Figure 1

Localization of integrated and transfected operator repeats containing no transcription units. Components labeled are indicated in the upper corners of the panels with their respective colors. (A) HEp-2-Op (derived from HEp-2 cells integrated with 10 kb = 256 operator repeats) probed by in situ hybridization for the location of the operator repeat insert; the operator DNA does not colocalize with PML. (B) HEp-2-Op (256 repeats) cells fused with HEp-2gfplacI cells. The single integrated operator repeat insert from 3 HEp-2-Op cell nuclei is found within PML-stained regions (yellow). The GFP-repressor is diffusely distributed in the three HEp-2gfplacI cell nuclei. (C) HEp-2-Op cells with 128 operator repeats were transfected with the GFP-repressor. Integrated operator repeats appear green (arrow). Insets show the color-separated small operator repeats at higher magnification. (D) HF cells probed by in situ hybridization for the location of the U2 gene locus relative to PML. The respective loci do not colocalize. (E) The same as (D), but showing a U2 locus localized adjacent to PML (arrow). (F) HEp-2 cells transfected with operator repeats and probed by in situ hybridization for operator location relative to PML. Operator plasmid aggregates do not colocalize with PML. (G) HEp-2 cells cotransfected with operator repeats and the GFP-repressor expression vector. The “green” operator/repressor complexes in the nucleus colocalize with PML. (H) MEF PML−/− cotransfected with operator repeats and the GFP-lac repressor expression vector and labeled for Daxx. Daxx colocalizes with the operator/repressor complex. The inset shows the area framed in blue at a higher magnification and with the colors separated. (I) MEF PML−/− cotransfected with operator repeats and the GFP-repressor expression vector and labeled for SUMO, which colocalizes with the operator/repressor complex. The inset shows the area framed in blue at a higher magnification and with the colors separated. (J) Mouse Daxx−/− cells cotransfected with operator repeats and the GFP-repressor expression vector and labeled for PML, which colocalizes with the operator/repressor complex. The inset shows the area framed in blue at a higher magnification and with the colors separated. (K) HEp-2 cells transfected with a plasmid-containing cellular heat shock promoter. In situ hybridization shows plasmid aggregates not colocalizing with PML. The inset shows the area framed in blue at a higher magnification and with the colors separated.