Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

Identification of the avian myeloblastosis virus genome. II. Restriction endonuclease analysis of DNA from lambda proviral recombinants and leukemic myeoblast clones.

Two lambda proviral DNA recombinants were characterized with a number of restriction endonucleases. One recombinant contained a complete presumptive avian myeloblastosis virus (AMV) provirus flanked by cellular sequences on either side, and the second recombinant contained 85% of a myeloblastosis-associated virus type 1 (MAV-1)-like provirus with cellular sequences adjacent to the 5' end of the provirus. Comparing the restriction maps for the proviral DNAs contained in each lambda hybrid showed that the putative AMV and MAV-1-like genomes shared identical enzyme sites for 3.6 megadaltons beginning at the 5' termini of the proviruses with respect to viral RNA. Two enzyme sites near the 3'-end of the MAV-1-like provirus were not present in the putative AMV genome. We also examined a number of leukemic myeloblast clones for proviral content and cell-provirus integration sites. The presumptive AMV provirus was present in all the leukemic myeloblast clones regardless of the endogenous proviral content of the target cells or the AMV pseudotype used for conversion. Multiple cellular sites were suitable for integration of the putative AMV genome and the helper genomes. The proviral genomes were all integrated colinearly with respect to linear viral DNA.     

Np95 interacts with de novo DNA methyltransferases,Dnmt3a and Dnmt3b,and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.     

Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.     

Mobility of endogenous ecotropic murine leukemia viral genomes within mouse chromosomal DNA and integration of a mink cell focus-forming virus-type recombinant provirus in the germ line

Characterization of endogenous ecotropic Akv proviruses in DNA of low and high leukemic mouse strains revealed the presence of one to six copies of the Akv genome per haploid genome equivalent integrated in the germ line. Low leukemic strains analyzed so far contained only one complete copy of the Akv proviral DNA. The site of integration varied among strains, although genetically related strains often carried the Akv proviral gene in the same chromosomal site. The different substrains of the AKR mouse displayed the presence of variable numbers (two to six) of Akv genomes. In all substrains one Akv genome was present in an identical chromosomal site; this locus probably comprised the progenitor genome. Closely related substrains had several Akv proviral DNAs integrated in common sites. The accumulation of Akv genomes in the germ line of the AKR/FuRdA strain is likely the result of independent integration events, since backcross studies with the Akv-negative 129 strain showed random segregation of all six proviral loci. The AKR/Cnb strain carried a recombinant provirus in the germ line. This provirus resembled in structure the AKR mink cell focus-forming viruses, which are generated by somatic recombination during leukemogenesis. Therefore, the germ-line amplification of Akv proviral DNAs occurs most likely through infection of embryonic cells by circulating virus.     

Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.