Bisulfite sequencing of the Mos gene. Open circles represent non-methylated cytosines in CpGs in the analyzed sequence, and the filled circles represent methylated cytosines. Rows of circles represent unique DNA molecules as described in the text. The gray area depicts CpGs within the cognate sequence. ZP3-TG, transgenic; WT, wild type.

Bisulfite sequencing of the RNAi transgene in tail DNA. The RNAi transgene is methylated even if it is not active. Tail sample, in which the Zp3 promoter is not active, was obtained from one of the transgenic females used for analysis of Mos methylation in transgenic oocytes (Figure 2). Open circles represent non-methylated cytosines in CpGs in the analyzed sequence, and the filled circles represent methylated cytosines. Rows of circles represent unique DNA molecules as described in the text. The Mos PCR amplified the central part of the inverted repeat (dotted line plus full line) but only the first 200 nt (full line) could be sequenced due to the nature of the template. EGFP, enhanced green fluorescent protein; IR, inverted repeat.

Schematic description of the model system. The transgene [described in detail in (15,17)] expresses a dsRNA hairpin. The length of the expressed dsRNA is ∼0.5 kb and this RNA is presumably capped and polyadenylated. The bottom shows the position of the homologous sequence and the amplified sequence in the Mos gene (Mos is an intronless gene). EGFP, enhanced green fluorescent protein; IR, inverted repeat. The coding sequence is red. The schematic diagram of the Mos gene is drawn to scale.

Quantification of bisulfite sequencing results. (A) Relative abundance of non-CpG cytosines per 100 cytosines in upstream and cognate sequences in the S1amplicon. Black bars, cognate sequence (256 nt of the amplicon, 63 non-CpG cytosines); white bars, upstream sequence (289 nt, 72 non-CpG cytosines). The data are expressed as the mean ± SEM. (B) Normalized frequency of methylated cytosines in the cognate sequence and upstream. The total number of methylated CpGs in a given region of a given sample was divided by the number of individual clones multiplied by the number of CpGs in the region. The resulting number thus represents the average DNA methylation frequency per CpG in the given region. The data are expressed as the mean ± SEM. TAIL WT, tail samples of wild-type animals; TAIL ZP3, tail samples from transgenic animals; OO WT, wild-type oocytes; OO WT*, same as previous sample, except one DNA clone with four cytosines was omitted for the analysis to demonstrate its effect on the outcome of the experiment; OO ZP3, oocytes from transgenic RNAi animals, in which dsRNA is expressed, PG ZP3, parthenogenotes obtained from spontaneous parthenogenetic activation of matured oocytes from transgenic animals.