E1A recruits hPaf1 to HAdV early gene promoters via hBre1.

<p>(A, B) hPaf1 is localized at the E2e, E3 and E4 promoters. A549 cells were infected with WT HAdV, ΔE1A HAdV or HAdV containing deletions within E1A for 20 hours. Chromatin immunoprecipitation (ChIP) was performed with the indicated antibodies and DNA was probed via qRT-PCR for the presence of HAdV early gene promoters or transcribed regions. hPaf1 localization requires residues 4–25 of E1A and s found on the E2e, E3 and E4 but not the E1A or E1B promoter regions. A statistically significant decrease from WT in A is indicated (* P<0.01). (C) hPaf1 colocalizes with E1A and hBre1 on the E3 and E4 promoters. hPaf1 ChIP was followed by re-ChIP with either hBre1 or E1A specific antibodies to determine co-occupancy. Data was normalized to ΔE1A HAdV and a non-specific antibody control. (D and E) hPaf1 recruitment to the E3 and E4 promoter requires hBre1. A549 cells were treated with a non-specific siRNA, or siRNA specific for hBre1 (D), or hPaf1 (E) prior to virus infection. ChIP assays were then performed using hBre1 or hPaf1 specific antibodies. hPaf1 and hBre1 occupancy was then determined at the HAdV E3 and E4 promoters as described above. n = 3.