FIGURE 7.

Down-regulation of BLV promoter activity by site-specific methylation. A, using site-directed mutagenesis we generated BLV promoter luciferase constructs with site-specific methylation at the −154 CpG or/and −129 CpG (pLTR-154me, pLTR-129me, and pLTR-154/-129me, respectively) as well as its unmethylated counterpart, pLTRwt-luc. By the same technique, we generated mutated BLV promoter luciferase constructs containing point mutation abolishing transcription factor binding to the CRE1 or CRE2 motif (pLTRmutCRE1 or pLTRmutCRE2, respectively). The products from these mutagenesis reactions (500 ng of DNA) were transfected directly into HeLa cells using the FuGENE procedure. Each transfection included 50 ng of the internal control plasmid, pRL-TK, in which the herpes simplex virus thymidine kinase promoter drives Renilla luciferase gene expression. Luciferase activities (firefly and Renilla) were measured in cell lysates 44 h after transfection. The results are expressed as luciferase_firefly/luciferase_Renilla. As an additional control, we generated a pLTRwt-luc derivative that was hypermethylated only in the LTR (i.e. in which every CpG of the BLV LTR, but no CpG elsewhere in the pLTRwt-luc plasmid, was methylated in vitro by SssI methyltransferase) and named pLTRmSssIme. The results are presented as histograms indicating the luciferase activity of each reporter construct relative to that measured with the pLTRwt-luc vector, which was arbitrarily assigned an activity value of 100%. The down-regulation of BLV promoter activity by methylation(s) or by mutation(s) is also indicated (-fold) at the top of the histogram representation. Means ± S.E. from five independent transfections performed with different DNA products are shown. B, HeLa cells were transiently cotransfected using the FuGENE procedure with 500 ng of pLTRwt-luc, pLTRmutCRE1, or pLTRmutCRE2 and 50 ng of the internal control plasmid, pRL-TK. Here, in contrast to the plasmids used in A, the reporter plasmids were amplified in bacteria prior to transfection. Luciferase activities (firefly and Renilla) were measured in cell lysates 44 h after transfection. The results are expressed as Luciferase_firefly/luciferase_Renilla. The results are presented as histograms indicating the luciferase activity of each reporter construct relative to that measured with the pLTRwt-luc vector, which was arbitrarily assigned a value of 100% of activity. The down-regulation of BLV promoter activity by mutations is indicated (in -fold) at the top of the histogram representation. Means ± S.E. from triplicate samples are represented. An experiment representative of two independent transfections is shown.