Mechanism of suppression of the long terminal repeat of Moloney leukemia virus in mouse embryonal carcinoma cells.

Abstract

Sequence-specific DNA-binding proteins that bind to the long terminal repeat (LTR) of Moloney leukemia virus in undifferentiated and differentiated mouse embryonal carcinoma (EC) cells were identified by gel retardation assay. The proteins that bind to the CCAAT box were present in both undifferentiated and differentiated EC cells. The amounts and the number of species of the proteins that bind to the enhancer and the GC-rich region were far lower in undifferentiated EC cells than in the differentiated counterparts. These proteins were supposed to be transcriptional activators. Proteins that bind upstream of the enhancer, namely, the -352 to -346 region and the -407 to -404 region, were identified. These proteins were designated the embryonic LTR-binding protein (ELP) and the LTR-binding protein, respectively. The ELP was present only in undifferentiated EC cell lines. The LTR-binding protein was detected in all cell lines tested. The mechanism of suppression of the LTR was investigated by the chloramphenicol acetyltransferase assay. The enhancer and the GC-rich region of the LTR functioned poorly in undifferentiated cells. When eight copies of ELP-binding sequences were inserted upstream of the enhancer region, expression of the chloramphenicol acetyltransferase gene was reduced about threefold in ECA2 cells. From these data, we concluded that two mechanisms, the shortage of activator proteins and the presence of a negative regulatory protein (ELP), are involved in the suppression of the LTR in undifferentiated EC cells.

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

“…12,15 In the Moloney murine leukemia virus (Mo-MuLV), a number of regulatory elements have been found to influence transcriptional down-regulation of viral genes from the LTR. 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.…”

“…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.…”

Stable transfection of MSCs by electroporation

“…We elected not to employ viral vectors because of their tendency to be inactivated through methylation, induce immune reactions, and impose a limit on the size of the exogenous gene that can be introduced. [6][7][8] Instead, we selected to develop improved conditions for transduction of MSCs by electroporation. Keating et al 9 previously used electroporation to create stable cell lines of human MSCs (hMSCs) by expressing the immortalizing SV40 large Tantigen.…”

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

“…The block to replication occurs after the viral DNA has integrated into the host genome and is caused by transcriptional silencing of the proviruses (2,4). This silencing is the result of several cumulative effects that include poor enhancer function of the 5Ј LTR (5-7), the binding of several cell-type specific transacting transcriptional repressors (6,8), and de novo DNA methylation (4). A critical target of this repression is the DNA element known as the repressor binding site (RBS) (2,5,9,10).…”