Kateřina Trejbalová

Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin

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.

The transcription factor EGR1 regulates metastatic potential of v-src transformed sarcoma cells.

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines—the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

Nonconserved Tryptophan 38 of the Cell Surface Receptor for Subgroup J Avian Leukosis Virus Discriminates Sensitive from Resistant Avian Species

ABSTRACT Subgroup J avian leukosis virus (ALV-J) is unique among the avian sarcoma and leukosis viruses in using the multimembrane-spanning cell surface protein Na+/H+ exchanger type 1 (NHE1) as a receptor. The precise localization of amino acids critical for NHE1 receptor activity is key in understanding the virus-receptor interaction and potential interference with virus entry. Because no resistant chicken lines have been described until now, we compared the NHE1 amino acid sequences from permissive and resistant galliform species. In all resistant species, the deletion or substitution of W38 within the first extracellular loop was observed either alone or in the presence of other incidental amino acid changes. Using the ectopic expression of wild-type or mutated chicken NHE1 in resistant cells and infection with a reporter recombinant retrovirus of subgroup J specificity, we studied the effect of individual mutations on the NHE1 receptor capacity. We suggest that the absence of W38 abrogates binding of the subgroup J envelope glycoprotein to ALV-J-resistant cells. Altogether, we describe the functional importance of W38 for virus entry and conclude that natural polymorphisms in NHE1 can be a source of host resistance to ALV-J.

DNA vaccination against v-src oncogene-induced tumours in congenic chickens

DNA vaccination is particularly efficient for induction of cytotoxic T-lymphocyte (CTL) response. In our experiments, we used MHC(B) congenic chicken lines CB and CC (regressors and progressors of v-src-induced tumours, respectively) and a mutated, non-oncogenic v-src gene construct as the DNA vaccine. A high degree of vaccine protection against oncogenic v-src challenge was achieved in the CB line chickens. CTL response was demonstrated in vitro and by adoptive transfer of immune cells to the syngeneic host and to the CC line chickens rendered tolerant to CB cells. In the CC line chickens we observed tumour growth retardation after a low-dose DNA vaccination administered to immature recipients while higher amounts of DNA vaccine in immunocompetent chickens exerted an enhancing effect.

Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A

ABSTRACT The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.

Proviral load and expression of avian leukosis viruses of subgroup C in long-term persistently infected heterologous hosts (ducks)

Summary.Proviral DNA load and expression of avian leukosis viruses of subgroup C (ALV-C) in ducks infected in mid embryogenesis were studied using quantitative PCR, RT-PCR, in situ hybridization employing ALV-specific riboprobe, and immunohistochemistry. A group of long-term surviving, non-reviremic ducks was selected for the study and compared to control reviremic animals in order to obtain information about persisting retroviruses in different duck tissues. A widespread distribution of proviruses in the tested tissues was found, but the proviral load was significantly lower in non-reviremic in comparison to reviremic animals. The only exception were brain and blood cells, in which no significant difference in the quantity of integrated proviruses was found between both categories of ducks, thus indicating an exceptional position of the brain and blood cells among all tested tissues. Contrary to reviremic, the proviruses were not transcribed in non-reviremic ducks, with the exception of brain and thymus. In the majority of non-reviremic ducks viral RNA was revealed in the brain, but no infectious virus could be recovered from this tissue. The opposite situation was observed in the thymus, where infectious virus was recovered but viral RNA remained below the detection limit of the assay. As revealed by in situ analysis, infected cells were either disseminated or focally distributed in tissues. From the long-term follow up of ALV-C in intraembryonally infected ducks we conclude that this model is suitable for the study of retrovirus persistence accompained both by the presence and absence of reviremias. The possible consequences of transmission and long-term persistence of retroviruses in the heterologous host for retroviral evolution are discussed.

Downregulation of HOPX Controls Metastatic Behavior in Sarcoma Cells and Identifies Genes Associated with Metastasis

Comparing the gene expression profiles of metastatic and nonmetastatic cells has the power to reveal candidate metastasis-associated genes, whose involvement in metastasis can be experimentally tested. In this study, differentially expressed genes were explored in the v-src-transformed metastatic cell line PR9692 and its nonmetastatic subclone PR9692-E9. First, the contribution of homeodomain only protein X (HOPX) in metastasis formation and development was assessed. HOPX-specific knockdown decreased HOPX expression in the nonmetastatic subclone and displayed reduced cell motility in vitro. Critically, HOPX knockdown decreased the in vivo metastatic capacity in a syngeneic animal model system. Genomic analyses identified a cadre of genes affected by HOPX knockdown that intersected significantly with genes previously found to be differentially expressed in metastatic versus nonmetastatic cells. Furthermore, 232 genes were found in both screens with at least a two-fold change in gene expression, and a number of high-confidence targets were validated for differential expression. Importantly, significant changes were demonstrated in the protein expression level of three metastatic-associated genes (NCAM, FOXG1, and ITGA4), and knockdown of one of the identified HOPX-regulated metastatic genes, ITGA4, showed marked inhibition of cell motility and metastasis formation. These data demonstrate that HOPX is a metastasis-associated gene and that its knockdown decreases the metastatic activity of v-src-transformed cells through altered gene expression patterns. Implications: This study provides new mechanistic insight into a HOPX-regulated metastatic dissemination signature. Mol Cancer Res; 11(10); 1235–47. ©2013 AACR.

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1‐3 enzymes and the level of their product, 5‐hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non‐seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real‐time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5‐methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography‐mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non‐seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This “anti‐methylator” phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.

Molecular events accompanying Rous sarcoma virus rescue from rodent cells and the role of viral gene complementation

ABSTRACT Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.

DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas

AbstractBackground 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.ResultsWe 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.ConclusionsWe 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.