Obox4 promotes zygotic genome activation upon loss of Dux.

Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux-knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here, we present multiple lines of evidence that the multicopy homeobox gene, Obox4, encodes a transcription factor that is highly expressed in mouse two-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4/Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to preimplantation development.

Transcription of MERVL retrotransposons is required for preimplantation embryo development.

Zygotic genome activation (ZGA) is a critical postfertilization step that promotes totipotency and allows different cell fates to emerge in the developing embryo. MERVL (murine endogenous retrovirus-L) is transiently upregulated at the two-cell stage during ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we show that full-length MERVL transcripts, but not encoded retroviral proteins, are essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. Both knockdown and CRISPRi-based repression of MERVL result in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that loss of MERVL transcripts led to retention of an accessible chromatin state at, and aberrant expression of, a subset of two-cell-specific genes. Taken together, our results suggest a model in which an endogenous retrovirus plays a key role in regulating host cell fate potential.

TDP-43 safeguards the embryo genome from L1 retrotransposition.

Transposable elements (TEs) are genomic parasites that propagate within the host genome and introduce mutations. Long interspersed nuclear element-1 (LINE-1 or L1) is the major TE class, which occupies nearly 20% of the mouse genome. L1 is highly active in mammalian preimplantation embryos, posing a major threat to genome integrity, but the mechanism of stage-specific protection against L1 retrotransposition is unknown. Here, we show that TAR DNA-binding protein 43 (TDP-43), mutations in which constitute a major risk factor for amyotrophic lateral sclerosis, inhibits L1 retrotransposition in mouse embryonic stem cells (mESCs) and preimplantation embryos. Knockdown of TDP-43 resulted in massive genomic L1 expansion and impaired cell growth in preimplantation embryos and ESCs. Functional analysis demonstrated that TDP-43 interacts with L1 open reading frame 1 protein (L1 ORF1p) to mediate genomic protection, and loss of this interaction led to derepression of L1 retrotransposition. Our results identify TDP-43 as a guardian of the embryonic genome.

Memory B Cells and Memory T Cells Induced by SARS-CoV-2 Booster Vaccination or Infection Show Different Dynamics and Responsiveness to the Omicron Variant.

Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

Generation of Stable Drosophila Ovarian Somatic Cell Lines Using the piggyBac System.

Transposable elements (TEs) constitute a large proportion of the genome in multiple organisms. Therefore, anti-transposable element machineries are essential to maintain genomic integrity. PIWI-interacting RNAs (piRNAs) are a major force to repress TEs in Drosophila ovaries. Ovarian somatic cells (OSC), in which nuclear piRNA regulation is functional, have been used for research on piRNA pathway as a cell culture system to elucidate the molecular mechanisms underlying the piRNA pathway. Analysis of piRNA pathway using a reporter system to monitor the gene regulation or overexpression of specific genes would be a powerful approach. Here, we present the technical protocol to establish stable cell lines using the piggyBac system, adopted for OSCs. This easy, consistent, and timesaving protocol may accelerate research on the piRNA pathway.

The emergence of SARS-CoV-2 variants threatens to decrease the efficacy of neutralizing antibodies and vaccines.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease (COVID-19) pandemic. As of August 2021, more than 200 million people have been infected with the virus and 4.3 million have lost their lives. Various monoclonal antibodies of human origin that neutralize the SARS-CoV-2 infection have been isolated from convalescent patients for therapeutic and prophylactic purposes. Several vaccines have been developed to restrict the spread of the virus and have been rapidly administered. However, the rollout of vaccines has coincided with the spread of variants of concern. Emerging variants of SARS-CoV-2 present new challenges for therapeutic antibodies and threaten the efficacy of current vaccines. Here, we review the problems faced by neutralizing antibodies and vaccines in the midst of the increasing spread of mutant viruses.

Potent mouse monoclonal antibodies that block SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies.

Piwi suppresses transcription of Brahma-dependent transposons via Maelstrom in ovarian somatic cells.

Drosophila Piwi associates with PIWI-interacting RNAs (piRNAs) and represses transposons transcriptionally through heterochromatinization; however, this process is poorly understood. Here, we identify Brahma (Brm), the core adenosine triphosphatase of the SWI/SNF chromatin remodeling complex, as a new Piwi interactor, and show Brm involvement in activating transcription of Piwi-targeted transposons before silencing. Bioinformatic analyses indicated that Piwi, once bound to target RNAs, reduced the occupancies of SWI/SNF and RNA polymerase II (Pol II) on target loci, abrogating transcription. Artificial piRNA-driven targeting of Piwi to RNA transcripts enhanced repression of Brm-dependent reporters compared with Brm-independent reporters. This was dependent on Piwi cofactors, Gtsf1/Asterix (Gtsf1), Panoramix/Silencio (Panx), and Maelstrom (Mael), but not Eggless/dSetdb (Egg)-mediated H3K9me3 deposition. The λN-box B-mediated tethering of Mael to reporters repressed Brm-dependent genes in the absence of Piwi, Panx, and Gtsf1. We propose that Piwi, via Mael, can rapidly suppress transcription of Brm-dependent genes to facilitate heterochromatin formation.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.

Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing.

Eggless/SETDB1 (Egg), the only essential histone methyltransferase (HMT) in Drosophila, plays a role in gene repression, including piRNA-mediated transposon silencing in the ovaries. Previous studies suggested that Egg is post-translationally modified and showed that Windei (Wde) regulates Egg nuclear localization through protein-protein interaction. Monoubiquitination of mammalian SETDB1 is necessary for the HMT activity. Here, using cultured ovarian somatic cells, we show that Egg is monoubiquitinated and phosphorylated but that only monoubiquitination is required for piRNA-mediated transposon repression. Egg monoubiquitination occurs in the nucleus. Egg has its own nuclear localization signal, and the nuclear import of Egg is Wde-independent. Wde recruits Egg to the chromatin at target gene silencing loci, but their interaction is monoubiquitin-independent. The abundance of nuclear Egg is governed by that of nuclear Wde. These results illuminate essential roles of nuclear monoubiquitination of Egg and the role of Wde in piRNA-mediated transposon repression.

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.

Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

Profiling Open Chromatin Structure in the Ovarian Somatic Cells Using ATAC-seq.

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) was recently established as a method to profile open chromatin, which overcomes the sample size limitations of the alternative methods DNase/MNase-seq. To investigate the role of Piwi in heterochromatin formation around transposable element loci, we have used ATAC-seq to examine chromatin accessibility at target transposable elements in a Drosophila cultured cell line, ovarian somatic cells (OSCs). In this chapter, we describe our method to profile open chromatin structure in OSCs using ATAC-seq.

Mitotic phosphorylation of CCCTC-binding factor (CTCF) reduces its DNA binding activity.

During mitosis, higher order chromatin structures are disrupted and chromosomes are condensed to achieve accurate chromosome segregation. CCCTC-binding factor (CTCF) is a highly conserved and ubiquitously expressed C2H2-type zinc finger protein which is considered to be involved in epigenetic memory through regulation of higher order chromatin architecture. However, the regulatory mechanism of CTCF in mitosis is still unclear. Here we found that the DNA-binding activity of CTCF is regulated in a phosphorylation-dependent manner during mitosis. The linker domains of the CTCF zinc finger domain were found to be phosphorylated during mitosis. The phosphorylation of linker domains impaired the DNA-binding activity in vitro. Mutation analyses showed that amino acid residues (Thr289, Thr317, Thr346, Thr374, Ser402, Ser461, and Thr518) located in the linker domains were phosphorylated during mitosis. Based on these results, we propose that the mitotic phosphorylation of the linker domains of CTCF is important for the dissociation of CTCF from mitotic chromatin.

Piwi Modulates Chromatin Accessibility by Regulating Multiple Factors Including Histone H1 to Repress Transposons.

PIWI-interacting RNAs (piRNAs) mediate transcriptional and post-transcriptional silencing of transposable element (TE) in animal gonads. In Drosophila ovaries, Piwi-piRNA complexes (Piwi-piRISCs) repress TE transcription by modifying the chromatin state, such as by H3K9 trimethylation. Here, we demonstrate that Piwi physically interacts with linker histone H1. Depletion of Piwi decreases H1 density at a subset of TEs, leading to their derepression. Silencing at these loci separately requires H1 and H3K9me3 and heterochromatin protein 1a (HP1a). Loss of H1 increases target loci chromatin accessibility without affecting H3K9me3 density at these loci, while loss of HP1a does not impact H1 density. Thus, Piwi-piRISCs require both H1 and HP1a to repress TEs, and the silencing is correlated with the chromatin state rather than H3K9me3 marks. These findings suggest that Piwi-piRISCs regulate the interaction of chromatin components with target loci to maintain silencing of TEs through the modulation of chromatin accessibility.

Oseltamivir expands quasispecies of influenza virus through cell-to-cell transmission.

The population of influenza virus consists of a huge variety of variants, called quasispecies, due to error-prone replication. Previously, we reported that progeny virions of influenza virus become infected to adjacent cells via cell-to-cell transmission pathway in the presence of oseltamivir. During cell-to-cell transmission, viruses become infected to adjacent cells at high multiplicity since progeny virions are enriched on plasma membrane between infected cells and their adjacent cells. Co-infection with viral variants may rescue recessive mutations with each other. Thus, it is assumed that the cell-to-cell transmission causes expansion of virus quasispecies. Here, we have demonstrated that temperature-sensitive mutations remain in progeny viruses even at non-permissive temperature by co-infection in the presence of oseltamivir. This is possibly due to a multiplex infection through the cell-to-cell transmission by the addition of oseltamivir. Further, by the addition of oseltamivir, the number of missense mutation introduced by error-prone replication in segment 8 encoding NS1 was increased in a passage-dependent manner. The number of missense mutation in segment 5 encoding NP was not changed significantly, whereas silent mutation was increased. Taken together, we propose that oseltamivir expands influenza virus quasispecies via cell-to-cell transmission, and may facilitate the viral evolution and adaptation.

Reconstitution of human rRNA gene transcription in mouse cells by a complete SL1 complex.

An important characteristic of the transcription of a ribosomal RNA gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I is its stringent species specificity. SL1/TIF-IB is a key complex for species specificity, but its functional complex has not been reconstituted. Here, we established a novel and highly sensitive monitoring system for Pol I transcription to reconstitute the SL1 activity in which a transcript harboring a reporter gene synthesized by Pol I is amplified and converted into translatable mRNA by the influenza virus RNA-dependent RNA polymerase. Using this monitoring system, we reconstituted Pol I transcription from the human rDNA promoter in mouse cells by expressing four human TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1 complex. The reconstituted SL1 also re-activated human rDNA transcription in mouse A9 cells carrying an inactive human chromosome 21 that contains the rDNA cluster. Chimeric SL1 complexes containing human and mouse TAFIs could be formed, but these complexes were inactive for human rDNA transcription. We conclude that four human TAFIs are necessary and sufficient to overcome the barrier of species specificity for human rDNA transcription in mouse cells.

Regulation of nucleolar chromatin by B23/nucleophosmin jointly depends upon its RNA binding activity and transcription factor UBF.

Histone chaperones regulate the density of incorporated histone proteins around DNA transcription sites and therefore constitute an important site-specific regulatory mechanism for the control of gene expression. At present, the targeting mechanism conferring this site specificity is unknown. We previously reported that the histone chaperone B23/nucleophosmin associates with rRNA chromatin (r-chromatin) to stimulate rRNA transcription. Here, we report on the mechanism for site-specific targeting of B23 to the r-chromatin. We observed that, during mitosis, B23 was released from chromatin upon inactivation of its RNA binding activity by cdc2 kinase-mediated phosphorylation. The phosphorylation status of B23 was also shown to strongly affect its chromatin binding activity. We further found that r-chromatin binding of B23 was a necessary condition for B23 histone chaperone activity in vivo. In addition, we found that depletion of upstream binding factor (UBF; an rRNA transcription factor) decreased the chromatin binding affinity of B23, which in turn led to an increase in histone density at the r-chromatin. These two major strands of evidence suggest a novel cell cycle-dependent mechanism for the site-specific regulation of histone density via joint RNA- and transcription factor-mediated recruitment of histone chaperones to specific chromosome loci.

Sp1-mediated transcription regulation of TAF-Ialpha gene encoding a histone chaperone.

TAF-I, one of histone chaperones, consists of two subtypes, TAF-Ialpha and TAF-Ibeta. The histone chaperone activity of TAF-I is regulated by dimer patterns of these subtypes. TAF-Ibeta is expressed ubiquitously, while the expression level of TAF-Ialpha with less activity than TAF-Ibeta differs among cell types. It is, therefore, assumed that the expression level of TAF-Ialpha in a cell is important for the TAF-I activity level. Here, we found that TAF-Ialpha and TAF-Ibeta genes are under the control of distinct promoters. Reporter assays and gel shift assays demonstrated that Sp1 binds to three regions in the TAF-Ialpha promoter and two or all mutaions of the three Sp1 binding regions reduced the TAF-Ialpha promoter activity. ChIP assays demonstrated that Sp1 binds to the TAF-Ialpha promoter in vivo. Furthermore, the expression level of TAF-Ialpha mRNA was reduced by knockdown of Sp1 using siRNA method. These studies indicated that the TAF-Ialpha promoter is under the control of Sp1.

Transcription regulation of the rRNA gene by a multifunctional nucleolar protein, B23/nucleophosmin, through its histone chaperone activity.

It is well established that the transcription rate of the rRNA gene is closely associated with profound alterations in the cell growth rate. Regulation of rRNA gene transcription is likely to be dependent on the dynamic conversion of the chromatin structure. Previously, we identified B23/nucleophosmin, a multifunctional nucleolar phosphoprotein, as a component of template activating factor III that remodels the chromatin-like structure of the adenovirus genome complexed with viral basic proteins. It has also been shown that B23 has histone chaperone activity. Here, we examined the effect of B23 on rRNA gene transcription. B23 was found to be associated with the rRNA gene chromatin. Small-interfering-RNA-mediated down-regulation of the B23 expression level resulted in reduction of the transcription rate of the rRNA gene. We constructed a B23 mutant termed B23DeltaC, which lacks the domain essential for the histone chaperone activity and inhibited the histone binding activity of B23 in a dominant-negative manner. Expression of B23DeltaC decreased rRNA gene transcription and the rate of cell proliferation. These results suggest that B23 is involved in the transcription regulation of the rRNA gene as a nucleolar histone chaperone.