FIG. 8.

Transduction of soluble Tat upregulates IL-2 and IL-2R and increases the acetylation status of IL-2 and IL-2Rα promoters in Jurkat T cells. (A) SDS-15% PAGE analysis of purified recombinant Tat proteins. Wild-type Tat (lane 5), Tat 1-48 (lane 3), Tat 40-72 (lane 4), and C22G mutant Tat (lane 7) were expressed as GST fusions in BL21 cells and purified as described in Materials and Methods. Tat (lane 6) and GST (lane 2) were obtained by thrombin cleavage. B. Recombinant GST-Tat (lane 1) and Tat (lane 2) proteins were transduced separately into Jurkat cells, and nuclear lysates were prepared as described in Materials and Methods. The nuclear lysates were resolved by electrophoresis in 15% SDS-polyacrylamide gels followed by Western blot analysis using anti-Tat antibody. (C) Flow cytometric (FACS) analysis of transduced cells. Cells incubated with 100 ng of recombinant Tat for 4 h were immunostained using rabbit IgG (upper histogram) or anti-Tat (lower histogram) and acquired on a flow cytometer as described in Materials and Methods. (D) RT-PCR analysis of mRNA from transduced cells. Total mRNA was isolated from Jurkat cells incubated with GST (lane 1), GST-Tat 40-72 (lane 2), and GAT-Tat (lane 3). RT-PCR analysis was performed using IL-2 (upper panel), IL-2R (middle panel), and GAPDH (lower panel) cDNA primers. (E) HDAC1 occupancy and promoter acetylation are inversely correlated. To monitor the acetylation status of IL-2 and IL-2R promoters independent of HIV-1 virion exposure, we transduced the wild-type Tat (lanes 2 and 8), GST (lanes 3 and 9), Tat-40-72 (lanes 4 and 10), Tat-C22G (lanes 5 and 11), and Tat-1-48 (lanes 6 and 12) proteins into Jurkat cells as described in Materials and Methods. Chromatin was then prepared and subjected to ChIP analysis using anti-SATB1, anti-HDAC1, and anti-H3 acetylated lysine 9 antibodies. As negative and positive controls we used rabbit IgG (R-IgG, lanes 1 and 7) and input chromatin (bottom panels), respectively.