• Klíčová slova
• syncytin-1,
• MeSH
• endogenní retroviry * MeSH
• genom MeSH
• lidé MeSH
• Retroviridae * genetika   MeSH
• retrovirové infekce MeSH
• Check Tag
• lidé MeSH

Syncytin-1 is a captive envelope glycoprotein encoded by one of human endogenous retroviruses W. It is expressed exclusively in the placental trophoblast where it participates in cell-to-cell fusion during differentiation of syncytiotrophobast. In other tissues, however, syncytin-1 expression must be kept in check because inadvertent cell fusion might be dangerous for tissue organization and integrity. We describe here an inverse correlation between CpG methylation of syncytin-1 5' long terminal repeat and its expression. Hypomethylation of the syncytin-1 5' long terminal repeat in the placenta and in the choriocarcinoma-derived cell line BeWo was detected. However, other analyzed primary cells and cell lines non-expressing syncytin-1 contain proviruses heavily methylated in this sequence. CpG methylation of syncytin-1 is resistant to the effect of the demethylating agent 5-azacytidine. The inhibitory role of CpG methylation is further confirmed by transient transfection of in-vitro-methylated syncytin-1 promoter-driven reporter construct. Altogether, we conclude that CpG methylation plays a principal role in the transcriptional suppression of syncytin-1 in non-placental tissues, and, in contrast, demethylation of the syncytin-1 promoter in trophoblast is a prerequisite for its expression and differentiation of multinucleated syncytiotrophoblast.

• MeSH
• buněčné linie MeSH
• CpG ostrůvky fyziologie   MeSH
• down regulace imunologie   MeSH
• financování organizované MeSH
• genové produkty env antagonisté a inhibitory   biosyntéza   genetika   MeSH
• HeLa buňky MeSH
• koncové repetice MeSH
• lidé MeSH
• metylace DNA MeSH
• placenta metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• těhotenské proteiny antagonisté a inhibitory   biosyntéza   genetika   MeSH
• transkripční faktory antagonisté a inhibitory   biosyntéza   genetika   MeSH
• trofoblasty metabolismus   MeSH
• Check Tag
• lidé MeSH

BACKGROUND: Syncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors. RESULTS: We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. CONCLUSIONS: We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.

• MeSH
• DNA virů metabolismus   MeSH
• endogenní retroviry genetika   MeSH
• epigeneze genetická MeSH
• genové produkty env biosyntéza   MeSH
• lidé MeSH
• metylace DNA MeSH
• regulace genové exprese * MeSH
• seminom patologie   virologie   MeSH
• těhotenské proteiny biosyntéza   MeSH
• testikulární nádory patologie   virologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. RESULTS: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. CONCLUSIONS: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

• MeSH
• buněčné linie MeSH
• fibroblasty virologie   MeSH
• fluorescence MeSH
• genové produkty env genetika   metabolismus   MeSH
• konfokální mikroskopie MeSH
• kur domácí MeSH
• lidé MeSH
• placenta virologie   MeSH
• těhotenské proteiny genetika   metabolismus   MeSH
• těhotenství MeSH
• transportní systém ASC pro aminokyseliny genetika   metabolismus   MeSH
• vedlejší histokompatibilní antigeny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Syncytin-1, a human fusogenic protein of retroviral origin, is crucial for placental syncytiotrophoblast formation. To mediate cell-to-cell fusion, Syncytin-1 requires specific interaction with its cognate receptor. Two trimeric transmembrane proteins, Alanine, Serine, Cysteine Transporters 1 and 2 (ASCT1 and ASCT2), were suggested and widely accepted as Syncytin-1 cellular receptors. To quantitatively assess the individual contributions of human ASCT1 and ASCT2 to the fusogenic activity of Syncytin-1, we developed a model system where the ASCT1 and ASCT2 double knockout was rescued by ectopic expression of either ASCT1 or ASCT2. We demonstrated that ASCT2 was required for Syncytin-1 binding, cellular entry, and cell-to-cell fusion, while ASCT1 was not involved in this receptor interaction. We experimentally validated the ASCT1-ASCT2 heterotrimers as a possible explanation for the previous misidentification of ASCT1 as a receptor for Syncytin-1. This redefinition of receptor specificity is important for proper understanding of Syncytin-1 function in normal and pathological pregnancy.

• MeSH
• antigeny CD98 - těžký řetězec MeSH
• fúze buněk * MeSH
• genové produkty env * metabolismus   genetika   MeSH
• lidé MeSH
• placenta * metabolismus   MeSH
• těhotenské proteiny * metabolismus   genetika   MeSH
• těhotenství MeSH
• transportní systém ASC pro aminokyseliny * metabolismus   genetika   MeSH
• transportní systémy pro neutrální aminokyseliny metabolismus   genetika   MeSH
• trofoblasty metabolismus   cytologie   MeSH
• vedlejší histokompatibilní antigeny metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Bude stanovena genová exprese syncytinu-1 a 2 na úrovni mRNA v nádorech mužské germinální linie, zejména seminomech, smíšených germinálních karcinomech a choriokarcinomech. Bude sledována korelace s histologickým obrazem nádorů, hlavně s přítomností mnohojaderných obřích buněk. Exprese syncytinů bude posouzena co do prediktivního a diagnostického významu pro klinickou praxi. Dále budeme sledovat sestřih syncytinové mRNA pomocí PCR se specifickými primery umístěnými v intronech nebo exonech. Pomocí bisulfitové reakce popíšeme rovněž pattern CpG methylace syncytinového promotoru a LTR. Na buněčných liniích odvozených od příslušných nádorů germinální linie budeme sledovat pomocí chromatinové imunoprecipitace histonové modifikace a další epigentické parametry syncytinových lokusů.; We will examine the gene expression of syncytin-1 and 2 at the level of mRNA in tumors of the male germ line, particularly seminomas, mixed germinal carcinomas, and choriocarcinomas. This will be correlated with the tumor histology, mainly with the representation of of multinuclear giant cells. Expression of syncytins will be assessed as to their predictive and diagnostic value for the clinics. Furthermore, we will follow the splicing of syncytin mRNAs using the PCR with splice-specific primers localized in exons and introns. Using the bisulfite technique, we will describe the patterns of CpG methylation of syncytin promoters and LTRs in aforementioned tumors. In the cell lines derived from respective germ line tumors, we will examine histone modifications and other epigenomic features of syncytin loci using the chromatin immunoprecipitation.

• MeSH
• choriokarcinom MeSH
• chromatinová imunoprecipitace MeSH
• exony MeSH
• exprese genu MeSH
• genové produkty env MeSH
• germinální a embryonální nádory MeSH
• histony MeSH
• mikro RNA analýza   MeSH
• polymerázová řetězová reakce MeSH
• Retroviridae - proteiny onkogenní MeSH
• seminom MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• andrologie
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Syncytin-1 and -2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. We studied the epigenetic suppression of ERVWE1 and ERVFRDE1 5'-long terminal repeats by DNA methylation and chromatin modifications. Immunoprecipitation of the provirus-associated chromatin revealed the H3K9 trimethylation at transcriptionally inactivated syncytins in HeLa cells. qRT-PCR analysis of non-spliced ERVWE1 and ERVFRDE1 mRNAs and respective env mRNAs detected efficient splicing of endogenously expressed RNAs in trophoblastic but not in non-placental cells. Pointing to the pathogenic potential of aberrantly expressed syncytin-1, we have found deregulation of transcription and splicing of the ERVWE1 in biopsies of testicular seminomas. Finally, ectopic expression experiments suggest the importance of proper chromatin context for the ERVWE1 splicing. Our results thus demonstrate that cell-specific retroviral splicing represents an additional epigenetic level controling the expression of endogenous retroviruses.

• MeSH
• buněčné linie MeSH
• endogenní retroviry MeSH
• epigeneze genetická MeSH
• genetická transkripce MeSH
• genové produkty env genetika   metabolismus   MeSH
• germinální a embryonální nádory genetika   metabolismus   MeSH
• glykoproteiny genetika   metabolismus   MeSH
• HeLa buňky MeSH
• histony metabolismus   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• proviry genetika   metabolismus   MeSH
• sestřih RNA MeSH
• těhotenské proteiny genetika   metabolismus   MeSH
• testis metabolismus   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• syncytin-1,
• MeSH
• genové produkty env genetika   MeSH
• komplikace těhotenství prevence a kontrola   MeSH
• lidé MeSH
• placenta metabolismus   MeSH
• těhotenské proteiny * MeSH
• těhotenství MeSH
• transportní systém ASC pro aminokyseliny MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH

Equilibrative ( SLC29A) and concentrative ( SLC28A) nucleoside transporters contribute to proper placental development and mediate uptake of nucleosides/nucleoside-derived drugs. We analyzed placental expression of SLC28A mRNA during gestation. Moreover, we studied in choriocarcinoma-derived BeWo cells whether SLC29A and SLC28A mRNA levels can be modulated by activity of adenylyl cyclase, retinoic acid receptor activation, CpG islands methylation, or histone acetylation, using forskolin, all- trans-retinoic acid, 5-azacytidine, and sodium butyrate/sodium valproate, respectively. We found that expression of SLC28A1, SLC28A2, and SLC28A3 increases during gestation and reveals considerable interindividual variability. SLC28A2 was shown to be a dominant subtype in the first-trimester and term human placenta, while SLC28A1 exhibited negligible expression in the term placenta only. In BeWo cells, we detected mRNA of SLC28A2 and SLC28A3. Levels of the latter were affected by 5-azacytidine and all- trans-retinoic acid, while the former was modulated by sodium valproate (but not sodium butyrate), all- trans-retinoic acid, 5-azacytidine, and forskolin that caused 25-fold increase in SLC28A2 mRNA; we documented by analysis of syncytin-1 that the observed changes in SLC28A expression do not correlate with the morphological differentiation state of BeWo cells. Upregulated SLC28A2 mRNA was reflected in elevated uptake of [3H]-adenosine, high-affinity substrate of concentrative nucleoside transporter 2. Using KT-5720 and inhibitors of phosphodiesterases, we subsequently confirmed importance of cAMP/protein kinase A pathway in SLC28A2 regulation. On the other hand, SLC29A genes exhibited constitutive expression and none of the tested compounds increased SLC28A1 expression to detectable levels. In conclusion, we provide the first evidence that methylation status and activation of retinoic acid receptor affect placental SLC28A2 and SLC28A3 transcription and substrates of concentrative nucleoside transporter 2 might be taken up in higher extent in placentas with overactivated cAMP/protein kinase A pathway and likely in the term placenta.

• MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• ekvilibrační proteiny přenášející nukleosidy genetika   metabolismus   MeSH
• gestační stáří * MeSH
• karbazoly farmakologie   MeSH
• lidé MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• nádorové buněčné linie MeSH
• placenta účinky léků   metabolismus   MeSH
• pyrroly farmakologie   MeSH
• těhotenství MeSH
• upregulace MeSH
• vývojová regulace genové exprese účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH