first functional prediction study at the cellular level. As previously reported, eutherian LEG1s can be classified into three paralogous groups. Pigs have all three LEG1 genes (pLEG1s), while humans and mice have retained only LEG1a. Hence, pLEG1s might represent an ideal model for studying LEG1 gene functions. RNA-seq was performed by the overexpression of pLEG1s and platypus MLP in HepG2 cells. Enrichment analysis showed that pLEG1a and pLEG1b might exhibit little function in liver cells; however, pLEG1c is probably involved in the endoplasmic reticulum (ER) stress response and protein folding. Additionally, gene set enrichment analysis revealed that platypus MLP shows antibacterial activity, confirming the functional study in platypus. Therefore, our study showed from the transcriptomic perspective that mammalian LEG1s have different functions in liver cells due to the subfunctionalization of paralogous genes.

Evolutionary and Molecular Characterization of liver-enriched gene 1.

Liver-enriched gene 1 (Leg1) is a newly identified gene with little available functional information. To evolutionarily and molecularly characterize Leg1 genes, a phylogenetic study was first conducted, which indicated that Leg1 is a conserved gene that exists from bacteria to mammals. During the evolution of mammals, Leg1s underwent tandem duplications, which gave rise to Leg1a, Leg1b, and Leg1c clades. Analysis of the pig genome showed the presence of all three paralogs of pig Leg1 genes (pLeg1s), whereas only Leg1a could be found in the human (hLeg1a) or mouse (mLeg1a) genomes. Purifying force acts on the evolution of Leg1 genes, likely subjecting them to functional constraint. Molecularly, pLeg1a and its coded protein, pig LEG1a (pLEG1a), displayed high similarities to its human and mouse homologs in terms of gene organization, expression patterns, and structures. Hence, pLeg1a, hLeg1a, and mLeg1a might preserve similar functions. Additionally, expression analysis of the three Leg1as suggested that eutherian Leg1as might have different functions from those of zebrafish and platypus due to subfunctionalization. Therefore, pLeg1a might provide essential information about eutherian Leg1a. Moreover, a preliminary functional study using RNA-seq suggested that pLeg1a is involved in the lipid homeostasis. In conclusion, our study provides some basic information on the aspects of evolution and molecular function, which could be applied for further validation of Leg1 using pig models.

Identification of ADPKD-Related Genes and Pathways in Cells Overexpressing PKD2.

Consistent with the gene dosage effect hypothesis, renal cysts can arise in transgenic murine models overexpressing either PKD1 or PKD2, which are causal genes for autosomal dominant polycystic kidney disease (ADPKD). To determine whether PKD gene overexpression is a universal mechanism driving cystogenesis or is merely restricted to rodents, other animal models are required. Previously, we failed to observe any renal cysts in a transgenic porcine model of PKD2 overexpression partially due to epigenetic silencing of the transgene. Thus, to explore the feasibility of porcine models and identify potential genes/pathways affected in ADPKD, LLC-PK1 cells with high PKD2 expression were generated. mRNA sequencing (RNA-seq) was performed, and MYC, IER3, and ADM were found to be upregulated genes common to the different PKD2 overexpression cell models. MYC is a well-characterized factor contributing to cystogenesis, and ADM is a biomarker for chronic kidney disease. Thus, these genes might be indicators of disease progression. Additionally, some ADPKD-associated pathways, e.g., the mitogen-activated protein kinase (MAPK) pathway, were enriched in the cells. Moreover, gene ontology (GO) analysis demonstrated that proliferation, apoptosis, and cell cycle regulation, which are hallmarks of ADPKD, were altered. Therefore, our experiment identified some biomarkers or indicators of ADPKD, indicating that high PKD2 expression would likely drive cystogenesis in future porcine models.

Essential roles of HDAC1 and 2 in lineage development and genome-wide DNA methylation during mouse preimplantation development.

Epigenetic modifications, including DNA methylation and histone modifications, are reprogrammed considerably following fertilization during mammalian early embryonic development. Incomplete epigenetic reprogramming is a major factor leading to poor developmental outcome in embryos generated by assisted reproductive technologies, such as somatic cell nuclear transfer. However, the role of histone modifications in preimplantation development is poorly understood. Here, we show that co-knockdown (cKD) of Hdac1 and 2 (but not individually) resulted in developmental failure during the morula to blastocyst transition. This outcome was also confirmed with the use of small-molecule HDAC1/2-specific inhibitor FK228. We observed reduced cell proliferation and increased incidence of apoptosis in cKD embryos, which were likely caused by increased acetylation of TRP53. Importantly, both RNA-seq and immunostaining analysis revealed a failure of lineage specification to generate trophectoderm and pluripotent cells. Among many gene expression changes, a substantial decrease of Cdx2 may be partly accounted for by the aberrant Hippo pathway occurring in cKD embryos. In addition, we observed an increase in global DNA methylation, consistent with increased DNA methyltransferases and UHRF1. Interestingly, deficiency of RBBP4 and 7 (both are core components of several HDAC1/2-containing epigenetic complexes) results in similar phenotypes as those of cKD embryos. Overall, HDAC1 and 2 play redundant functions required for lineage specification, cell viability and accurate global DNA methylation, each contributing to critical developmental programmes safeguarding a successful preimplantation development.

Aminopeptidase N-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus.

Swine enteric diseases have caused significant economic loss and have been considered as the major threat to the global swine industry. Several coronaviruses, including transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV), have been identified as the causative agents of these diseases. Effective measures to control these diseases are lacking. The major host cells of transmissible gastroenteritis virus and porcine epidemic diarrhea virus have thought to be epithelial cells on small intestine villi. Aminopeptidase-N (APN) has been described as the putative receptor for entry of transmissible gastroenteritis virus and porcine epidemic diarrhea virus into cells in vitro. Recently, Whitworth et al. have reported that APN knockout pigs are resistant to TGEV but not PEDV after weaning. However, it remains unclear if APN-null neonatal pigs are protected from TGEV. Here we report the generation of APN-null pigs by using CRISPR/Cas9 technology followed by somatic cell nuclear transfer. APN-null pigs are produced with normal pregnancy rate and viability, indicating lack of APN is not embryonic lethal. After viral challenge, APN-null neonatal piglets are resistant to highly virulent transmissible gastroenteritis virus. Histopathological analyses indicate APN-null pigs exhibit normal small intestine villi, while wildtype pigs show typical lesions in small intestines. Immunochemistry analyses confirm that no transmissible gastroenteritis virus antigen is detected in target tissues in APN-null piglets. However, upon porcine epidemic diarrhea virus challenge, APN-null pigs are still susceptible with 100% mortality. Collectively, this report provides a viable tool for producing animals with enhanced resistance to TGEV and clarifies that APN is dispensable for the PEDV infection in pigs.