Endogenous HP1β and HP1γ are enriched at the condensed transgenic locus but depleted upon transcriptional activation. At 24 h posttransfection with EYFP-LacR (A to D) (silent) or a combination of EYFP-LacR-VP16 and hTet-Off (E to H) (double activation), cells were immunofluorescently labeled with antibodies against HP1β (B and F) or HP1γ (D and H). The transgenic locus is highly condensed in silent cells (A and C; marked by EYFP-LacR), but significantly decondensed upon double activation (E and G; marked by EYFP-LacR-VP16). The images shown are optical sections taken with an ApoTome. Scale bar, 5 μm.

The CMVm promoter and transcribed CFP regions of the transgene are associated with HP1β, HP1γ, and H3K9me3. Cells mock transfected with pBluescriptII KS(−) (silent, indicated as OFF) or transcriptionally activated by hTet-Off (indicated as ON) were subjected to ChIP assays using antibodies against HP1β, HP1γ, and H3K9me3. The IP DNA and input DNA were analyzed by quantitative real-time PCR. The relative enrichment was determined by normalizing the immunoprecipitation percentage of transgene sequences (CMVm, black bars; CFP, gray bars), defined as the ratio of IP DNA to input DNA, against that of the active β-actin promoter region (white bars). The mean values and standard deviations from three independent experiments are shown.

Northern blot analyses of transgene-derived small RNAs. Low-stringency Northern blot hybridization using radiolabeled DNA probes homologous to the lac operator (lanes 1 to 4), TRE (lanes 5 to 8), CMVm promoter (lanes 9 to 12), and CFP (lanes 13 to 16) sequences in the transgene was performed to detect transgene-specific small RNAs. Small RNA fractions isolated from parental BHK cells (7.5 μg/lane for lanes 1, 5, 9, and 13), clone 2 cells (7.5 μg/lane for lanes 2, 6, 10, and 14), or clone 2 cells transfected with pSilencer plasmids (0.5 μg/lane for lanes 3, 4, 7, 8, 11, 12, 15, and 16) were loaded for probing. The closed triangles (lanes 3, 7, 11, and 15) represent transiently expressed siRNAs. The electrophoretic mobilities of the digoxigenin-labeled 22-nt and 25-nt DNA oligonucleotides are indicated. N, pSilencer-negative.

Dicer knockdown does not cause up-regulation of transgene expression. Control or Dicer-depleted cells were either mock transfected with pBluescriptII KS(−) (A) (silent) or transfected with phTet-Off and incubated for 24 h in the presence (B) (weak activation) or absence (C) (strong activation) of Dox. The relative CFP mRNA levels were determined as the ratios of CFP mRNA to β-actin mRNA. The mean values and standard deviations from three independent experiments using the average value of triplicate RT-PCRs are shown. The black bars represent the data from RT-PCRs, while the gray bars in panel A represent the data from direct PCRs without the reverse transcription step.

Effects of overexpressed transgene-directed siRNAs on transgene expression. Cells were activated by transfection with phTet-Off, together with the indicated pSilencer plasmids. The relative CFP mRNA levels in cells expressing transgene-directed siRNAs were determined by normalizing the ratios of CFP mRNA to β-actin mRNA against that of control cells cotransfected with pSilencer-negative. The mean values and standard deviations from three independent experiments using the average value of triplicate RT-PCRs are shown.

ECFP-HP1α and ECFP-SUV39H1 localize to the condensed transgenic locus. Cells were transiently transfected with combinations of plasmids encoding proteins as follows: EYFP-LacR and ECFP-HP1α (A and B) (silent); EYFP-LacR-VP16, ECFP-HP1α, and hTet-Off (C and D) (double activation); EYFP-LacR and ECFP-HP1α V22M (E and F) (silent); EYFP-LacR and ECFP-SUV39H1 (G and H) (silent); and EYFP-LacR, ECFP-SUV39H1, and hTet-Off (I and J) (single activation). The left panels show the transgenic loci, and the right panels show the distribution patterns of the ECFP fusion proteins. The cytoplasmic punctate fluorescence (D and J) represents peroxisomal CFP signals in the activated cells. The triangles indicate the sites of the transgenic loci. Scale bar, 5 μm.

Bisulfite genomic sequencing analysis of CpG methylation in the transgene array. (A) Distribution of 20 CpG dinucleotides in the CMVm promoter and 5′ untranslated region of the transgene. (B) Percentage of methylated clones for each CpG. Genomic DNA isolated from cells mock transfected with pBluescriptII KS(−) (silent, black bars) or activated by hTet-Off (gray bars) was used for bisulfite sequencing. A total of 20 individual clones were sequenced to reveal the methylation pattern of the 20 CpG sites over the amplified region.

RNAi-mediated Dicer knockdown. (A) Quantitative real-time RT-PCR analysis of the Dicer mRNA levels. The relative Dicer mRNA levels in siRNA-transfected cells were determined by normalizing the ratios of Dicer mRNA to β-actin mRNA against that of a nontransfected sample. An siRNA targeting the silkworm ACE gene was used as a negative control. The mean values and standard deviations from three independent experiments using the average value of triplicate RT-PCRs are shown. (B) Western blot analysis of the Dicer protein levels. Total cell lysates were resolved by 8% SDS-PAGE and detected with an anti-Dicer antibody. The levels of catalase are shown as a loading control. The indicated number (0.21) in the Dicer siRNA-transfected cells was determined by normalizing the ratio of Dicer to catalase against that of the control siRNA-transfected cells. (C) Dicer knockdown caused a defect in siRNA generation from shRNA precursors. Top, Western blot analysis of Dicer knockdown. Bottom, Northern blot analysis of the siRNA expression levels. Following transfection of pSilencer-lac operator into control and Dicer-depleted cells, small RNAs were detected by Northern blot analysis using radiolabeled probes homologous to the lac operator sequence. The levels of 5S rRNA stained with ethidium bromide are shown as a loading control. The indicated number (0.39) in the Dicer-depleted cells was determined by normalizing the ratio of siRNA to 5S rRNA against that of control cells.

Dicer knockdown does not cause deconstruction of the heterochromatic transgenic locus. ECFP-HP1α (B) and ECFP-SUV39H1 (D) localized to the condensed locus (A and C) in Dicer siRNA-transfected cells. Enrichment of endogenous HP1β (F) and HP1γ (H) at the condensed locus (E and F) in Dicer siRNA-transfected cells was detected by immunofluorescence staining. The transgenic locus is marked by EYFP-LacR (A, C, E, and G). Scale bar, 5 μm. (I) ChIP analysis of H3K9me3 in the CMVm promoter region (black bars) and transcribed CFP region (gray bars), in control or Dicer siRNA-transfected cells. The mean values and standard deviations from three independent experiments are shown.

HP1 tethering represses VP16-induced transgene activation in a Dicer-independent manner. (A) Real-time RT-PCR analysis of CFP mRNA levels. Cells consecutively transfected with control (black bars) or Dicer (gray bars) siRNA were cotransfected with plasmids as indicated. The relative CFP mRNA levels were determined by normalizing the ratios of CFP mRNA to β-actin mRNA against those of cells cotransfected with control siRNA, phTet-On-FLAG-NLS-VP16, and pBluescriptII KS(−). The remaining levels of Dicer mRNA and protein in Dicer-depleted cells relative to that of control siRNA-transfected cells are shown in parentheses (left, mRNA; right, protein). The mean values and standard deviations from three independent experiments are shown. (B) Western blot analysis of FLAG-tagged fusion proteins in control siRNA-transfected cells. Total cell lysates were resolved by 10% SDS-PAGE and detected with an anti-FLAG antibody. pBlue, pBluescriptII KS(−); VP16, hTet-On-FLAG-NLS-VP16 (bands indicated by arrows in lanes 1 to 5); ΔVP, hTet-On-FLAG-NLS-ΔVP (band indicated by open triangle in lane 2); HP1, hTet-On-FLAG-HP1 (bands indicated by closed triangles in lanes 3 to 5).