Figure 1.

SUV39H1 and HP1γ mediate repression of the HIV-1 LTR in a chromatin-dependent manner. (A) HeLa LTR-Luc and HeLa cells were transfected twice with siRNA as indicated. For HeLa cells, LTR-Luc construct was cotransfected with siRNA in the second round of transfection. Twenty-four hours after the second round of transfection, cells were transduced with GST or GST-Tat as indicated. Expression of Suv39H1 and GAPDH mRNA was analyzed by RT–PCR using specific oligonucleotides (top panel). Twenty-four hours after transduction, extracts were prepared and luciferase activity was measured (lower panels). Results are presented as fold activation relative to GST-treated cells. The mean relative luciferase activities were obtained from three independent experiments. (B) Experiment was performed as in (A), except that siRNA specific for each isoform of HP1 was used. Expression levels of HP1α, β (Supplementary Figure S1) and γ were analyzed by Western blotting using specific antibodies (top panel). Results are presented as fold activation relative to GST-treated cells. The mean relative luciferase activities were obtained from three independent experiments. (C) Total RNA was prepared from cells transfected with control siRNA (Sc) or HP1γ-specific siRNA (experiment shown in (B)). RT–PCR was performed using oligonucleotides specific for the indicated genes. PCR products were analyzed on agarose gels and visualized by ethidium bromide.