DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells.

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Lipid droplets are formed in 2-cell-like cells.

Embryonic stem (ES) cells, derived from the inner cell mass of a blastocyst, are believed to pluripotent cells and give rise to embryonic, but not extraembryonic, tissues. In mice, totipotent 2-cell stage embryo-like (2-cell-like) cells, which are identified by reactivation of murine endogenous retrovirus with leucin transfer RNA primer (MuERV-L), arise at a very few frequencies in ES cell cultures. Here, we found that a lipid droplet forms during the transition from ES cells to 2-cell-like cells, and we propose that 2-cell-like cells utilize a unique energy storage and production pathway.

De novo DNA methylation through the 5'-segment of the H19 ICR maintains its imprint during early embryogenesis.

Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gamete-specific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, whereas it was not methylated in sperm, suggesting that its gametic and postfertilization modifications were separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here, we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal DNMT3A- and DNMT3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice and found that deletion of the 5' segment of the endogenous paternal H19 ICR decreased its methylation after fertilization and attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo postfertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis.

PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos.

The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell differentiation and development through epigenetic gene regulation. 5mC can be converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet methylcytosine dioxygenase (Tet) family of enzymes. Notably, the balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment. We have previously reported that the maternal factor PGC7 (also known as Dppa3, Stella) is required for the maintenance of DNA methylation in early embryogenesis, and protects 5mC from conversion to 5hmC in the maternal genome. Here we show that PGC7 protects 5mC from Tet3-mediated conversion to 5hmC by binding to maternal chromatin containing dimethylated histone H3 lysine 9 (H3K9me2) in mice. In addition, imprinted loci that are marked with H3K9me2 in mature sperm are protected by PGC7 binding in early embryogenesis. This type of regulatory mechanism could be involved in DNA modifications in somatic cells as well as in early embryos.

DNA hypomethylation circuit of mouse rDNA repeats in the germ cell lineage.

DNA methylation is dynamically reprogrammed at two developmental periods, in primordial germ cells and pre-implantation embryos, via distinct phases of DNA demethylation and de novo methylation. Here we show that ribosomal DNA (rDNA) promoters are hypomethylated in sperm and oocytes; this hypomethyaltion was maintained during pre-implantation development. A DNA methylation analysis of embryonic and extra-embryonic cells on embryonic day 7.5 (E7.5) revealed that the rDNA promoter was slightly methylated in embryonic and extra-embryonic regions. Intriguingly, this hypomethylated status was observed throughout germ cell development on E13.5 and E18.5. In contrast, fetal somatic cells in gonad and liver acquired methylation on E13.5, which was maintained in adult tissues. These findings indicate a unique rDNA methylation signature in the germ cell lineage.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos.

In mammals, the structure of the pericentromeric region alters from a ring structure to a dot-like structure during the 2-cell stage. This structural alteration is termed chromocenter formation (CF) and is required for preimplantation development. Although reverse transcripts of major satellite repeats at pericentromeric regions are known to play roles in CF, its underlying mechanism is not fully understood. We previously reported that Stella (also known as PGC7 and Dppa3) deficiency led to developmental arrest at the preimplantation stage, accompanied by frequent chromosome segregation. In this study, we further investigated the effect of Stella deficiency on chromatin reorganization. The Stella-null embryos exhibited impaired CF and reduced expression of the reverse strand of major satellite repeats. In addition, the accumulation of H3.3, a histone H3 variant associated with transcriptional activation, at the pericentromeric regions and expression of the H3.3-specific chaperone Daxx were reduced in Stella-null embryos. These abnormalities were restored by the enforced expression of Daxx in Stella-null embryos. Thus, Stella controls the expression of Daxx and ensures chromatin reorganization in early embryos.

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

[A STUDY OF SURGICAL TREATMENT FOR PATIENTS WITH MYCOBACTERIUM ABSCESSUS PULMONARY DISEASE AND A COMPARATIVE EXAMINATION OF MYCOBACTERIUM AVIUM COMPLEX DISEASE].

OBJECTIVE: This is a retrospective study on six surgical cases of Mycobacterium abscessus pulmonary disease, including a comparison with M. avium complex (MAC) disease. SUBJECTS AND METHODS: We performed surgery for six cases of M. abscessus pulmonary disease between July 2012 and June 2014. In all the cases, video-assisted thoracic surgery alone was performed. Age, sex, bacillus identification method, disease type, preoperative anti-glycopeptidolipid core immunoglobulin A antibody value, preoperative chemotherapy, preoperative chemotherapy period, adaptation of the operation, surgical method, result of the bacillus culture of an organization that was extracted at operation, postoperative hospitalization period, surgical complications, and postoperative relapse were examined for the six cases of M. abscessus pulmonary disease. In addition, the cases were compared with 36 cases of MAC disease for which operation was performed during the same period. RESULT: None of the patients had major surgical complications or in-hospital death. Although three patients survived for more than 1 postoperative year and completed chemotherapy, relapses are not accepted in all cases at present. In the comparison with MAC disease, the mean preoperative chemotherapy period for M. abscessus pulmonary disease was 5.5 months, which was 18.9 months shorter than that for MAC disease, with a statistically significant difference. CONCLUSION AND CONSIDERATION: Surgery for M. abscessus pulmonary disease may be considered a safe and effective therapeutic procedure. Moreover, some physicians believe that surgical treatment is required at an earlier stage of M. abscessus pulmonary disease compared with MAC disease.

Inhibition of maintenance DNA methylation by Stella.

DNA methylation is a key epigenetic regulator in mammals, and the dynamic balance between methylation and demethylation impacts various processes, from development to disease. DNA methylation is erased during replication when DNA methyltransferase 1 (DNMT1) fails to methylate the daughter strand, in a process known as passive DNA demethylation. We found that the enforced expression of Stella (also known as PGC7, Dppa3), a maternal factor required for the maintenance of DNA methylation in early embryos, induced global DNA demethylation in NIH3T3 cells. This demethylation was caused by the binding of Stella to Np95 (also known as Uhrf1, ICBP90) and the subsequent inhibition of DNMT1 recruitment. Considering that impaired DNA methylation profiles are associated with various developmental or disease phenomena, Stella may be a powerful tool with which to study the biological effects of global DNA hypomethylation.

PGC7/Stella protects against DNA demethylation in early embryogenesis.

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.

RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos.

Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.

Effects of dppa3 on DNA methylation dynamics during primordial germ cell development in mice.

DNA methylation is a central epigenetic event that regulates cellular differentiation, reprogramming, and pathogenesis. Genomewide DNA demethylation occurs in preimplantation embryos and in embryonic germ cell precursors called primordial germ cells (PGCs). We previously showed that Dppa3, also known as Stella and PGC7, protects the maternal genome from tet methylcytosine dioxygenase 3 (Tet3)-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in zygotes. Here, we demonstrated that retrotransposon genes, such as long interspersed nuclear element-1 (Line-1) and intracisternal A particle (IAP), showed higher 5mC levels in Dppa3-null PGCs. In contrast, oxidative bisulfite sequence analysis revealed that the amounts of 5hmC in Line-1 and IAP were slightly reduced in the Dppa3-deficient PGCs. From our findings, we propose that Dppa3 is involved in the Tet-mediated active demethylation process during reprogramming of PGCs.

Quercetin protects against pulmonary oxidant stress via heme oxygenase-1 induction in lung epithelial cells.

The lung is a primary target for oxygen toxicity because of its constant exposure to high oxygen levels and environmental oxidants. Quercetin is one of the most commonly found dietary flavonoids, and it provides cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways. In the present study, we showed that quercetin increased the levels of heme oxygenase (HO)-1 expression and protected against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in lung epithelial cell lines. Quercetin suppressed H(2)O(2)-induced apoptotic events, including hypodiploid cells, activation of caspase 3 enzyme activity and lactate dehydrogenase release. This cytoprotective effect was attenuated by the addition of the HO inhibitor, tin protoporphyrin IX. In addition, the end products of heme metabolites catalyzed by HO-1, carbon monoxide and bilirubin, protect against H(2)O(2)-induced cytotoxicity in LA-4 cells. Quercetin may well be one of the promising substances to attenuate oxidative epithelial cell injury in lung inflammation.

Essential role of DPPA3 for chromatin condensation in mouse oocytogenesis.

Dynamic alterations in chromatin configuration occur in mammalian oocytogenesis. Based on chromatin configuration patterns, fully grown oocytes are classified into two types. One is surrounded nucleolus (SN)-type and the other is nonsurrounded nucleolus (NSN)-type oocytes. Although chromatin condensation during the transition from NSN- to SN-type oocytes is a prerequisite for normal early embryonic development, the molecular mechanisms remain unclear. In this study, we analyzed the role of DPPA3 (also known as PGC7/Stella) in this transition using Dppa3-null oocytes. The NSN-to-SN transition was significantly impaired, and transcriptional repression was incomplete in the Dppa3-null oocytes. Additionally, we revealed that prior transcriptional repression was necessary for the NSN-to-SN transition. These findings demonstrate that DPPA3 is an essential factor for the production of functional oocytes through transcriptional repression and chromatin condensation.

Pramef12 enhances reprogramming into naïve iPS cells.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Somatic cell nuclear transfer can also be utilized to reprogram somatic cells into totipotent embryos, suggesting that factors present in oocytes potentially enhance the efficiency of iPS cell generation. Here, we showed that preferentially expressed antigen of melanoma family member 12 (Pramef12), which is highly expressed in oocytes, enhances the generation of iPS cells from mouse fibroblasts. Overexpression of Pramef12 during the early phase of OKSM-induced reprogramming enhanced the efficiency of iPS cell derivation. In addition, overexpression of Pramef12 also enhanced expression of naïve pluripotency-associated genes, Gtl2 located within the Dlk1-Dio3 imprinted region essential for full pluripotency, glycolysis-associated genes, and oxidative phosphorylation-associated genes, and it promoted mesenchymal-to-epithelial transition during iPS cell generation. Furthermore, Pramef12 greatly activated ß-catenin during iPS cell generation. These observations suggested that Pramef12 enhances OKSM-induced reprogramming via activation of the Wnt/ß-catenin pathway.

Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP.

Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135-210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173-210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS-PAGE, dynAP migrates anomalously at ~ 45 kDa, much larger than expected (22.5 kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135-210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.

Attenuation of transforming growth factor-ß-stimulated collagen production in fibroblasts by quercetin-induced heme oxygenase-1.

Quercetin is a flavonoid with a wide variety of cytoprotective and modulatory functions. Heme oxygenase-1 (HO-1) is an inducible enzyme. Its reaction product, carbon monoxide (CO), confers cellular protection in a number of conditions and diseases associated with oxidative or inflammatory lung injury. Furthermore, quercetin was reported to be a potent inducer of HO-1 in several cell types. We hypothesized that quercetin suppresses the production of collagen in fibroblasts via the induction of HO-1. Here, we showed that quercetin suppresses transforming growth factor-ß (TGF-ß)-induced collagen production in NIH3T3 cells and in normal human lung fibroblasts. This suppressive effect of quercetin was mediated by quercetin-induced HO-1. The suppression of collagen production was conferred by the reaction product of HO-1, CO, but not by bilirubin. Furthermore, the translocation of the nuclear factor E2-related factor-2 (Nrf2), an important transcription factor that regulates the expression of HO-1 from the cytoplasm to the nuclei, was demonstrated in NIH3T3 cells by exposure to quercetin. Assessment of the signal transduction pathway involved in TGF-ß signaling showed that quercetin stimulated the Smad and mitogen-activated protein kinase pathway to varying degrees. Our results demonstrate that quercetin exerts suppressive effects on the expression of collagen by the induction of HO-1. Idiopathic pulmonary fibrosis is the most lethal diffuse fibrosing lung disease, and is characterized by the deposition of extracellular matrix. Given that HO-1 is one of the important molecules emerging as a central player in diseases, quercetin or its derivatives, which effectively induced HO-1, will lead to new therapeutic strategies for promoting antifibrotic therapy in respiratory diseases.

A large-sized bubbling appearance of the glomerular basement membrane in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis.

We report an unusual pathological finding, a large-sized bubbling appearance of the glomerular basement membrane (GBM), in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis. The first renal biopsy specimen from 10 years ago, when systemic lupus erythematosus was diagnosed, demonstrated mild mesangial proliferation and subepithelial deposits (WHO classification: III + V). Light microscopy of the current biopsy using periodic acid methenamine silver (PAMS) stain demonstrated a large-sized bubbling appearance of the GBM; however, very weak immunoglobulin and complement deposition was observed in immunofluorescence studies. Routine electron microscopy demonstrated partial subendothelial expansion with electron-lucent materials, but no electron-dense deposits or amyloid fibrils. Electron microscopy with PAMS stain revealed electron-lucent endothelial scalloping, including some cellular components and microspheres in the GBM; however, it is not clear if these materials are derived from endothelial cells. One possibility is that these unique findings represent a recovery phase of lupus membranous nephritis; another is that these findings correspond to a new disease entity.