Similarity and dissimilarity in alterations of the gene expression profile associated with inhalational anesthesia between sevoflurane and desflurane.

Although sevoflurane is one of the most commonly used inhalational anesthetic agents, the popularity of desflurane is increasing to a level similar to that of sevoflurane. Inhalational anesthesia generally activates and represses the expression of genes related to xenobiotic metabolism and immune response, respectively. However, there has been no comprehensive comparison of the effects of sevoflurane and desflurane on the expression of these genes. Thus, we used a next-generation sequencing method to compare alterations in the global gene expression profiles in the livers of rats subjected to inhalational anesthesia by sevoflurane or desflurane. Our bioinformatics analyses revealed that sevoflurane and, to a greater extent, desflurane significantly activated genes related to xenobiotic metabolism. Our analyses also revealed that both anesthetic agents, especially sevoflurane, downregulated many genes related to immune response.

MAX controls meiotic entry in sexually undifferentiated germ cells.

Meiosis is a specialized type of cell division that occurs physiologically only in germ cells. We previously demonstrated that MYC-associated factor X (MAX) blocks the ectopic onset of meiosis in embryonic and germline stem cells in culture systems. Here, we investigated the Max gene's role in mouse primordial germ cells. Although Max is generally ubiquitously expressed, we revealed that sexually undifferentiated male and female germ cells had abundant MAX protein because of their higher Max gene expression than somatic cells. Moreover, our data revealed that this high MAX protein level in female germ cells declined significantly around physiological meiotic onset. Max disruption in sexually undifferentiated germ cells led to ectopic and precocious expression of meiosis-related genes, including Meiosin, the gatekeeper of meiotic onset, in both male and female germ cells. However, Max-null male and female germ cells did not complete the entire meiotic process, but stalled during its early stages and were eventually eliminated by apoptosis. Additionally, our meta-analyses identified a regulatory region that supports the high Max expression in sexually undifferentiated male and female germ cells. These results indicate the strong connection between the Max gene and physiological onset of meiosis in vivo through dynamic alteration of its expression.

Identification of novel oncogenes in oral cancer among elderly nonsmokers.

OBJECTIVES: In recent years, an increase in oral cancer among elderly nonsmokers has been noted. The aim of this study was to identify novel oncogenes in oral cancer in older nonsmokers. MATERIAL AND METHODS: Whole-exome sequencing (WES) data from 324 oral cancer patients were obtained from The Cancer Genome Atlas. Single nucleotide variants (SNVs) and insertions/deletions (INDELs) were extracted from the WES data of older patients. Fisher's exact test was performed to determine the specificity of variants in these genes. Finally, SNVs and INDELs were identified by target enrichment sequencing. RESULTS: Gene ontology analysis of 112 genes with significant SNVs or INDELs in nonsmokers revealed that nonsynonymous SNVs in HECTD4 were significantly more frequent in nonsmokers than in smokers by target enrichment sequencing (p = .02). CONCLUSIONS: Further investigation of the function of HECTD4 variants as oncogenes in older nonsmokers is warranted.

Alternative splicing for germ cell-specific Mga transcript can be eliminated without compromising mouse viability or fertility.

The stimulated by retinoic acid gene 8 (STRA8)/MEIOSIN complex and polycomb repressive complex (PRC) 1.6, a PRC1 subtype, are believed to be positive and negative regulators of meiotic onset, respectively. During meiotic initiation, the transcription repressive activity of PRC1.6 must be attenuated so that meiosis-related genes can be effectively activated by the STRA8/MEIOSIN complex. However, the molecular mechanisms that control the impairment of PRC1.6 function remain unclear. We recently demonstrated that the Mga gene, which encodes a scaffolding component of PRC1.6, produces variant mRNA by alternative splicing specifically during meiosis. Furthermore, the anomalous MGA protein encoded by the variant mRNA bears an intrinsic ability to function as a dominant negative regulator against the construction of PRC1.6 and is therefore assumed to be, at least in part, involved in impairment of the complex. Therefore, to unequivocally evaluate the physiological significance of Mga variant mRNA production in gametogenesis, we examined the consequences of a genetic manipulation that renders mice unable to produce Mga variant mRNA. Our data revealed that mutant mice were equivalent to wild-type mice in terms of viability and fertility. Our detailed examination of spermatogenesis also revealed that this genetic alteration is not associated with any apparent abnormalities in testis size, spermatogenic cycle, timing of meiotic onset, or marker gene expression of spermatogonia and spermatocytes. Taken together, these data indicate that the production of germ cell-specific Mga variant mRNA is dispensable not only for viability but also for gametogenesis.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline "genome-defence" (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.

Two DNA binding domains of MGA act in combination to suppress ectopic activation of meiosis-related genes in mouse embryonic stem cells.

Although the physiological meaning of the high potential of mouse embryonic stem cells (ESCs) for meiotic entry is not understood, a rigid safeguarding system is required to prevent ectopic onset of meiosis. PRC1.6, a non-canonical PRC1, is known for its suppression of precocious and ectopic meiotic onset in germ cells and ESCs, respectively. MGA, a scaffolding component of PRC1.6, bears two distinct DNA-binding domains termed bHLHZ and T-box. However, it is unclear how this feature contributes to the functions of PRC1.6. Here, we demonstrated that both domains repress distinct sets of genes in murine ESCs, but substantial numbers of meiosis-related genes are included in both gene sets. In addition, our data demonstrated that bHLHZ is crucially involved in repressing the expression of Meiosin, which plays essential roles in meiotic entry with Stra8, revealing at least part of the molecular mechanisms that link negative and positive regulation of meiotic onset.

Effects of Pparγ1 deletion on late-stage murine embryogenesis and cells that undergo endocycle.

Peroxisome proliferator-activated receptor (PPAR) γ1, a nuclear receptor, is abundant in the murine placenta during the late stage of pregnancy (E15-E16), although its functional roles remain unclear. PPARγ1 is encoded by two splicing isoforms, namely Pparγ1canonical and Pparγ1sv, and its embryonic loss leads to early (E10) embryonic lethality. Thus, we generated knockout (KO) mice that carried only one of the isoforms to obtain a milder phenotype. Pparγ1sv-KO mice were viable and fertile, whereas Pparγ1canonical-KO mice failed to recover around the weaning age. Pparγ1canonical-KO embryos developed normally up to 15.5 dpc, followed by growth delays after that. The junctional zone of Pparγ1canonical-KO placentas severely infiltrated the labyrinth, and maternal blood sinuses were dilated. In the wild-type, PPARγ1 was highly expressed in sinusoidal trophoblast giant cells (S-TGCs), peaking at 15.5 dpc. Pparγ1canonical-KO abolished PPARγ1 expression in S-TGCs. Notably, the S-TGCs had unusually enlarged nuclei and often occupied maternal vascular spaces, disturbing the organization of the fine labyrinth structure. Gene expression analyses of Pparγ1canonical-KO placentas indicated enhanced S-phase cell cycle signatures. EdU-positive S-TGCs in Pparγ1canonical-KO placentas were greater in number than those in wild-type placentas, suggesting that the cells continued to endoreplicate in the mutant placentas. These results indicate that PPARγ1, a known cell cycle arrest mediator, is involved in the transition of TGCs undergoing endocycling to the terminal differentiation stage in the placentas. Therefore, PPARγ1 deficiency, induced through genetic manipulation, leads to placental insufficiency.

Identification of germ cell-specific Mga variant mRNA that promotes meiosis via impediment of a non-canonical PRC1.

A non-canonical PRC1 (PRC1.6) prevents precocious meiotic onset. Germ cells alleviate its negative effect by reducing their amount of MAX, a component of PRC1.6, as a prerequisite for their bona fide meiosis. Here, we found that germ cells produced Mga variant mRNA bearing a premature termination codon (PTC) during meiosis as an additional mechanism to impede the function of PRC1.6. The variant mRNA encodes an anomalous MGA protein that lacks the bHLHZ domain and thus functions as a dominant negative regulator of PRC1.6. Notwithstanding the presence of PTC, the Mga variant mRNA are rather stably present in spermatocytes and spermatids due to their intrinsic inefficient background of nonsense-mediated mRNA decay. Thus, our data indicate that meiosis is controlled in a multi-layered manner in which both MAX and MGA, which constitute the core of PRC1.6, are at least used as targets to deteriorate the integrity of the complex to ensure progression of meiosis.

Predicting the radiosensitivity of HPV-negative oropharyngeal squamous cell carcinoma using miR-130b.

BACKGROUND: Human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinoma shows a higher rate of radiation resistance than HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). Radioresistant HPV-negative OPSCC is associated with unfavourable outcomes, but validated prognostic biomarkers remain lacking. AIMS/OBJECTIVES: This study investigated biomarkers for radioresistant HPV-negative OPSCC. MATERIAL AND METHODS: The Cancer Genome Atlas included miRNA sequence and mRNA sequence data from 528 HNSCC tumours. Of these, we used gene expression data for HPV-negative head and neck squamous cell carcinoma for which data were available on the effects of radiation, and compared miRNA sequence and mRNA sequence data between radioresistant and radiosensitive groups. We subsequently estimated downstream miRNA from the results. Finally, we validated miRNAs related to the outcomes of radiotherapy in our clinical cases. RESULTS: Investigation of miRNA sequence revealed expression of miR-130b as the greatest difference between radiosensitive and radioresistant groups. We subsequently evaluated miR-130b expression in our clinical OPSCC cases. Values of miR-130b >5.372 (low expression), determined from receiver operating characteristic curve analyses, were associated with significantly longer progression-free survival and overall survival (p = .006, p = .04, respectively). CONCLUSIONS AND SIGNIFICANCE: Our results suggest that miR-130b has potential as a biomarker for the radiosensitivity of HPV-negative OPSCC.

Transformation of normal cells by aberrant activation of YAP via cMyc with TEAD.

YAP (also known as YAP1 or YAP65) is a transcriptional coactivator that interacts with a number of transcription factors including RUNX and TEAD and plays a pivotal role in controlling cell growth. YAP is classified as a proto-oncogene. However, the mechanism by which activated YAP induces cancerous changes is not well known. Here we demonstrate that overexpression of YAP in NIH3T3 cells was sufficient for inducing tumorigenic transformation of cells. Mechanistically, YAP exerts its function in cooperation with the TEAD transcription factor. Our data also show that cMYC is a critical factor that acts downstream of the YAP/TEAD complex. Furthermore, we also found that aberrant activation of YAP is sufficient to drive tumorigenic transformation of non-immortalized mouse embryonic fibroblasts. Together our data indicate that YAP can be categorized as a new type of proto-oncogene distinct from typical oncogenes, such as H-RAS, whose expression in non-immortalized cells is tightly linked to senescence.

Identification and characterization of splenic adherent cells forming densely-packed colonies.

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.

DNMTs and SETDB1 function as co-repressors in MAX-mediated repression of germ cell-related genes in mouse embryonic stem cells.

In embryonic stem cells (ESCs), the expression of development-related genes, including germ cell-related genes, is globally repressed. The transcription factor MAX represses germ cell-related gene expression in ESCs via PCGF6-polycomb repressive complex 1 (PRC1), which consists of several epigenetic factors. However, we predicted that MAX represses germ cell-related gene expression through several additional mechanisms because PCGF6-PRC1 regulates the expression of only a subset of genes repressed by MAX. Here, we report that MAX associated with DNA methyltransferases (DNMTs) and the histone methyltransferase SETDB1 cooperatively control germ cell-related gene expression in ESCs. Both DNA methylation and histone H3 lysine 9 tri-methylation of the promoter regions of several germ cell-related genes were not affected by knockout of the PRC1 components, indicating that the MAX-DNMT and MAX-SETDB1 pathways are independent of the PCGF6-PRC1 pathway. Our findings provide insights into our understanding of MAX-based repressive mechanisms of germ cell-related genes in ESCs.

A CRISPR knockout screen identifies SETDB1-target retroelement silencing factors in embryonic stem cells.

In mouse embryonic stem cells (mESCs), the expression of provirus and endogenous retroelements is epigenetically repressed. Although many cellular factors involved in retroelement silencing have been identified, the complete molecular mechanism remains elusive. In this study, we performed a genome-wide CRISPR screen to advance our understanding of retroelement silencing in mESCs. The Moloney murine leukemia virus (MLV)-based retroviral vector MSCV-GFP, which is repressed by the SETDB1/TRIM28 pathway in mESCs, was used as a reporter provirus, and we identified more than 80 genes involved in this process. In particular, ATF7IP and the BAF complex components are linked with the repression of most of the SETDB1 targets. We characterized two factors, MORC2A and RESF1, of which RESF1 is a novel molecule in retroelement silencing. Although both factors are recruited to repress provirus, their roles in repression are different. MORC2A appears to function dependent on repressive epigenetic modifications, while RESF1 regulates repressive epigenetic modifications associated with SETDB1. Our genome-wide CRISPR screen cataloged genes which function at different levels in silencing of SETDB1-target retroelements and provides a useful resource for further molecular studies.

Identification of the Coiled-Coil Domain as an Essential Methyl-CpG-Binding Domain Protein 3 Element for Preserving Lineage Commitment Potential of Embryonic Stem Cells.

Embryonic stem cells (ESCs) exhibit two salient features beneficial for regenerative medicine: unlimited self-renewal and pluripotency. Methyl-CpG-binding domain protein 3 (Mbd3), a scaffolding component of the nucleosome remodeling deacetylase complex, is a specific regulator of pluripotency, as ESCs lacking Mbd3 are defective for lineage commitment potential but retain normal self-renewal properties. However, functional similarities and dissimilarities among the three Mbd3 isoforms (a, b, and c) have not been intensively explored. Herein, we demonstrated that Mbd3c, which lacks an entire portion of the MBD domain, exerted equivalent activity for counteracting the defective lineage commitment potential of Mbd3-knockout ESCs. Our analyses also revealed that the coiled-coil domain common to all three MBD3 isoforms, but not the MBD domain, plays a crucial role in this activity. Mechanistically, our data demonstrate that the activity of the coiled-coil domain is exerted, at least in part, through recruitment of polycomb repressive complex 2 to a subset of genes linked to development and organogenesis, thus establishing stable transcriptional repression. Stem Cells 2018;36:1355-1367.

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci.

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.

PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes.

The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.

Does MAX open up a new avenue for meiotic research?

Meiosis is a central event of sexual reproduction. Like somatic cells, germ cells conduct mitosis to increase their cell number, but unlike somatic cells, germ cells switch their cell division mode from mitosis to meiosis at a certain point in gametogenesis. However, the molecular basis of this switch remains elusive. In this review article, we give an overview of the onset of mammalian meiosis, including our recent finding that MYC Associated Factor X (MAX) prevents ectopic and precocious meiosis in embryonic stem cells (ESCs) and germ cells, respectively. We present a hypothetical model of a MAX-centered molecular network that regulates meiotic entry in mammals and propose that inducible Max knockout ESCs provide an excellent platform for exploring the molecular mechanisms of meiosis initiation, while excluding other aspects of gametogenesis.