FIG. 5.

Sp1 mediates c-Myc regulation of HIV-1 LTR expression. Sp1 sites are required for c-Myc-mediated repression, and Sp1 and c-Myc coexist within a complex at the LTR. (A) Expression plasmids pCMV-empty vector, pCMV-Tat, and pcDNA-c-Myc and the wild-type (wt) pILIC-CAT, pSpA-CAT, and pNFA-CAT reporter plasmids were cotransfected into HeLa cells using Lipofectamine 2000 reagent. CAT expression was analyzed by real-time RT-PCR. Tat-activated LTR RNA expression is displayed, with normalization to the cells transfected with pCMV-Tat for each of the reporter assays. PCR products for the GAPDH loading control and transfected Tat are shown. Error bars indicate standard errors of the means for three independent experiments. (B) Sequential ChIP assays were performed after an initial IP with anti-Sp1. A protein-DNA complex near Nuc-1 was recovered after a second IP with anti-Sp1, anti-c-Myc, or anti-HDAC1 (P < 0.01) but not at the downstream region or with isotype IgG or antibody (Ab) against the methyltransferase Dnmt3a. Real-time PCR quantitation of DNA IP, as detailed in Materials and Methods, is indicated.