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

Abstract

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 m 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.Moloney murine leukemia virus (M-MuLV) does not productively infect early embryonic stem cells (22) or murine embryonal carcinoma (EC) cells (17,40,42). EC cells share many properties with early embryonic stem cells (24) and are a convenient cell culture system with which to study MMuLV restriction. The block in the viral life cycle occurs at the stage of viral RNA accumulation, subsequent to proviral DNA integration (11,41). At least two cis-acting elements are responsible for the block in RNA expression: the MMuLV enhancer and an M-MuLV intragenic element. The M-MuLV enhancer is inactive in EC cells (29,30), possibly due to an enhancer-specific repressor (19) or the lack of specific positive enhancer-binding factors (36). The intragenic element spans the M-MuLV proline tRNA primerbinding site (PBS) and inhibits RNA expression from MMuLV recombinants specifically in EC cells (13,30,47). Competition assays indicate that the inhibition is mediated by a trans-acting factor. The mechanism of action is probably at the transcriptional level, since inhibition is independent of the orientation of the intragenic element (31). In addition, a point mutation in the tRNA PBS of an M-MuLV host range mutant, termed B2, allows virus replication (4) and expression (47) in EC cells.In this report, we precisely define the borders of the intragenic inhibitory element, identify the element as a transcriptional silencer sequence, and correlate in vitro factor-binding activity at the tRNA PBS to EC-specific repression.MATERUILS AND METHODS Cell lines. The PC13 (2), F9 (5), and C2 cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics. The F9 and PC13 EC cells were grown on gelatin-coated plasticware.Recombinant constructions. The mutagenized recombinants used to map the negative regulatory element (NRE) were derived by synthesis of the indicated oligonucleotides with a SaI 4-base-pair (bp) overhang and a BgilI restriction * Corresponding author. site at the 5' and 3' termini, respectively. The annealed oligonucleotides were ligated with MLV.4...

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

“…These include the enhancer repeat unit, 16,17 the negatively acting cis element in the upstream conserved region (UCR) that binds the YY1 transcription factor, 18,19 and the primer binding site (PBS). 17,20,21 Methylation of cytosine bases in vector sequences has been associated with the observed transcriptional down-regulation. [22][23][24][25] However, the precise role of methylation in mediating the repression of gene expression is not clear.…”

TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells

“…Previous work from many laboratories has documented the presence of strong sequence-specific DNA-binding activity for the PBS Pro of MLV in lysates of embryonic cells (9,12,13,24). Similar tests for DNA-binding activity in F9 EC cell extracts, when performed using the DNA sequence of PBS Lys-1,2 , the PBS used by retroviruses such as visna, spuma, and Mason-Pfizer monkey virus (20)(21)(22), yielded a complex detected by EMSA with a shift indicative of similar size as the complex that binds to the PBS Pro sequence (9) (Fig.…”

No Evidence for Infection of Human Embryonic Stem Cells by Feeder Cell–Derived Murine Leukemia Viruses

“…The fact that neither human HEK293 cells nor hESCs had been infected by MEF-derived MuLV and that productive infection of hESCs was achieved after coculture with MLV-X strongly argues against a specific inhibition of MuLV expression and replication in hESCs as the underlying reason, like previously described for murine ESCs and embryonic carcinoma cells [25][26][27][28]. Although our experiments supplied evidence for humantropic MuLV mRNA expression and virion release, it is much more likely that ICR-derived feeder cells do not release infectious and replication-competent humantropic virions, or at least not to a degree capable to infect and replicate.…”

“…6). Moreover, robust MuLV mRNA expression was detected in hESCs after coculture with MLV-X, suggesting that in contrast to murine embryonic carcinoma cells [25][26][27][28], MuLV expression is not significantly repressed in hESCs on the transcription level (Fig. 6).…”

“…Whereas MuLV usually replicate efficiently in differentiated somatic cells, there is some evidence that MuLV expression in embryonic cells is depressed [25][26][27][28]. So far, it has not been investigated whether MuLV expression is inhibited in embryonic/fetal fibroblasts.…”