Expression In Embryonal Carcinoma Cells Research Articles

CD24 targeting with NK-CAR immunotherapy in testis, prostate, renal and (luminal-type) bladder cancer and identification of direct CD24 interaction partners.

Alternative therapeutic options targeting urologic malignancies, such as germ cell tumours, as well as urothelial, renal and prostate carcinomas, are still urgently needed. The membrane protein CD24 represents a promising immunotherapeutical approach. The present study aimed to decipher the molecular function of CD24 in vitro and evaluate the cytotoxic capacity of a third-generation natural killer (NK) cell chimeric antigen receptor (CAR) against CD24 in urologic tumour cell lines. Up to 20 urologic tumour cell lines and several non-malignant control cells were included. XTT viability assays and annexin V/propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co-immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of CD24 expression by SOX2. N-and O-glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. The study demonstrates that SOX2 transactivates CD24 expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding and post-translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumour cells with NK-CD24-CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24+ tumour cells. Limitations of the study include the in vitro setting, which still has to be confirmed in vivo. In conclusion, we show that CD24 is a promising novel target for immune therapeutic approaches targeting urologic malignancies.

Abstract 2175: Role of p38 MAPK in regulating Cripto-1 expression and signaling in human embryonal carcinoma cells

Abstract Cripto-1 (CR-1) is a cell-membrane protein that is overexpressed in several different types of human carcinomas. We have previously shown that TGFβ1 and Activins can enhance CR-1 expression while BMP2 and BMP4 can inhibit CR-1 expression in NTERA-2 embryonal carcinoma cells and in LS174T colon cancer cells. Nevertheless, there is still a very incomplete picture as to the nature of the epigenetic, transcriptional and hormonal factors that might be involved in regulating Cripto-1 expression under different conditions. Previous studies have demonstrated that activation of p38 MAPK can regulate expression of Nodal, a Cripto-1 ligand, during early embryogenesis and is necessary for the specification of mouse cardiomyocytes from embryonic stem cells, which depends upon a Nodal-dependent signaling pathway involving Cripto-1. We therefore evaluated if activation of p38 MAPK might be involved in regulating Cripto-1 expression and Cripto-1 dependent signaling in human embryonal carcinoma cells. In undifferentiated NTERA-2 and NCCIT embryonal carcinoma cells two p38 MAPK inhibitors PD169316 and SB20358 completely inhibited the basal levels of CR-1 mRNA and protein expression in both cell lines. A JNK inhibitor SP600125 had only minor effects on CR-1 mRNA and protein expression whereas the p44/42 MAPK inhibitor U0126 showed no effect. Transfection of specific siRNA against p38 MAPK was also able to significantly interfere with CR-1 mRNA and protein expression in NTERA2 and NCCIT embryonal carcinoma cells. In contrast, knockdown of p44/42 MAPK or JNK with specific siRNA did not significantly affect CR-1 expression in embryonal carcinoma cells. We therefore evaluated whether CR-1 might trigger activation of p38 MAPK in embryonal carcinoma cells and in mouse mammary epithelial cells. Recombinant CR-1 protein was found to stimulate in a delayed fashion (after 50-60 minutes) p38 MAPK phosphorylation in mouse mammary epithelial cells and in embryonal carcinoma cells suggesting a feed-forward activation loop. Whether p38 MAPK functions in undifferentiated embryonal carcinoma cells to regulate CR-1 expression and/or signaling involving regulation of invasion, migration and epithelial to mesenchymal transition in human embryonal carcinoma cells and in mouse mammary epithelial cells is under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2175. doi:10.1158/1538-7445.AM2011-2175

Abstract 4943: Regulation of human Cripto-1 gene expression by nuclear receptors and DNA promoter methylation

Abstract Human Cripto-1 (CR-1) is a membrane bound protein that belongs to the epidermal growth factor (EGF)-CFC family. CR-1 plays an important role in regulating embryonic development while also having significant implications in various forms of carcinogenesis. Previous studies have shown that germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily of transcription factors, inhibits CR-1 expression in human embryonal carcinoma NTERA-2 cells following retinoic acid-induced differentiation due to direct binding to a DR0 element within the CR-1 promoter. In the present study, we investigated the effects on CR-1 gene expression of another member of the nuclear receptor superfamily, liver receptor homologue-1 (LRH-1), which can bind to the same GCNF-binding site within the promoter of several genes. NTERA-2 cells transfected with a CR-1 promoter luciferase reporter vector showed substantially increased CR-1 promoter activity in the presence of LRH-1 and decreased CR-1 promoter activity in the presence of GCNF. Retinoic acid treatment of NTERA-2 cells induced downregulation of CR-1 and LRH-1 mRNA and protein expression. In contrast, a strong increase in GCNF was detected in NTERA-2 cells that have been treated with retinoic acid. Additionally, chromatin immunoprecipitation assay displayed direct binding of LRH-1 to the CR-1 promoter in NTERA-2 cells. Real-time PCR and Western blot analysis of NTERA-2 cells transfected with LRH-1 siRNA revealed a significant decrease in CR-1 mRNA and protein expression, suggesting that LRH-1 is required for mantaining high levels of CR-1 expression in embryonal carcinoma cells. Real time PCR analysis for GCNF and LRH-1 in human embryonal carcinoma cells and in a panel of human breast cancer cell lines, expressing very low levels of CR-1, revealed that GCNF is highly expressed in MCF7, T47D and ZR75-1 breast cancer cells as compared to NTERA-2 and NCCIT embryonal carcinoma cells. Since one of the mechanisms used by GCNF to repress gene expression is through promoter DNA methylation, we investigated the role of epigenetic modification of the CR-1 promoter in embryonal and breast carcinoma cells. While NTERA-2 and NCCIT cells showed very low levels of CR-1 promoter methylation, in human breast cancer cells CR-1 promoter was highly methylated in agreement with low levels of CR-1 expression in these cell lines. Finally, treatment of various breast cancer cell lines with the demethylating agent 5-aza-2′-deoxycytidine alone or in combination with the histone deacetylase inhibitors trichostatin A or valproic acid induced a significant increase in CR-1 expression, thus overcoming the repressive effects of GCNF. Collectively, these findings offer promising insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4943.

660. The Effect of the B2 Mutation on Hematopoietic Reconstitution in the French X- SCID Gene Therapy Trial

Top of pageAbstract The B2 mutation arose in Murine Leukemia Virus (MLV) vectors selected for expression in embryonal carcinoma cells, and is postulated to prevent silencing resulting from the binding of undetermined factors to the integrated proviral DNA (Virology 193:690|[ndash]|699, 1993). This B2 mutation is also one of the few differences between the otherwise strikingly similar common |[gamma]|-chain retrovirus vectors that were used in the two well-known French and British X-SCID clinical trials. To date, three leukemias have occurred in the French trial, and none have occurred in the British trial. Although leukemias may still occur in the British trial, their absence to date impels us to investigate methodological differences between the two trials. An unusual property of the B2 mutation is derived from the fact that it resides embedded within the tRNA primer binding site of the retroviral genome, which causes it to revert at high frequency. This reversion occurs mechanistically when the tRNA primer binding site is reverse transcribed both from the full length genomic RNA during minus strand synthesis, and subsequently when the tRNA primer (covalently linked to the 5' end of minus strand cDNA) is copied into plus sense DNA. These DNAs (one copied from the B2 mutated RNA genome and one copied from the tRNA primer) later form a duplex prior to elongation of the 3' ends and synthesis of the remainder of the proviral genome. As a result, the proviral genome from B2 mutant retroviral vectors contains a single base pair mismatch at the site of the mutation. Progeny cells from such integrations show roughly equal distribution of wild-type and mutant proviral genomes, as would be predicted by equal probabilities of mismatch repair and/or segregation of the two strands into daughter cells. Although the B2 mutation has been shown to prevent silencing in murine systems, the evidence for an effect on gene expression in human cells is considerably weaker. If the level of gene expression in human cells is increased by the B2 mutation, and if the resulting increase in common |[gamma]|-chain gene expression increases proliferation and the efficiency of hematopoietic reconstitution after retrovirally transduced autologous X-SCID bone marrow transplantation, then we would expect that reconstituted blood samples from the French X-SCID gene therapy trial would show a bias in the fraction of cells that contained the B2 mutation relative to those that had reverted to wild type. With this in mind, we analyzed the proportion of B2 mutated genomes in peripheral blood mononuclear cells from 2 successfully treated X-SCID gene therapy patients which have not contracted leukemia, and compared them with control infections of HeLa cells using similar MFG-B2-|[gamma]|-chain vectors. Although we did observe frequent reversion of the B2 mutation, as expected, the data do not support the hypothesis that cells with B2 mutated vector genomes improve reconstitution significantly. In fact, B2 mutant genomes constituted approximately 30|[ndash]|45% of the total, suggesting that the B2 mutated genomes may have been occasionally selected against during hematopoietic reconstitution.

The Ras/Erk Pathway Induces Primitive Endoderm but Prevents Parietal Endoderm Differentiation of F9 Embryonal Carcinoma Cells

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

High-resolution analysis of cytosine methylation in the 5ĺong terminal repeat of retroviral vectors.

Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.

NF-Y binds to the CCAAT box motif of the FGF-4 gene and promotes FGF-4 expression in embryonal carcinoma cells.

FGF-4 appears to be the first fibroblast growth factor (FGF) expressed during embryogenesis, and its expression is critical for early mammalian development. FGF-4 is expressed in the embryonic cell lines, F9, D3, and NT2/D1; but its expression in these cells is repressed upon differentiation. Transcription of the FGF-4 gene in embryonic cells is regulated by an enhancer in the third exon and by a positive regulatory region upstream of the transcription start site. A CCAAT box motif within the positive regulatory region has been shown to support FGF-4 expression, but the factor that binds to this site in vivo has not been identified. In this report, we demonstrate that the transcription factor complex NF-Y binds to the FGF-4 CCAAT box motif when nuclear extracts from each of the embryonic cell lines and their differentiated cells were examined by gel mobility shift analyses. Importantly, we demonstrate that expression of a dominant-negative NF-YA mutant protein reduces the expression of FGF-4 promoter/reporter gene constructs in F9 EC cells. Hence, we provide strong evidence that the transcription factor NF-Y is involved in the expression of the FGF-4 gene.

Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites.

Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. We have previously shown that fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. We now demonstrate that increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer "spacing mutants" which failed to activate transcription were also compromised in their ability to form the Oct* complexes even though they could still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes.

NF‐Y binds to the CCAAT box motif of the FGF‐4 gene and promotes FGF‐4 expression in embryonal carcinoma cells

FGF-4 appears to be the first fibroblast growth factor (FGF) expressed during embryogenesis, and its expression is critical for early mammalian development. FGF-4 is expressed in the embryonic cell lines, F9, D3, and NT2/D1; but its expression in these cells is repressed upon differentiation. Transcription of the FGF-4 gene in embryonic cells is regulated by an enhancer in the third exon and by a positive regulatory region upstream of the transcription start site. A CCAAT box motif within the positive regulatory region has been shown to support FGF-4 expression, but the factor that binds to this site in vivo has not been identified. In this report, we demonstrate that the transcription factor complex NF-Y binds to the FGF-4 CCAAT box motif when nuclear extracts from each of the embryonic cell lines and their differentiated cells were examined by gel mobility shift analyses. Importantly, we demonstrate that expression of a dominant-negative NF-YA mutant protein reduces the expression of FGF-4 promoter/reporter gene constructs in F9 EC cells. Hence, we provide strong evidence that the transcription factor NF-Y is involved in the expression of the FGF-4 gene. Mol. Reprod. Dev. 48:301–309, 1997. © 1997 Wiley-Liss, Inc.

A Dynamic Balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and Retinoic Acid Receptor:Retinoid X Receptor Heterodimers Regulates Oct-3/4 Expression in Embryonal Carcinoma Cells

The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.

Regulatory sequences required for hst-1 expression in embryonal carcinoma cells

The hst-1 gene, which is implicated in mammalian embryonic development and morphological transformation of NIH3T3 cells, is expressed in undifferentiated F9 cells, but not in differentiated F9 and other well-differentiated cells, such as PYS-2, NIH3T3 and HeLa cells. An octamer element present in the 3' untranslated region acts as an enhancer. Although Oct3 is down-regulated when F9 cells are differentiated, transient expression of Oct3 did not enhance the hst-1 promoter activity in HeLa, NIH3T3 or PYS-2 cells. Thus, the role of Oct3 on hst-1 expression remains elusive, and an additional transcription factor which interacts may regulate hst-1 transcription in association with Oct1, Oct3 or both.

Characterization of the Moloney Murine Leukemia Virus Stem Cell-Specific Repressor Binding Site

The Moloney murine leukemia virus (M-MuLV) repressor binding site (RBS) mediates cell-type-specific repression in embryonal carcinoma (EC) cells of expression from several different promoters, including the M-MuLV long terminal repeat promoter. Silencing has been shown to depend on an element normally located in the proviral 5′ noncoding region and occurs at the DNA level in the absence of retroviral proteins. Using fragments of the RBS region, we now show that the minimal size of the silencer corresponds to M-MuLV nt 147-163 and overlaps with the retroviral primer binding site region by 17 of its 18 bp. A panel of point mutations within the RBS has been examined to yield a consensus RBS sequence which is consistent with the notion that a previously identified nuclear factor (binding factor A) mediates RBS repression. Viral vectors using neomycin, β-galactosidase, and luciferase reporters have been employed to show that RBS-mediated repression occurs in EC and embryonal stem, but not in other tested cell types. Repression was observed to occur within 48 hr of infection, prior to when global methylation of proviruses has been reported to occur. Repression also occurred after azacytidine treatment of EC cells, supporting the notion that the RBS functions independently of provirus methylation. However, levels of provirus methylation in selected cells were increased in the presence of a wild-type RBS, and methylation correlated with a secondary stage of virus repression. Thus, the M-MuLV RBS acts directly to control expression in EC cells and also appears to trigger a secondary level of repression which is coincident with provirus methylation.

Polyomavirus enhancer requirements for expression in embryonal carcinoma cells.

Wild type polyomavirus expression is suppressed in embryonal carcinoma (EC) cell lines. This suppression is alleviated when the EC cells are induced to differentiate. Several characterized host range mutants of polyoma overcome suppression and are able to express and replicate in the undifferentiated EC cells. These previously described isolates were obtained by serial passage of a wild type strain through PCC4 or F9 EC lines. We present a new pyPCC4 isolate (LPT) derived without selection in EC cells. Isolates with host range specificity for a given EC line have been reported to share several common rearrangements and features. These features are also observed in LPT. We report a novel feature shared by these mutants, including LPT, capable of expression in the EC cell line PCC4. In 8 of 10 isolates a novel sequence is created within the enhancer region by rearrangement junctions with near perfect homology to the AP-1 core consensus sequence, 'TGACT(C/A)A'. That the precise location of these junctions varies among these isolates suggest a functional role for this conserved sequence. Our goal is to understand the function of various mutations in host range mutants of polyoma. In order to understand the rearrangements necessary for expression and replication of polyoma in PCC4 cells, we have further characterized the limits of the B enhancer in these cells as compared to those described in permissive cell systems. We have been able to locate the origin proximal limit of the B enhancer for replication close to nt 5189 and distinguish it from the origin proximal limit of the B enhancer for transcription near nt 5215. The two B enhancer cores overlap but do not coincide and are conserved in both cell lines.

A Stem Cell-Specific Silencer in the Primer-Binding Site of a Retrovirus

Retrovirus expression in embryonal carcinoma (EC) cells is blocked at a postintegration stage of the viral life cycle, in part because of the inadequate function of the viral long terminal repeat promoter in this cell type. However, selection for retrovirus expression in EC cells has identified mutations in Moloney murine leukemia virus (M-MuLV) located in the tRNA primer-binding site (PBS) region which relieve the EC cell-specific repression. We have found that exchanging the M-MuLV proline PBS for a glutamine one in a recombinant virus permits expression in EC cells. By using the recombinant virus as a backbone, the EC cell-specific repressor-binding site (RBS) element has been mapped to M-MuLV nucleotides 147 to 174. The RBS does not require precise positioning downstream of the M-MuLV promoter and can function in either orientation and in an intron, indicating that the regulatory effect is probably at the DNA, rather than RNA, level. We also show that the RBS element can repress heterologous promoters from an upstream position. Our results indicate that the RBS acts as a silencer that its inhibitory effect is mediated by a trans-acting factor, and that the mechanism of action is probably at the level of transcription. Through in vitro binding assays we have identified a binding factor which specifically recognizes the wild-type RBS sequence (binding factor A). The binding characteristics of factor A suggest that it is a stem cell repressor which acts at the M-MuLV RBS. Our DNA-binding assays also have identified a unique binding factor (binding factor Hp) which specifically recognizes a hemimethylated form of the wild-type RBS. This factor may play a role in methylation mediated control of retrovirus expression in EC cells.

A stem cell-specific silencer in the primer-binding site of a retrovirus.

Retrovirus expression in embryonal carcinoma (EC) cells is blocked at a postintegration stage of the viral life cycle, in part because of the inadequate function of the viral long terminal repeat promoter in this cell type. However, selection for retrovirus expression in EC cells has identified mutations in Moloney murine leukemia virus (M-MuLV) located in the tRNA primer-binding site (PBS) region which relieve the EC cell-specific repression. We have found that exchanging the M-MuLV proline PBS for a glutamine one in a recombinant virus permits expression in EC cells. By using the recombinant virus as a backbone, the EC cell-specific repressor-binding site (RBS) element has been mapped to M-MuLV nucleotides 147 to 174. The RBS does not require precise positioning downstream of the M-MuLV promoter and can function in either orientation and in an intron, indicating that the regulatory effect is probably at the DNA, rather than RNA, level. We also show that the RBS element can repress heterologous promoters from an upstream position. Our results indicate that the RBS acts as a silencer that its inhibitory effect is mediated by a trans-acting factor, and that the mechanism of action is probably at the level of transcription. Through in vitro binding assays we have identified a binding factor which specifically recognizes the wild-type RBS sequence (binding factor A). The binding characteristics of factor A suggest that it is a stem cell repressor which acts at the M-MuLV RBS. Our DNA-binding assays also have identified a unique binding factor (binding factor Hp) which specifically recognizes a hemimethylated form of the wild-type RBS. This factor may play a role in methylation mediated control of retrovirus expression in EC cells.

Determinants of retrovirus gene expression in embryonal carcinoma cells

The expression of Moloney murine leukemia virus is restricted in embryonal carcinoma (EC) cells. To characterize specific mutations necessary for expression of retroviruses in EC cells, we analyzed the expression of retrovirus mutants and recombinants thereof in EC cell lines F9 and PCC4. DNA sequence comparison and functional studies allowed us to define three point mutations in the enhancer region of the viral mutants at positions -345, -326, and -166 and two point mutations within the 5'-untranslated region of the viral genome at positions +164 and +165 that were essential for retrovirus expression in EC cells. DNA fragments derived from either the wild type or mutant viruses were used to search for sequence-specific DNA-binding factors in nuclear extracts from undifferentiated PCC4 cells. A cellular factor was found to bind strongly to sequences within the enhancer region (-354 to -306) of wild-type viruses but only weakly to sequences derived from mutant viruses. This factor was named ECF-I (for EC cell factor I). Retroviral expression in EC cells correlates with decreased binding affinity for ECF-I.

Evidence for a Stem Cell-Specific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.

Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells.

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.

Deletions in a recombinant retrovirus genome associated with its expression in embryonal carcinoma cells.

We analyzed embryonal carcinoma cell lines infected with a recombinant Moloney murine leukemia virus. Lines that carried but did not express the neo gene retained a provirus of LTR-gag-pol-neo-LTR, where LTR is a long terminal repeat, whereas all G418-resistant lines deleted regions that included the primer binding site and the splicing donor site. This suggested the presence of multiple inhibitory elements.

Identification of new human repetitive sequences: characterization of the corresponding cDNAs and their expression in embryonal carcinoma cells

We have identified new repeated interspersed DNA sequences by analysis of homologous RNA transcripts from a human teratocarcinoma cell line (NTERA-2 clone D1). The abundance of transcripts varies upon retinoic acid induced differentiation of NTERA-2/D1 cells, and it is highest when the cells display the embryonal carcinoma phenotype. The expression of these novel repeated sequences appears to be tissue specific as no detectable expression was found in various cell lines of different embryological derivation. Characterization of the RNA transcripts by analysis of recombinant cDNA clones indicated that transcripts of different genomic units are present in undifferentiated embryonal teratocarcinoma cells. Nucleotide sequencing of the cloned cDNAs reveals a complex structure composed by unique and tandemly repeated sub-elements.