The AP-1 transcription factors c-Fos, JunB and JunD are recruited <i>in vivo</i> to the 5103 fragment region.

<p>HeLa cells were transiently transfected with the wild-type pHIV-1 or the mutated pHIV-1-AP-1#totmut construct. Twenty-four hours post-transfection, cells were mock-treated (-) or treated with PMA (+). Twenty-four hours post-induction, cells were cross-linked for 10 min at room temperature with 1% formaldehyde. To detect chromosomal flanking regions, pellets were sonicated to obtain DNA fragments of an average size of 400 bp. Chromatin immunoprecipitations were performed with specific antibodies directed against c-Fos, JunB, Fra-1 or JunD. To test aspecific binding to the beads, a purified IgG was used as a control for immunoprecipitation. Quantitative PCR reactions were performed with oligonucleotide primers hybridizing either in the nuc-1 region (termed nuc-1), or in a region overlapping the three AP-1 binding sites of the 5103 fragment (termed 5103 fragment), or in the <i>vpr</i> gene (termed <i>vpr</i> gene) where no AP-1 binding sites have been previously reported. Fold enrichments were calculated as percentages of immunoprecipitated DNA following the formula “Immunoprecipitated DNA (IP)*100/Input DNA (INP)”. Values represent the means of triplicate samples and standard errors of the means are indicated. An experiment representative of three independent ChIP assays is shown. (<b>B</b>) <b>Mutations in the intragenic AP-1 sites affect the PMA-inducible </b><b><i>in vivo</i></b><b> recruitment of RNA polymerase II to the HIV-1 5′LTR region.</b> Chromatin immunoprecipitations were performed with a specific antibody directed against RNAPII and a purified IgG as a control. Quantitative PCR reactions were performed with the same oligonucleotide primers and fold enrichments were calculated as in panel (A). Means and standard errors of the means from one experiment representative of three independent ChIP assays are shown.