Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Susceptibilities of Human ACE2 Genetic Variants in Coronavirus Infection.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 235 million cases worldwide and 4.8 million deaths (October 2021), with various incidences and mortalities among regions/ethnicities. The coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize the angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2. We then applied an integrated approach of genetics, biochemistry, and virology to explore the capacity of select ACE2 variants to bind coronavirus spike proteins and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity. IMPORTANCE There is considerable variation in disease severity among patients infected with SARS-CoV-2, the virus that causes COVID-19. Human genetic variation can affect disease outcome, and the coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize human ACE2 as the receptor to enter cells. We found that several missense ACE2 single-nucleotide variants (SNVs) that showed significantly altered binding with the spike proteins of SARS-CoV, SARS-CoV-2, and NL63-HCoV. We identified an ACE2 SNP, D355N, that restricts the spike protein-ACE2 interaction and consequently has the potential to protect individuals against SARS-CoV-2 infection. Our study highlights that ACE2 polymorphisms could impact human susceptibility to SARS-CoV-2, which may contribute to ethnic and geographical differences in SARS-CoV-2 spread and pathogenicity.

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.

A role for host cell exocytosis in InlB-mediated internalisation of Listeria monocytogenes.

The bacterial surface protein InlB mediates internalisation of Listeria monocytogenes into human cells through interaction with the host receptor tyrosine kinase, Met. InlB-mediated entry requires localised polymerisation of the host actin cytoskeleton. Apart from actin polymerisation, roles for other host processes in Listeria entry are unknown. Here, we demonstrate that exocytosis in the human cell promotes InlB-dependent internalisation. Using a probe consisting of VAMP3 with an exofacial green fluorescent protein tag, focal exocytosis was detected during InlB-mediated entry. Exocytosis was dependent on Met tyrosine kinase activity and the GTPase RalA. Depletion of SNARE proteins by small interfering RNA demonstrated an important role for exocytosis in Listeria internalisation. Depletion of SNARE proteins failed to affect actin filaments during internalisation, suggesting that actin polymerisation and exocytosis are separable host responses. SNARE proteins were required for delivery of the human GTPase Dynamin 2, which promotes InlB-mediated entry. Our results identify exocytosis as a novel host process exploited by Listeria for infection.

The HLA-II immunopeptidome of SARS-CoV-2.

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks, yet the design of these vaccines requires a comprehensive knowledge of viral immunogens. Here, we report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides that are naturally processed and loaded onto human leukocyte antigen-II (HLA-II) complexes in infected cells. We identify over 500 unique viral peptides from canonical proteins as well as from overlapping internal open reading frames. Most HLA-II peptides colocalize with known CD4+ T cell epitopes in coronavirus disease 2019 patients, including 2 reported immunodominant regions in the SARS-CoV-2 membrane protein. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and nonstructural and noncanonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize vaccine effectiveness.

SARS-CoV-2 nonstructural protein 6 from Alpha to Omicron: evolution of a transmembrane protein.

We recently reported that mutations in both the spike glycoprotein and nonstructural protein 6 (nsp6) were associated with attenuation of the SARS-CoV-2 Omicron BA.1 variant. While mutations in spike allow evasion of neutralizing antibodies and promote specific modes of viral entry, the role of nsp6 mutations in pathogenesis is less clear. Nsp6 is essential for modifying the endoplasmic reticulum and generating double-membrane vesicles, the site of viral RNA replication. To investigate the evolution of nsp6, we evaluated 91,596 high-confidence human SARS-CoV-2 whole-genome sequences across 19 variants and lineages. While nsp6 of early variants of concern, such as Alpha, Beta, and Gamma, carried a triple amino acid deletion (106-108, termed ΔSGF), the Delta, Epsilon, and Mu lineages retained the ancestral nsp6 sequence. For nsp6 in the emerging Omicron variants, we report a transition from an amino acid 105-107 ΔLSG deletion in BA.1 to increased dominance of the ΔSGF in BA.2 and subsequent lineages. Our findings indicate that deletion within nsp6 was independently selected in multiple lineages of SARS-CoV-2, both early and late in the pandemic. Analysis of SARS-CoV-2-related coronaviruses in bats and pangolins revealed nsp6 sequences similar to the ancestral SARS-CoV-2 virus, indicating that the deletion in nsp6 may be an adaptation to replication in humans. Analysis of nsp6 sequences from multiple coronaviruses predicts a multipass transmembrane protein with a conserved C-terminal domain. Monitoring and evaluating changes in nsp6 and other nonstructural proteins will contribute to our understanding of factors associated with the attenuation of pandemic coronaviruses. IMPORTANCE There is an ongoing need to evaluate genetic changes in SARS-CoV-2 for effects on transmission and pathogenesis. We recently reported an unexpected role for replicase component nsp6, in addition to changes in spike, in the attenuation of Omicron BA.1. In this commentary, we document a triple-amino-acid deletion in a predicted lumenal domain of nsp6 that was found in multiple, independent variants of SARS-CoV-2, including all recent Omicron lineages. Furthermore, we modeled the predicted structure of nsp6, implicating a multipass transmembrane architecture as conserved in members of the Coronaviridae family. This information can guide future studies investigating the role of nsp6 in the pathogenesis of existing and emerging coronaviruses.

Plant flavonoid inhibition of SARS-CoV-2 main protease and viral replication.

Plant-based flavonoids have been evaluated as inhibitors of ß-coronavirus replication and as therapies for COVID-19 on the basis of their safety profile and widespread availability. The SARS-CoV-2 main protease (Mpro) has been implicated as a target for flavonoids in silico. Yet no comprehensive in vitro testing of flavonoid activity against SARS-CoV-2 Mpro has heretofore been performed. We screened 1,019 diverse flavonoids for their ability to inhibit SARS-CoV-2 Mpro. Multiple structure-activity relationships were identified among active compounds such as enrichment of galloylated flavonoids and biflavones, including multiple biflavone analogs of apigenin. In a cell-based SARS-CoV-2 replication assay, the most potent inhibitors were apigenin and the galloylated pinocembrin analog, pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). Molecular dynamic simulations predicted that PGHG occludes the S1 binding site via a galloyl group and induces a conformational change in Mpro. These studies will advance the development of plant-based flavonoids-including widely available natural products-to target ß-coronaviruses.

The HLA-II immunopeptidome of SARS-CoV-2.

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks; yet the design of these vaccines requires a comprehensive knowledge of viral immunogens, including T-cell epitopes. Having previously mapped the SARS-CoV-2 HLA-I landscape, here we report viral peptides that are naturally processed and loaded onto HLA-II complexes in infected cells. We identified over 500 unique viral peptides from canonical proteins, as well as from overlapping internal open reading frames (ORFs), revealing, for the first time, the contribution of internal ORFs to the HLA-II peptide repertoire. Most HLA-II peptides co-localized with the known CD4+ T cell epitopes in COVID-19 patients. We also observed that two reported immunodominant regions in the SARS-CoV-2 membrane protein are formed at the level of HLA-II presentation. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and non-structural and non-canonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize the vaccine effectiveness.

High-resolution photocatalytic mapping of SARS-CoV-2 spike interactions on the cell surface.

Identifying virus-host interactions on the cell surface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the causative agent of the COVID-19 disease, uses ACE2 as a receptor to enter cells. Yet the full repertoire of cell surface proteins that contribute to viral entry is unknown. We developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) to probe the molecular neighborhood of the SARS-CoV-2 spike protein on the human cell surface. Application of ViraMap to ACE2-expressing cells captured ACE2, the established co-receptor NRP1, and several novel cell surface proteins. We systematically analyzed the relevance of these candidate proteins to SARS-CoV-2 entry by knockdown and overexpression approaches in pseudovirus and authentic infection models and identified PTGFRN and EFNB1 as bona fide viral entry factors. Our results highlight additional host targets that participate in SARS-CoV-2 infection and showcase ViraMap as a powerful platform for defining viral interactions on the cell surface.

Cell culture systems for isolation of SARS-CoV-2 clinical isolates and generation of recombinant virus.

A simple and robust cell culture system is essential for generating authentic SARS-CoV-2 stocks for evaluation of viral pathogenicity, screening of antiviral compounds, and preparation of inactivated vaccines. Evidence suggests that Vero E6, a cell line commonly used in the field to grow SARS-CoV-2, does not support efficient propagation of new viral variants and triggers rapid cell culture adaptation of the virus. We generated a panel of 17 human cell lines overexpressing SARS-CoV-2 entry factors and tested their ability to support viral infection. Two cell lines, Caco-2/AT and HuH-6/AT, demonstrated exceptional susceptibility, yielding highly concentrated virus stocks. Notably, these cell lines were more sensitive than Vero E6 cells in recovering SARS-CoV-2 from clinical specimens. Further, Caco-2/AT cells provided a robust platform for producing genetically reliable recombinant SARS-CoV-2 through a reverse genetics system. These cellular models are a valuable tool for the study of SARS-CoV-2 and its continuously emerging variants.

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron.

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

Production of cloned transgenic cow expressing omega-3 fatty acids.

n-3 Polyunsaturated fatty acids (n-3 PUFA) are important for human health. Alternative resources of n-3 PUAFs created by transgenic domestic animals would be an economic approach. In this study, we generated a mfat-1 transgenic cattle expressed a Caenorhabditis elegans gene, mfat-1, encoding an n-3 fatty acid desaturase. Fatty acids analysis of tissue and milk showed that all of the examined n-3 PUAFs were greatly increased and simultaneously the n-6 PUAFs decreased in the transgenic cow. A significantly reduction of n-6/n-3 ratios (P<0.05) in both tissue and milk were observed.

HLA-I immunopeptidome profiling of human cells infected with high-containment enveloped viruses.

Immunopeptidome profiling of infected cells is a powerful technique for detecting viral peptides that are naturally processed and loaded onto class I human leukocyte antigens (HLAs-I). Here, we provide a protocol for preparing samples for immunopeptidome profiling that can inactivate enveloped viruses while still preserving the integrity of the HLA-I complex. We detail steps for lysate preparation of infected cells followed by HLA-I immunoprecipitation and virus inactivation. We further describe peptide purification for mass spectrometry outside a high-containment facility. For complete details on the use and execution of this protocol, please refer to Weingarten-Gabbay et al. (2021).1.

Optimized ACE2 decoys neutralize antibody-resistant SARS-CoV-2 variants through functional receptor mimicry and treat infection in vivo.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolves to escape natural antibodies, it also loses sensitivity to therapeutic antibody drugs. By contrast, evolution selects for binding to ACE2, the cell-surface receptor required for SARS-CoV-2 infection. Consistent with this, we find that an ACE2 decoy neutralizes antibody-resistant variants, including Omicron, with no loss in potency. To identify design features necessary for in vivo activity, we compare several enzymatically inactive, Fc effector-silenced ACE2-Fc decoys. Inclusion of the ACE2 collectrin-like domain not only improves affinity for the S protein but also unexpectedly extends serum half-life and is necessary to reduce disease severity and viral titer in Syrian hamsters. Fc effector function is not required. The activity of ACE2 decoy receptors is due, in part, to their ability to trigger an irreversible structural change in the viral S protein. Our studies provide a new understanding of how ACE2 decoys function and support their development as therapeutics to treat ACE2-dependent coronaviruses.

Detecting SARS-CoV-2 3CLpro expression and activity using a polyclonal antiserum and a luciferase-based biosensor.

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.

The in-vitro effect of famotidine on sars-cov-2 proteases and virus replication.

The lack of coronavirus-specific antiviral drugs has instigated multiple drug repurposing studies to redirect previously approved medicines for the treatment of SARS-CoV-2, the coronavirus behind the ongoing COVID-19 pandemic. A recent, large-scale, retrospective clinical study showed that famotidine, when administered at a high dose to hospitalized COVID-19 patients, reduced the rates of intubation and mortality. A separate, patient-reported study associated famotidine use with improvements in mild to moderate symptoms such as cough and shortness of breath. While a prospective, multi-center clinical study is ongoing, two parallel in silico studies have proposed one of the two SARS-CoV-2 proteases, 3CLpro or PLpro, as potential molecular targets of famotidine activity; however, this remains to be experimentally validated. In this report, we systematically analyzed the effect of famotidine on viral proteases and virus replication. Leveraging a series of biophysical and enzymatic assays, we show that famotidine neither binds with nor inhibits the functions of 3CLpro and PLpro. Similarly, no direct antiviral activity of famotidine was observed at concentrations of up to 200 µM, when tested against SARS-CoV-2 in two different cell lines, including a human cell line originating from lungs, a primary target of COVID-19. These results rule out famotidine as a direct-acting inhibitor of SARS-CoV-2 replication and warrant further investigation of its molecular mechanism of action in the context of COVID-19.

The NBS1 genetic polymorphisms and the risk of the systemic lupus erythematosus in Taiwanese patients.

INTRODUCTION: Systemic lupus erythematosus (SLE), a multisystemic autoimmune disease, is characterized by the production of a range of autoantibodies against nuclear constituents and other self-antigens. The studies in DNA repair deficiencies in SLE patients have been recently investigated. AIMS: Few studies have been conducted on DNA repair gene polymorphisms and their role in autoimmune diseases. Our study purpose was to examine and compare NBS1 genotype distributions in a group of Taiwanese SLE patients and controls in Taiwan. PATIENTS AND METHODS: Participants were Taiwanese SLE patients and healthy controls. We studied associations among NBS1 polymorphisms--rs1061302, rs709816, and rs1805794--considering clinical features for the entire group and stratified subgroups. No statistically significant differences between the patients and controls were noted. However, we observed significant decreases in Ht1-GGG, Ht2-AAC, and Ht3-AGC in the SLE patients (Ht1-GGG, OR = 0.26, 95% CI: 0.16-0.41; Ht2-AAC, OR = 0.30, 95% CI: 0.17-0.53; Ht3-AGC, OR = 0.35, 95% CI: 0.19-0.71) and significant increases in Ht4-AAG, Ht5-AGG, and Ht8-GGC among the SLE patients. Combined, these results suggest an association between NBS1 genetic polymorphisms and Taiwanese SLE patients.

The histone demethylase JMJD2C is stage-specifically expressed in preimplantation mouse embryos and is required for embryonic development.

Epigenetic modifications play a pivotal role in embryonic development by dynamically regulating DNA methylation and chromatin modifications. Although recent studies have shown that core histone methylation is reversible, very few studies have investigated the functions of the newly discovered histone demethylases during embryonic development. In the present study, we investigated the expression characteristics and function of JMJD2C, a histone demethylase that belongs to the JmjC-domain-containing histone demethylases, during preimplantation embryonic development of the mouse. We found that JMJD2C is stage-specifically expressed during preimplantation development, with the highest activity being observed from the two-cell to the eight-cell stage. Depletion of JMJD2C in metaphase II oocytes followed by parthenogenetic activation causes a developmental arrest before the blastocyst stage. Moreover, consistent with a previous finding in embryonic stem (ES) cells, depletion of JMJD2C causes a significant down-regulation of the pluripotency gene Nanog in embryos. However, contrary to a previous report in ES cells, we observed that other pluripotency genes, Pou5f1 and Sox2, are also significantly down-regulated in JMJD2C-depleted embryos. Furthermore, the depletion of JMJD2C in early embryos also caused significant down-regulation of the Myc and Klf4 genes, which are associated with cell proliferation. Our data suggest that the deregulation of these critical genes synergistically causes the developmental defects observed in JMJD2C-depleted embryos.