RESUMEN

Cell fusion in the placenta is tightly regulated. Suppressyn is a human placental endogenous retroviral protein that inhibits the profusogenic activities of another well-described endogenous retroviral protein, syncytin-1. In this study, we aimed to elucidate the mechanisms underlying suppressyn's placenta-specific expression. We identified the promoter region and a novel enhancer region for the gene encoding suppressyn, ERVH48-1, and examined their regulation via DNA methylation and their responses to changes in the oxygen concentration. Like other endogenous retroviral genes, the ERVH48-1 promoter sequence is found within a characteristic retroviral 5' LTR sequence. The novel enhancer sequence we describe here is downstream of this LTR sequence (designated EIEs: ERV internal enhancer sequence) and governs placental expression. The placenta-specific expression of ERVH48-1 is tightly controlled by DNA methylation and further regulated by oxygen concentration-dependent, hypoxia-induced transcription factors (HIF1α and HIF2α). Our findings highlight the involvement of (1) tissue specificity through DNA methylation, (2) expression specificity through placenta-specific enhancer regions, and (3) the regulation of suppressyn expression in differing oxygen conditions by HIF1α and HIF2α. We suggest that these regulatory mechanisms are central to normal and abnormal placental development, including the development of disorders of pregnancy involving altered oxygenation, such as preeclampsia, pregnancy-induced hypertension, and fetal growth restriction.

Asunto(s)

Retrovirus Endógenos , Trofoblastos , Femenino , Humanos , Embarazo , Fusión Celular , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Productos del Gen env/genética , Productos del Gen env/metabolismo , Oxígeno/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo

RESUMEN

We previously identified suppressyn (SUPYN), a placental protein that negatively regulates the cell fusion essential for trophoblast syncytialization via binding to the trophoblast receptor for syncytin-1, ASCT2, and hypothesized that SUPYN may thereby regulate cell-cell fusion in the placenta. Here, we redefine in vivo SUPYN localization using specific monoclonal antibodies in a rare early placental sample, showing SUPYN localization in villous and extravillous trophoblast subtypes, the decidua and even in placental debris in the maternal vasculature. In human trophoblast cell lines, we show SUPYN alters ASCT2 glycosylation within the secretory pathway and that this binding is associated with inhibition of cell fusion. Using newly-optimized trophoblast isolation protocols that allow tracking of ex vivo cell fusion, we present transcription and translation dynamics of fusion-related proteins over 96 hours in culture and the effects of changes in ambient oxygen levels on these processes. We report converse syncytin-1 and SUPYN transcriptional and translational responses to surrounding oxygen concentrations that suggest both are important in the effects of hypoxia and hyperoxia on placental syncytialization. Our results suggest that SUPYN's anti-fusogenic properties may be exerted at several sites in the maternal body and its dysregulation may be associated with diseases of abnormal placentation.

Asunto(s)

Productos del Gen env/metabolismo , Placentación/fisiología , Proteínas Gestacionales/metabolismo , Trofoblastos/metabolismo , Adulto , Sistema de Transporte de Aminoácidos ASC/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicosilación , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Embarazo , Adulto Joven

RESUMEN

While common in viral infections and neoplasia, spontaneous cell-cell fusion, or syncytialization, is quite restricted in healthy tissues. Such fusion is essential to human placental development, where interactions between trophoblast-specific human endogenous retroviral (HERV) envelope proteins, called syncytins, and their widely-distributed cell surface receptors are centrally involved. We have identified the first host cell-encoded protein that inhibits cell fusion in mammals. Like the syncytins, this protein, called suppressyn, is HERV-derived, placenta-specific and well-conserved over simian evolution. In vitro, suppressyn binds to the syn1 receptor and inhibits syn1-, but not syn2-mediated trophoblast syncytialization. Suppressyn knock-down promotes cell-cell fusion in trophoblast cells and cell-associated and secreted suppressyn binds to the syn1 receptor, ASCT2. Identification of the first host cell-encoded inhibitor of mammalian cell fusion may encourage improved understanding of cell fusion mechanisms, of placental morphogenesis and of diseases resulting from abnormal cell fusion.

Asunto(s)

Retrovirus Endógenos/metabolismo , Productos del Gen env/metabolismo , Placenta/metabolismo , Proteínas Gestacionales/metabolismo , Trofoblastos/fisiología , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos ASC/metabolismo , Animales , Secuencia de Bases , Western Blotting , Fusión Celular , Línea Celular , Línea Celular Tumoral , Retrovirus Endógenos/genética , Femenino , Productos del Gen env/genética , Productos del Gen env/farmacología , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/farmacología , Unión Proteica , Interferencia de ARN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Trofoblastos/citología , Trofoblastos/efectos de los fármacos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/farmacología

RESUMEN

Genetic and epigenetic factors can potentially alter susceptibility to psychiatric disorders such as schizophrenia. In order to explore the effect of epigenetics on the pathogenesis of schizophrenia, we examined the global methylation level of leukocyte DNA from 210 patients with schizophrenia (124 males and 86 females) and 237 healthy subjects (108 males and 129 females). Methylated deoxycytidine (mC) content in peripheral leukocyte DNA was measured by high performance liquid chromatography (HPLC). We confirmed in the healthy subjects our previous finding that there are sex-dependent differences in mC content (males>females; beta=0.319, p<0.001), in addition to the effect of age (beta=-0.141, p=0.022). We therefore used multiple regression to analyze the data from all subjects by sex, with age as a co-variant. In males, a tendency was observed toward lower mC content in patients than in controls (beta=-0.115, p=0.075), with a significant effect of age (beta=-0.212, p<0.001). This difference was more prominent in younger individuals. In females, no effect of age or disease status on mC content was observed. These results established that there is significant sex-dependent difference in the mC content of human peripheral leukocyte DNA, and raise the possibility that alterations in DNA methylation state are present in patients with schizophrenia.

Asunto(s)

Metilación de ADN , Epigénesis Genética , Leucocitos/metabolismo , Esquizofrenia/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Niño , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores Sexuales

RESUMEN

BACKGROUND: We previously found that there is a subtle difference in the global methylation state of blood leukocyte DNA between male subjects with and without schizophrenia. The aim of the current study was to determine whether this difference was a primary effect of the disease state, or a secondary effect of antipsychotics administered to these patients. METHODS: We examined the methyl cytosine (mC) content of DNA from the leukocytes, brain, and liver of rats using high performance liquid chromatography. A total of 40 male and female rats received for 21 days daily injection of haloperidol or vehicle solution alone. RESULTS: In control rats injected with buffer only, there was a sex-dependent difference in mC content in leukocyte DNA (male > female; P = 0.028, n = 10), similar to our previous observations in human peripheral leukocytes. No difference in mC content between the sexes was observed in the brain or liver in buffer-treated animals. Haloperidol treatment slightly decreased the mC content of leukocytes in male rats, but unexpectedly, increased the mC content of leukocytes in females. We observed a trend toward a higher level of mC in the liver in both sexes following haloperidol treatment, compared to buffer-treated animals. In contrast, haloperidol treatment resulted in a decrease in mC content in the brain in females, and this difference was statistically significant (P = 0.026). CONCLUSION: These results indicate that haloperidol can affect DNA methylation states in the brain, as well as in certain other tissues, and raise the possibility that antipsychotic drugs play a role in the observed disparity in mC content in male subjects with and without schizophrenia.

RESUMEN

The purpose of this study was to know a role of confined placental mosaicism (CPM) in perinatal outcome and postnatal growth and development of infants with intrauterine growth restriction (IUGR). We selected 50 infants with IUGR (<-2.0 SD) from 3,257 deliveries in a regional medical center during the past 10-year period, and carried out cytogenetic and molecular analyses in their placenta and cord blood. Of the 50 infants, 8 had CPM (CPM group) and were composed of five single (CPM2, 7, 13, 22, and 22), one double (CPM7/13), and one quadruple trisomy (CPM2/7/15/20), and one partial monosomy [del(2)(p16)]. The origin of an extra chromosome of trisomy was maternal in six cases of CPM, paternal in one, and undetermined in one. Uniparental disomy in disomic cell lines was ruled out in all these mosaics. We also compared clinical parameters for perinatal outcome between CPM group and infants without evidence of CPM (non-CPM group), such as maternal and gestational age, birth weight, Apgar score, cord blood pH, gender, and uterine artery patterns by Doppler ultrasonography, as well as weight, height, and developmental quotient (DQ) by Denver Developmental Screening Test at age 12 months. Phenotypic abnormalities were noted in two infants with CPM and three infants of non-CPM group: One with CPM22 had ASD and hypospadias, one with CPM7/13 had Russell-Silver syndrome (RSS), and one without CPM had polydactyly, and two without CPM had RSS. All but one infant with CPM are alive at age 12 months. Among the clinical parameters, the detection rate of a notch waveform pattern of the uterine artery was significantly higher in the CPM group (P < 0.05). However, no significant difference was noted in perinatal outcome of pregnancy and in DQ at age 12 months between the two groups. Interestingly, short stature (<-2 SD) at age 12 months was more frequently seen in CPM group (7/8 infants with CPM vs. 8/15 infants without CPM), although no statistically significant difference was obtained. The information obtained will be useful for perinatal care and genetic counseling for infants with IUGR and CPM.

Asunto(s)

Retardo del Crecimiento Fetal/diagnóstico , Mosaicismo , Placenta/ultraestructura , Adulto , Femenino , Retardo del Crecimiento Fetal/etiología , Humanos , Lactante , Recién Nacido , Cariotipificación , Masculino , Embarazo , Resultado del Embarazo , Diagnóstico Prenatal , Estudios Retrospectivos , Trisomía

RESUMEN

The purpose of the present study was to understand the tissue specificity of DNA methylation and the relationship between methylation and expression of genes with essential roles in neurodevelopment and brain function. We chose dopamine receptor genes (DRD1 and DRD2), NCAM, and COMT as examples of genes with CpG islands around the promoter region, and serotonin receptor genes (HTR2A and HTR3A), HCRT, and DRD3 as genes without CpG islands. Methylation states were investigated in fetal brain, fetal liver, placenta, and in adult peripheral leukocytes from three individuals by Southern blot and bisulfite-modified DNA sequencing. A repetitive sequence, human endogenous retrovirus (HERV)-K was also examined. All genes examined were almost completely unmethylated in brains. The genes with CpG islands were unmethylated regardless of their expression state. In contrast, genes without CpG islands showed various methylation patterns, which did not necessarily reflect the transcriptional activity of the genes. Most HERV-K loci were methylated, but some loci showed relatively low methylation in the placenta and liver. Interestingly, we found inter-individual differences in methylation levels in HTR2A and HCRT in the placenta and in some loci of HERV-K in the placenta and liver. The sample with the lowest methylation levels in the two unique genes showed higher methylation of HERV-K loci than the other samples. These results provide detailed information about the methylation states of the genes analyzed and evidence for inter-individual variations in methylation in both unique and repetitive sequences.

Asunto(s)

Metilación de ADN , Retrovirus Endógenos/genética , Neuropéptidos/genética , Receptores de Amina Biogénica/genética , Encéfalo/citología , Encéfalo/enzimología , Encéfalo/metabolismo , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/metabolismo , Islas de CpG/genética , Retrovirus Endógenos/enzimología , Retrovirus Endógenos/metabolismo , Expresión Génica , Humanos , Modelos Genéticos , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Especificidad de Órganos , Regiones Promotoras Genéticas , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The GATM gene encodes L-arginine:glycine amidinotransferase, which catalyzes the conversion of L-arginine into guanidinoacetate, the rate-limiting step in the synthesis of creatine. Since, deficiencies in creatine synthesis and transport lead to certain forms of mental retardation in human, the human GATM gene appears to be involved in brain development. Recently it has been demonstrated that the mouse Gatm is expressed during development and is imprinted with maternal expression in the placenta and yolk sac, but not in embryonic tissues. We investigated the imprinting status of the human GATM by analyzing its expression in four human placentas. GATM was biallelically expressed, thus suggesting that this gene escapes genomic imprinting in placentas, differently from what has been reported in mouse extra-embryonic tissues.

Asunto(s)

Humanos , Animales , Femenino , Amidinotransferasas , Impresión Genómica , Glicina , Creatinina , Placenta , Polimorfismo Genético

RESUMEN

The evolutional and biological roles of human endogenous retroviruses (HERVs) are less recognized compared to those of L1. In the present study, we focused on the transcriptional activity of HERVs in normal human tissues and found five HERV loci that are actively expressed in normal tissues. All but one showed tissue specificity of expression: one was expressed in stomach and small intestine and three were in placenta. We subsequently examined by TaqMan-based RT-PCR assays the temporal expression profiles of the three placenta-specific HERVs along with syncytin and syncytin 2 and observed three patterns. Syncytin and HERV-Fb showed almost constant expression through gestations. Syncytin 2 gradually decreased as pregnancy proceeded. In contrast, expression from the HERV-H/F and HERV-K(HML-6) loci increased remarkably in term placentas. Term placentas in general showed larger interindividual differences in HERV expression levels. Our results suggest that HERVs might have more diverse effects than currently thought.

Asunto(s)

Retrovirus Endógenos/metabolismo , Feto/virología , Regulación del Desarrollo de la Expresión Génica , Productos del Gen env , Transcripción Genética , Bases de Datos Genéticas , Retrovirus Endógenos/genética , Etiquetas de Secuencia Expresada , Femenino , Productos del Gen env/genética , Productos del Gen env/metabolismo , Humanos , Hibridación in Situ , Especificidad de Órganos/genética , Placenta/virología , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular

RESUMEN

Genetic studies of neuropsychiatric disorders have often produced conflicting results, which might partly result from the involvement of epigenetic modifications. We intended to explore the possible implication of DNA methylation and human endogenous retroviruses (HERVs) in neuropsychiatric disorders. In the present study, we identified two HERV loci that are expected to retain the transcriptional activity in the brain. One was located on chromosome 1q21-q22 and the other on 22q12. Interestingly, these regions were overlapped with or included in those of schizophrenia-susceptible loci, SCZD9 and SCZD4, respectively. Particularly, the HERV on 22q12 was located in the opposite direction 4 kb downstream of the Synapsin III gene. These HERV loci could afford clear targets for methylation and expression analyses in postmortem brains of patients with psychiatric disorders such as schizophrenia. In addition, we confirmed our previous finding that only a few of particular HERV-K loci were activated among a number of highly homologous loci in teratocarcinoma cell lines. These activated loci included ones common to all teratocarcinoma cell lines analyzed and depending on their male or female origin.

Asunto(s)

Encéfalo/virología , Retrovirus Endógenos/genética , Trastornos Mentales/virología , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 22 , Metilación de ADN , Retrovirus Endógenos/aislamiento & purificación , Elementos de Facilitación Genéticos , Etiquetas de Secuencia Expresada , Femenino , Humanos , Masculino , Regiones Promotoras Genéticas , ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esquizofrenia/genética , Transcripción Genética

RESUMEN

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.

Asunto(s)

Impresión Genómica , Receptores de Esteroides/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Islas de CpG/genética , ADN/química , ADN/genética , ADN/metabolismo , Metilación de ADN , Femenino , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sintenía

RESUMEN

The mouse achaete-scute homolog-2 gene (Ascl2 or Mash2) encodes a transcription factor playing a role in the development of the trophoblast. The Ascl2 is an imprinted gene with maternal expression and assigned to an imprinting gene cluster region (ICR) at a distal region of mouse chromosome 7. We previously isolated a phage clone carrying the human homolog, ASCL2, and mapped it to human chromosome 11p15.5, a human ICR. In the present study, we demonstrate the expression patterns of the human ASCL2 in the fetus at a stage between first and second trimesters and in the placental tissues. In addition, it has been shown that the human ASCL2 gene escapes genomic imprinting.

Asunto(s)

Proteínas de Unión al ADN/genética , Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica/genética , Impresión Genómica/genética , Factores de Transcripción , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Northern Blotting , ADN/química , ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Impresión Genómica/fisiología , Humanos , Hibridación in Situ , Placenta/metabolismo , Polimorfismo de Longitud del Fragmento de Restricción , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Técnica del ADN Polimorfo Amplificado Aleatorio