De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.

Abstract

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection ofDNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression ofproviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.In eukaryotes, methylation of cytosine residues at the 5' position has been extensively studied as a possible mechanism involved in the control ofgene expression and these studies have shown that an inverse correlation exists between gene expression and the level of methylation (1-3).As a means to study gene expression in mammals, we have introduced the murine retrovirus Moloney murine leukemia virus (M-MuLV) into the germ line of mice. A number of substrains, each carrying one M-MuLV genome at a different chromosomal position, have been derived and the expression of the M-MuLV genome has been shown to differ between some of them (4). A previous report (5) showed that the germ line-transmitted copy of the viral genome was methylated and noninfectious, whereas the somatically acquired copies, derived from the germ-line copy, were hypomethylated and infectious. Furthermore, cloning of the germ-line gene, which removes the methyl group from the cytosine bases, rendered the germ-line copy infectious (6). Because the proviral DNA introduced into the mouse embryos to derive the Mov-substrains was nonmethylated, de novo methylation of the integrated proviral genome must have occurred at some point during derivation ofthe substrains. This may have occurred either during the development of the infected embryo or as a consequence of transmitting the proviral genome through the germ line to the next generation.To (20) were used for virus production. All cells were grown as described (21) in Dulbecco's modified Eagle's medium with 10% fetal calf serum (heat-inactivated at 56WC for 30 min before use).Infection of the Cells with M-MuLV. Two protocols were used to infect cells with M-MuLV. (i) F9 EC cells were infected by cocultivation with mitomycin C-treated (10 ,g/ml ...

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

“…However, de novo methylation was seen in several symmetrically unmethylated DNA sequences introduced into mammalian cells and in host DNA sequences hypomethylated by treatment with 5-azacytidine (13)(14)(15)(16)(17).…”

A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells

“…A summary of the methylation status of CCGG sites in transfected plasmids is presented in Fig. 3 (28). This differs from the inefficient de novo methylation activity observed in most somatic cells (19,28) and the sequence-specific de novo methylation observed in Yl adrenocortical cells (29 (34).…”

“…De novo activity is much higher in embryonal carcinoma cells than in differentiated cells (19,28), but there is no evidence for any additional species of methylase (2) What is the contribution of sequence diversity to the generation of HpaII tiny-fragment islands? Methylation-free islands may reflect the substrate specificity of de novo methylation during embryogenesis.…”

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

“…[11][12][13][14] The retroviral vector driving the stable gene expression in the mouse brain is required in the adult brain of the organism to be studied. In primary neural stem cells, the gene expression from the CE vector containing the cellular promoter was shut down more easily than those from MT, MS and MP vectors containing viral LTRs.…”

“…Moreover, when the same titer of the CE and MS vectors was coinjected into the embryonic brain, the MS vector drove the expression of the transgenes more stably than the CE vector in the adult mouse brain. It has previously been thought that the main reason for the retroviral silencing was the de novo methylation of CpG sequence in viral promoters, 11,12,14 and thus the use of a cellular promoter as an internal promoter had been expected to confer resistance to silencing. However, this does not seem to be the case for the CE vector.…”