Seiki Haraguchi

NCYM, a Cis-Antisense Gene of MYCN, Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Dependence receptor UNC5D mediates nerve growth factor depletion–induced neuroblastoma regression

Spontaneous regression of neuroblastoma (NB) resembles the developmentally regulated programmed cell death (PCD) of sympathetic neurons. Regressing tumor cells express high levels of the nerve growth factor (NGF) receptors TRKA and p75NTR and are dependent on NGF for survival; however, the underlying molecular mechanism remains elusive. Here, we show that UNC5D, a dependence receptor that is directly targeted by p53 family members, is highly expressed in favorable NBs. NGF withdrawal strongly upregulated UNC5D, E2F1, and p53 in human primary favorable NBs. The induced UNC5D was cleaved by caspases 2/3, and the released intracellular fragment translocated into the nucleus and interacted with E2F1 to selectively transactivate the proapoptotic target gene. The cleavage of UNC5D and its induction of apoptosis were strongly inhibited by addition of netrin-1. Unc5d(-/-) mice consistently exhibited a significant increase in dorsal root ganglia neurons and resistance to NGF depletion-induced apoptosis in sympathetic neurons compared with wild-type cells. Our data suggest that UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence-mediated PCD during NB regression.

Muscle Atrophy and Motor Neuron Degeneration in Human NEDL1 Transgenic Mice

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease. Approximately 20% cases of familial ALS show the mutation in the superoxide dismutase-1 (SOD1) gene. We previously demonstrated that homologue to E6AP carboxyl terminus- (HECT-) type ubiquitin protein E3 ligase (NEDL1) physically bind to mutated SOD1 protein but not wild-type SOD1 and promote the degradation of mutated SOD1 protein through ubiquitin-mediated proteasome pathway. To further understand the role of NEDL1 involved in the pathogenesis of familial ALS, we generated transgenic mice with human NEDL1 cDNA. The transgenic mice with human NEDL1 expression showed motor dysfunctions in rotarod, hanging wire, and footprint pattern examination. Histological studies indicated degeneration of neurons in the lumbar spinal cord and muscle atrophy. The number of activated microglia in the spinal cord of transgenic mice was significantly higher than that of wild-type mice, suggesting that inflammation might be observed in the spinal cord of transgenic mice. In conclusion, these findings suggest that the human NEDL1 transgenic mice might develop ALS-like symptoms, showing signs of motor abnormalities, accompanied with significant reduction in muscle strength.

A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse

Background The TH-MYCN transgenic mouse is the most widely used murine model of human neuroblastoma, in which a human MYCN oncogene is targeted to neuroectodermal cells of developing mice under the influence of the rat tyrosine hydroxylase promoter. So far, homozygous transgenic mice have been identified by either Southern blot or quantitative real-time PCR. Principal Findings To establish a simple and reliable genotyping method by conventional PCR, we confirmed the integration of the transgene in the TH-MYCN transgenic mouse by Southern blot and inverse PCR analyses. Our results showed that either five or six copies were found to be inserted in a head-to-tail tandem configuration at a single locus. The MYCN transgene/host DNA junction was sequenced and the integration site was identified at chromosome 18qE4. Finally, we succeeded in designing rapid, simple and reliable genotyping method by common PCR using primers flanking the integrated TH-MYCN transgene. Conclusion We established a simple and reliable genotyping PCR method for determining the integration site of the TH-MYCN transgene that enables all possible genotypes to be distinguished within several hours. TH-MYCN mice are excellent model for human neuroblastoma study, thus our results will largely be useful for facilitating the pace of neuroblastoma study, including in the study of the tumourigenic process, and in the development of therapies to treat patients suffering from neuroblastoma.

Downregulation of Histone Methyltransferase Genes SUV39H1 and SUV39H2 Increases Telomere Length in Embryonic Stem-like Cells and Embryonic Fibroblasts in Pigs

Abstract Telomere is a nucleoprotein structure at the ends of chromosomes that helps to protect the ends of chromosomes from being fused with other chromosomes. Knockout of histone methyltransferases Suv39h1 and Suv39h2 increases the telomere length in murine cells, whereas downregulation of SUV39H1 and SUV39H2 genes decreases the telomere length in human cells, suggesting that telomere biology is different among mammalian species. However, epigenetic regulation of the telomere has not been studied in mammals other than the human and mouse. In the present study, the effect of knockdown of SUV39H1 and SUV39H2 genes on telomere length was examined in porcine embryonic stem-like cells (pESLCs) and porcine embryonic fibroblasts (PEFs). The telomeres in SUV39H1 and SUV39H2 knockdown (SUV39KD) pESLCs (37.1 ± 0.9 kb) were longer (P<0.05) compared with those of the control (33.0 ± 0.7 kb). Similarly, SUV39KD PEFs had longer telomeres (22.1 ± 0.4 kb; P<0.05) compared with the control (17.8 ± 1.1 kb). Telomerase activities were not different between SUV39KD pESLCs (10.4 ± 1.7) and the control (10.1 ± 1.7) or between SUV39KD PEFs (1.0 ± 0.3) and the control (1.0 ± 0.4), suggesting that telomerase activities did not contribute to the telomere elongation in SUV39KD pESLCs and SUV39KD PEFs. Relative levels of trimethylation of histone H3 lysine 9 and expressions of DNMT1, DNMT3A and DNMT3B were decreased in SUV39KD cells, suggesting that telomere lengthening in SUV39KD pESLCs and SUV39KD PEFs might be not only related to the loss of histone modification marks but also linked to the decrease in DNA methyltransferase in pigs.

Development of single blastomeres derived from two-cell embryos produced in vitro in pigs

The objective was to investigate development of single blastomeres derived from IVP two-cell porcine embryos. There was no difference (P > 0.05) in blastocyst rates among intact two-cell embryos (IN), zona-free two-cell embryos (ZF), and single blastomere (SB) groups (50.0 ± 9.7, 57.4 ± 5.7, and 45.1 ± 7.2%, respectively; mean ± SEM). However, blastocyst yield for the SB group (90.2 ± 14.4%, based on the original number of two-cell embryos before blastomere separation) was higher (P < 0.05) than those of IN and ZF groups. Although the number of inner cell mass (ICM) and trophectoderm (TE) cells in SB blastocysts (6.2 ± 0.8 and 15.5 ± 1.1, respectively) was lower (P < 0.05) than those in IN (12.4 ± 1.3 and 26.0 ± 3.8) and ZF blastocysts (10.7 ± 1.6 and 26.4 ± 3.4), ICM:TE ratios did not differ significantly among groups. Expressions of transcripts associated with cellular organization (TUBA1 and TUBB) were reduced (P < 0.05) in SB versus IN blastocysts. However, there was no significant difference among groups for expression of transcripts associated with responses to stress (HSPE1, HSPD1, and HSPCA) or glucose catabolism (ENO1, COX6C, COX7B, NDUFA4, NDUFA13, UCRC, and UQCRFS1) in blastocysts. The percentage of the sister blastomere pairs in which both cells developed to blastocysts (36.6 ± 5.3%) or both degenerated (46.3 ± 10.3%) were higher (P < 0.05) than that of the pairs in which one developed to blastocyst while the other degenerated (17.1 ± 7.8%). When both pairs developed to blastocysts, one blastocyst had more (P < 0.05) ICM and TE cells (8.2 ± 1.2 and 20.2 ± 2.1, respectively) than the other (5.2 ± 0.9 and 13.5 ± 1.1), although ICM:TE cell ratios were not significantly different. In conclusion, blastomere separation at the two-cell stage significantly increased blastocyst yield from IVP porcine embryos. This might be a useful approach for conservation of rare pig breeds, in which low numbers of embryos limited the success of embryo transfer.

Telomere Elongation During Morula-to-Blastocyst Transition in Cloned Porcine Embryos

Although telomeres are elongated during morula-to-blastocyst transition in cloned embryos, it is still unknown whether donor cell types have any effect on this elongation. In the present study, we examined the changes of telomere length during morula-to-blastocyst transition in cloned porcine embryos using different types of donor cells. Porcine embryonic stem-like cells (pESLCs), porcine cumulus cells (PCs), and porcine embryonic fibroblasts at passages 7 and 10 (PEF7s and PEF10s, respectively) were used as donor cells. Telomere lengths of pESLCs (35.8±1.5 kb), PCs (24.4±0.5 kb), PEF7s (18.7±0.6 kb), and PEF10s (17.2±0.1 kb) were significantly different. In contrast, telomere length in morulae derived from pESLCs (18.2±0.3 kb), PC (17.8±0.7 kb), PEF7 (18.5±0.3 kb), and PEF10 (18.4±0.4 kb) did not differ significantly. Likewise, telomeres in blastocysts derived from pESLCs (22.3±1.5 kb), PCs (23.5±2.6 kb), PEF7s (20.2±1.0 kb), and PEF10s (20.9±1.0 kb) had similar lengths. However, telomeres in blastocysts were significant longer (p<0.05) compared with morulae in each group. Relative telomerase activities of morulae derived from pESLCs (4.2±0.4), PCs (4.0±0.5), PEF7s (5.1±0.4), and PEF10s (4.9±0.4) were significantly lower (p<0.01) than those of blastocysts derived from pESLCs (8.2±1.1), PCs (8.6±0.6), PEF7s (12.5±2.9), and PEF10s (8.3±1.1). In conclusion, the telomere elongation in cloned pig embryos that occurred during morula-to-blastocyst transition may be related to the rise of telomerase activity. The telomere elongation may also be independent of the type and telomere length of the donor cell.

Naloxone increases maturation rate and ratio of inner cell mass to total cells in blastocysts in pigs

The purposes of the present study were to examine the effect of naloxone, a mu-opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M-II) porcine oocytes, one-, four-cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10(-8) mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10(-4) mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10(-3) mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10(-8 ) mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10(-6) mol/L and 10(-8) mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.

Oncogenic Lmo3 cooperates with Hen2 to induce hydrocephalus in mice

We previously reported that LMO3 and HEN2 act as oncogenes in neuroblastoma development through up-regulating MASH1 transcription by interfering with HES1. To confirm these results in vivo, we generated transgenic mice of these genes. Lmo3 or Hen2 was expressed under the control of Wnt1 promoter, which is expressed in the central nervous system and neural crest of the sympathoadrenal lineage from which neuroblastoma develops. Heterozygous Lmo3 and Hen2 transgenic mice (Tg (Lmo3) and Tg (Hen2)) developed hydrocephalus at higher frequency than for the wild type mice, and all heterozygous double-transgenic mice (Tg (Lmo3; Hen2)) developed hydrocephalus. Therefore, Lmo3 and Hen2 may be involved in and have synergistic effects on hydrocephalus development. Although aqueduct stenosis occurred in all genotypes, it was mild in Tg (Lmo3; Hen2) mice. Furthermore, hydrocephalus was detected at E18.5 in Tg (Lmo3; Hen2). These results suggest that the causes of hydrocephalus are not only aqueduct stenosis but also disorder of neocortical development. A similar phenotype was reported in Robo1/2−/− mice, in which Hes1 expression level was decreased in ventricular zone progenitors. Thus, it is suggested that the expression levels of Lmo3 and/or Hen2 could determine the fate of stem cells by inhibiting Hes1 function during nervous system development and might be a trigger of aberrant neurogenesis in vivo.