Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression.

OBJECTIVES: Mammalian DNA methyltransferases are essential to re-establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)-a zinc finger type of MBP-is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear. MATERIALS AND METHODS: This study used the Cre-loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real-time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms. RESULTS: Germline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity. CONCLUSIONS: These findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Diphtheria toxin-mediated transposon-driven poly (A)-trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells.

Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

PD-1: Its Discovery, Involvement in Cancer Immunotherapy, and Beyond.

On December 10, 2018, I was sitting among the big crowd of audience, as one of the invited guests to the ceremony, in the Stockholm Concert Hall. When King of Sweden Carl XVI Gustaf bestowed the diploma and medal of Nobel Prize of Physiology or Medicine 2018 on Dr. Tasuku Honjo and shook his hand for a while, surrounded by the thunderous applause and energetically blessing orchestral music, I thought that it had been a long journey for the molecule that we had first isolated in the early 1990s. Although it was truly a commemorable moment in the history of the programmed death-1 (PD-1) research, I believe we still have a long way to go. In this review article, I will explain why I think so, particularly by focusing on the potential role(s) that PD-1 appears to play in self-nonself discrimination by the immune system.

Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis.

Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.

CIBZ Regulates Mesodermal and Cardiac Differentiation of by Suppressing T and Mesp1 Expression in Mouse Embryonic Stem Cells.

The molecular mechanisms underlying mesodermal and cardiac specification from embryonic stem cells (ESCs) are not fully understood. Here, we showed that the BTB domain-containing zinc finger protein CIBZ is expressed in mouse ESCs but is dramatically downregulated during ESC differentiation. CIBZ deletion in ESCs induced specification toward mesoderm phenotypes and their differentiation into cardiomyocytes, whereas overexpression of CIBZ delayed these processes. During ESC differentiation, CIBZ loss-and-gain-of-function data indicate that CIBZ negatively regulates the expressions of Brachyury (T) and Mesp1, the key transcriptional factors responsible for the specification of mammalian mesoderm and cardiac progenitors, respectively. Chromatin immunoprecipitation assays showed that CIBZ binds to T and Mesp1 promoters in undifferentiated ESCs, and luciferase assays indicate that CIBZ suppresses T and Mesp1 promoters. These findings demonstrate that CIBZ is a novel regulator of mesodermal and cardiac differentiation of ESCs, and suggest that CIBZ-mediated cardiac differentiation depends on the regulation of these two genes.

Low-cost three-dimensional gait analysis system for mice with an infrared depth sensor.

Three-dimensional (3D) open-field gait analysis of mice is an essential procedure in genetic and nerve regeneration research. Existing gait analysis systems are generally expensive and may interfere with the natural behaviors of mice because of optical markers and transparent floors. In contrast, the proposed system captures the subjects shape from beneath using a low-cost infrared depth sensor (Microsoft Kinect) and an opaque infrared pass filter. This means that we can track footprints and 3D paw-tip positions without optical markers or a transparent floor, thereby preventing any behavioral changes. Our experimental results suggest with healthy mice that they are more active on opaque floors and spend more time in the center of the open-field, when compared with transparent floors. The proposed system detected footprints with a comparable performance to existing systems, and precisely tracked the 3D paw-tip positions in the depth image coordinates.

An atypical tubulin kinase mediates stress-induced microtubule depolymerization in Arabidopsis.

BACKGROUND: As sessile organisms, plants adapt to adverse environmental conditions by quickly adjusting cell physiology and metabolism. Transient depolymerization of interphase microtubules is triggered by various acute stresses and biotic interactions with pathogenic organisms. Although rapid remodeling of plant microtubule arrays in response to external stresses is an intriguing phenomenon, the underlying molecular mechanisms and the advantages of this response to plant performance are poorly understood. RESULTS: A domain with weak homology to the slime mold actin-fragmin kinase in the Arabidopsis mitogen-activated protein kinase phosphatase PROPYZAMIDE-HYPERSENSITIVE 1 (PHS1) is a Mn2+-dependent kinase. This atypical kinase domain phosphorylates Thr349 of α-tubulin at the longitudinal interdimer interface, thereby generating a polymerization-incompetent isoform, and effectively depolymerizes microtubule arrays when ectopically expressed in plant or animal cells. The intrinsic tubulin kinase activity is normally suppressed by the phosphatase activity of PHS1 but is unmasked immediately after osmotic stress. In the phs1 null mutant, stress-induced microtubule depolymerization does not occur. CONCLUSIONS: The rapid and reversible modification of tubulin subunits by PHS1-mediated phosphorylation enables dynamic remodeling of the plant microtubule cytoskeleton in response to external stimuli. Suppression of the potent tubulin kinase activity by the juxtaposed phosphatase domain tightly controls this stress-activated microtubule regulator.

Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.

Evidence that the Upf1-related molecular motor scans the 3'-UTR to ensure mRNA integrity.

Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation-terminating ribosome and then with a downstream exon-junction complex (EJC), which is deposited at exon-exon junctions during splicing. Although the helicase activity of Upf1 is indispensable for NMD, its roles and substrates have yet to be fully elucidated. Here we show that stable RNA secondary structures between a PTC and a downstream exon-exon junction increase the levels of potential NMD substrates. We also demonstrate that a stable secondary structure within the 3'-untranslated region (UTR) induces the binding of Upf1 to mRNA in a translation-dependent manner and that the Upf1-related molecules are accumulated at the 5'-side of such a structure. Furthermore, we present evidence that the helicase activity of Upf1 is used to bridge the spatial gap between a translation-termination codon and a downstream exon-exon junction for the activation of NMD. Based on these findings, we propose a model that the Upf1-related molecular motor scans the 3'-UTR in the 5'-to-3' direction for the mRNA-binding factors including EJCs to ensure mRNA integrity.

Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells.

Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually and conditionally knockout the remaining 'difficult-to-trap' genes. Here, we show that transcriptionally silent genes in ES cells are severely underrepresented among the randomly trapped genes in KOMP. Our conditional poly(A)-trapping vector with a common retroviral backbone also has a strong bias to be integrated into constitutively transcribed genome loci. Most importantly, conditional gene disruption could not be successfully accomplished by using the retrovirus vector because of the frequent development of intra-vector deletions/rearrangements. We found that one of the cut and paste-type DNA transposons, Tol2, can serve as an ideal platform for gene-trap vectors that ensures identification and conditional disruption of a broad spectrum of genes in ES cells. We also solved a long-standing problem associated with multiple vector integration into the genome of a single cell by incorporating a mixture of differentially tagged Tol2 transposons. We believe our strategy indicates a straightforward approach to mass-production of conditionally disrupted alleles for genes in the target cells.

CtBP-interacting BTB zinc finger protein (CIBZ) promotes proliferation and G1/S transition in embryonic stem cells via Nanog.

Mouse embryonic stem cells (ESCs) require transcriptional regulation to ensure rapid proliferation that allows for self-renewal. However, the molecular mechanism by which transcriptional factors regulate this rapid proliferation remains largely unknown. Here we present data showing that CIBZ, a BTB domain zinc finger transcriptional factor, is a key transcriptional regulator for regulation of ESC proliferation. Here we show that deletion or siRNA knockdown of CIBZ inhibits ESC proliferation. Cell cycle analysis shows that loss of CIBZ delays the progression of ESCs through the G1 to S phase transition. Conversely, constitutive ectopic expression of exogenous CIBZ in ESCs promotes proliferation and accelerates G1/S transition. These findings suggest that regulation of the G1/S transition explains, in part, CIBZ-associated ESC proliferation. Our data suggest that CIBZ acts through the post-transcriptionally regulates the expression of Nanog, a positive regulator of ESC proliferation and G1/S transition, but does not affect Oct3/4 and Sox2 protein expression. Notably, constitutive overexpression of Nanog partially rescued the proliferation defect caused by CIBZ knockdown, indicating the role of CIBZ in ESC proliferation and G1/S transition at least in part depends on the Nanog protein level.

The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression.

Postnatal growth and regeneration of skeletal muscle are carried out mainly by satellite cells, which, upon stimulation, begin to express myogenin (Myog), the critical determinant of myogenic differentiation. DNA methylation status has been associated with the expression of Myog, but the causative mechanism remains almost unknown. Here, we report that the level of CIBZ, a methyl-CpG-binding protein, decreases upon myogenic differentiation of satellite-derived C2C12 cells, and during skeletal muscle regeneration in mice. We present data showing that the loss of CIBZ promotes myogenic differentiation, whereas exogenous expression of CIBZ impairs it, in cultured cells. CIBZ binds to a Myog promoter-proximal region and inhibits Myog transcription in a methylation-dependent manner. These data suggest that the suppression of myogenic differentiation by CIBZ is dependent, at least in part, on the regulation of Myog. Our data show that the methylation status of this proximal Myog promoter inversely correlates with Myog transcription in cells and tissues, and during postnatal growth of skeletal muscle. Notably, induction of Myog transcription by CIBZ suppression is independent of the demethylation of CpG sites in the Myog promoter. These observations provide the first reported molecular mechanism illustrating how Myog transcription is coordinately regulated by a methyl-CpG-binding protein and the methylation status of the proximal Myog promoter.

Down-regulation of CIBZ, a novel substrate of caspase-3, induces apoptosis.

We previously identified and characterized a murine BTB domain-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by small interfering RNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/propidium iodide labeling, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53(-/-) mouse embryonic fibroblast cells also activated caspase-3 and cleavage of poly(ADP-ribose) polymerase, indicating that CIBZ-associated apoptosis is mediated by a p53-independent pathway; however, because both common and distinct targets are regulated by CIBZ- and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3 and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.

Induction of murine leukemia and lymphoma by dominant negative retinoic acid receptor alpha.

Acute promyelocytic leukemia (APL) is invariably associated with chromosomal translocation to retinoic acid receptor alpha (RARalpha) locus. In a vast majority of cases, RARalpha translocates to and fuses with the promyelocytic leukemia (PML) gene. It was thought that the fusion protein PML-RARalpha acts as a double dominant negative mutant to inhibit the PML and RARalpha signaling. In an attempt to study the physiological role of retinoic acid in mammary gland development, we created a transgenic model system expressing a dominant negative RARalpha under the regulation of murine mammary tumor viral promoter. We found that the transgene was also targeted to the lymphoid system in addition to mammary gland. Here we showed that dominant negative RARalpha induced acute lymphoblastic leukemia and lymphoma development in the transgenic mice. Retinoic acid blocked tumor development ex vivo through induction of apoptosis. Thus, our results suggested that disruption of RARalpha signaling was the first essential step in the development of APL in vivo.

Identification of a novel BTB-zinc finger transcriptional repressor, CIBZ, that interacts with CtBP corepressor.

The transcriptional corepressor C-terminal binding protein (CtBP) is thought to be involved in development and oncogenesis, but the regulation of its corepressor activity is largely unknown. We show here that a novel BTB-zinc finger protein, CIBZ (CtBP-interacting BTB zinc finger protein; a mouse ortholog of rat ZENON that was recently identified as an e-box/dyad binding protein), redistributes CtBP to pericentromeric foci from a diffuse nuclear localization in interphase cells. CIBZ physically associates with CtBP via a conserved CtBP binding motif, PLDLR. When heterologously targeted to DNA, CIBZ represses transcription via two independent repression domains, an N-terminal BTB domain and a PLDLR motif-containing RD2 region, in a histone deacetylase-independent and -dependent manner, respectively. Mutation in the PLDLR motif abolishes the CIBZ-CtBP interaction and transcriptional repression activity of RD2, but does not affect the repression activity of the BTB domain. Furthermore, this PLDLR-mutated CIBZ cannot target CtBP to pericentromeric foci, although it is localized to the pericentromeric foci itself. These results suggest that at least one repression mechanism mediated by CIBZ is recruitment of the CtBP/HDAC complex to pericentromeric foci, and that CIBZ may regulate pericentromeric targeting of CtBP.

Cross-linking of CD45 on suppressive/regulatory T cells leads to the abrogation of their suppressive activity in vitro.

CD4(+)CD25(+) T cells have immunoregulatory and suppressive functions and are responsible for suppressing self-reactive cells and maintaining self-tolerance. In addition to CD4(+)CD25(+) T cells, there is some evidence that a fraction of CD4(+)CD25(-) T cells exhibit suppressive activity in vitro or in vivo. We have shown, using aged mice, that aging not only leads to a decline in the ability to mount CD4(+)CD25(-) T cell responses, but, at the same time, renders aged CD4(+)CD25(-) T cells suppressive. In this study we report two newly established mAbs that could abrogate the suppressive function of aged CD4(+)CD25(-) T cells. These mAbs recognized the same protein, the transmembrane phosphatase CD45. Cross-linking of CD45 on aged CD4(+)CD25(-) T cells was required for the disruption of their suppressive activity. Surprisingly, these mAbs also abrogated the suppressive action of CD4(+)CD25(+) T cells in vitro. Our results demonstrate an unexpected function of CD45 as a negative regulator neutralizing the suppressive activity of aged CD4(+)CD25(-) and young CD4(+)CD25(+) T cells.

Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells.

An international collaborative project has been proposed to inactivate all mouse genes in embryonic stem (ES) cells using a combination of random and targeted insertional mutagenesis techniques. Random gene trapping will be the first choice in the initial phase, and gene-targeting experiments will then be carried out to individually knockout the remaining 'difficult-to-trap' genes. One of the most favored techniques of random insertional mutagenesis is promoter trapping, which only disrupts actively transcribed genes. Polyadenylation (poly-A) trapping, on the other hand, can capture a broader spectrum of genes including those not expressed in the target cells, but we noticed that it inevitably selects for the vector integration into the last introns of the trapped genes. Here, we present evidence that this remarkable skewing is caused by the degradation of a selectable-marker mRNA used for poly-A trapping via an mRNA-surveillance mechanism, nonsense-mediated mRNA decay (NMD). We also report the development of a novel poly-A-trap strategy, UPATrap, which suppresses NMD of the selectable-marker mRNA and permits the trapping of transcriptionally silent genes without a bias in the vector-integration site. We believe the UPATrap technology enables a simple and straightforward approach to the unbiased inactivation of all mouse genes in ES cells.

Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis.

DNA arrays are capable of profiling the expression patterns of many genes in a single experiment. After finding a gene of interest in a DNA array, however, labor-intensive gene-targeting experiments sometimes must be performed for the in vivo analysis of the gene function. With random gene trapping, on the other hand, it is relatively easy to disrupt and retrieve hundreds of genes/gene candidates in mouse embryonic stem (ES) cells, but one could overlook potentially important gene-disruption events if only the nucleotide sequences and not the expression patterns of the trapped DNA segments are analyzed. To combine the benefits of the above two experimental systems, we first created approximately 900 genetrapped mouse ES cell clones and then constructed arrays of cDNAs derived from the disrupted genes. By using these arrays, we identified a novel gene predominantly expressed in the mouse brain, and the corresponding ES cell clone was used to produced mice homozygous for the disrupted allele of the gene. Detailed analysis of the knockout mice revealed that the gene trap vector completely abolished gene expression downstream of its integration site. Therefore, identification of a gene or novel gene candidate with an interesting expression pattern by using this type of DNA array immediately allows the production of knockout mice from an ES cell clone with a disrupted allele of the sequence of interest.