Abstract
Although DNA methylation and transcriptional repression are generally associated, a causal role for DNA methylation in silencing of retroviral vectors has not been established. The newer generation murine stem cell virus retroviral vector (MSCV) lacks many of the repressive cis-acting DNA sequences identified in Moloney murine leukemia virus but remains sensitive to transcriptional silencing in various cell types. To determine the contribution of cytosine methylation to MSCV silencing, we mutated CpG dinucleotides located in the MSCV long terminal repeat (LTR) that are clustered in the U3 region and directly spanning the transcription start site in the R region. Effects of the CpG mutations on MSCV silencing were assessed in murine embryonic stem cells. An analysis of numerous clonal proviral integrants showed that mutation of CpGs in both clusters eliminated proviral integrants that were completely silenced. Variegated expression was shown to represent a substantial component of intraclonal silencing and was independent of the presence of CpGs in the LTR. Treatment of transduced cells with 5-azadeoxycytidine delayed establishment of the silenced state but had only a modest effect on expression of some proviral integrants at late times post-transduction. These results are direct evidence for a causal contribution of DNA methylation in the LTR to MSCV silencing and define the promoter region CpGs as a repressive element in embryonic stem cells. Furthermore, distinct mechanisms are suggested for establishment and maintenance of the silenced proviral state.
Highlights
Because of their ability to stably integrate into their host cell genomes, murine oncoretroviruses are among the most commonly used vectors for gene delivery into mammalian cells
The newer generation murine stem cell virus retroviral vector (MSCV) lacks many of the repressive cis-acting DNA sequences identified in Moloney murine leukemia virus but remains sensitive to transcriptional silencing in various cell types
The newer generation MSCV retroviral vector investigated in this study lacks many of the known cis-acting repressive elements present in Moloney murine leukemia virus (MMLV) [21], it remains sensitive to transcriptional silencing in embryonic stem (ES) cells and hematopoietic stem cells (HSC) [2, 3, 22] and has been shown to harbor repressive cis-acting sequences in the long terminal repeat (LTR) and untranslated region [23]
Summary
Retroviral Vectors—The green fluorescent protein (GFP) coding sequence optimized for human codon usage [26] was inserted into the MSCV retroviral vector [21] in place of the phosphoglycerate kinaseneomycin cassette. Confirmation of CpG mutations in the 5Ј-LTRs of integrated provirus was confirmed by DNA sequencing of PCR-amplified provirus (data not shown). Integration Site Analysis—Genomic DNA purified from ES cell clones was digested with BamHI, which is present as a unique restriction site in MSCV located 3Ј of the GFP-coding sequence, and analyzed for the presence of proviral DNA by Southern blotting using a radiolabeled GFP sequence probe. SNARF-1 Labeling of Cells—For tracking cell divisions, transduced ES cells were sorted for proviral GFP expression as described above, incubated in PBS containing 5 M seminaphthorhodafluor-1 at 37 °C for 15 min (Molecular Probes, Eugene, OR), and re-introduced into culture following removal of free seminaphthorhodafluor-1. Amplifed DNA was sequenced with the primer 5Ј-GGATTTGAAAATGATTTTGTG-3Ј (rϭAϩG wobble)
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