p400 is required for E1A to stabilize Myc. (A) 12S E1A is depicted, showing conserved regions 1 (CR1) and 2 (CR2). The positions of adenovirus E1A deletion mutants dl1504 and dl1101–dl1110 are shown above, and residues required for interaction with p400 are shown below. (B) U2OS cells were infected with adenovirus deletion mutants, or dl520, and incubated for 6 h, and CHX chase was performed. WB was used to monitor levels of E1A and endogenous Myc. (C) U2OS cells were transduced with retroviral expression constructs encoding (i) a short hairpin directed against p400 (sh p400), or vector control, and (ii) WT 12S E1A, or vector control (−). CHX chase followed by WB analysis was used to determine levels and stability of Myc and E1A. (D) WB analysis was used to determine levels of Myc and p400 in control (−) and p400 hairpin (+)-expressing cells. (E) U2OS cells were transiently transfected with HA-tagged Myc, in the absence (−) or presence of an expression construct encoding p400. Levels of Myc and p400 were determined by immunoblotting. (F) U2OS cells were transfected with expression vectors encoding either Myc or p53, together with FLAG-tagged p400 (or vector control), and a plasmid expressing His-tagged Ub (20). Ub conjugates were recovered by Ni-NTA chromatography, and ubiquitylated Myc and p53 proteins were detected by WB.

Myc is a downstream effector of E1A. (A) IMR90 cells were transduced by retroviral vectors expressing 12S E1A, the E1A Δ26–35 mutant, or Myc. RNA was harvested, and levels of Rcl1 and Cad cDNAs were determined by quantitative PCR. (B) IMR90 cells were transduced with the indicated retroviruses, and then transfected with either nontargeting siControl, or siMyc RNA, duplexes for 48 h. RNA was then harvested and levels of Rcl cDNA were analyzed by quantitative PCR. Fold induction is normalized to an actin control for each sample. (C) As in B but assaying for levels of AHCY cDNA. (D) WT (TGR-1 cells) or congenic myc^−/− Rat-1 cells (HO.15.19 cells) were transduced with retroviruses expressing, E1A, E1A Δ26–35, or empty vector, and the resulting cell populations were treated with adriamycin and assessed for viability after 24 h by trypan blue exclusion. The myc−/−; Myc panel represents myc^−/− cells where full-length Myc was reintroduced by retroviral gene transfer. (E) WB analysis of samples derived from C showing expression of caspase-7, E1A, or tubulin. (F) IMR90 cells were transduced with retroviral expression constructs encoding E1A (•), E1A Δ26–35 (▴), E1A ΔCR2 (♦), Myc (□), E1A Δ26–35 + Myc (○), E1A ΔCR2 + Myc (▵), or an empty vector (■). Cell populations were treated with the indicated doses of adriamycin for 24 h, and viability was determined by trypan blue exclusion. (G) BJ fibroblasts were retrovirally transduced with the indicated constructs. The resulting cells were assayed for colony formation in soft agar.

Adenovirus E1A stabilizes Myc. (A) U2OS cells were infected with adenovirus dl520 and harvested at the indicated time points. Levels of Myc, E1A, and actin were determined by WB. (B) U2OS cells were infected with adenovirus, as in A, and RNA was harvested. Levels of c-Myc cDNAs were analyzed by quantitative PCR and normalized to those of an actin control. (C) U2OS cells were either infected with dl520 or a control virus expressing β-gal, mock-infected, or treated with the proteasome inhibitor, MG132, and incubated for 6 h. CHX was added to inhibit protein synthesis, and protein samples were taken at the indicated times (CT; hours). WB analysis shows the levels of Myc, E1A, and actin. (D) U2OS cells were transduced with a retroviral expression construct encoding WT 12S E1A (pLPC E1A). CHX chase, followed by WB, was used to analyze Myc turnover in E1A expressing cells and control (vector) cells. (E) RNA was isolated from cells in D, and levels of c-Myc cDNAs relative to actin, determined by quantitative PCR. (F) U2OS cells were transiently transfected with a plasmid encoding unstable GFP (U-GFP) and incubated for ≈20 h. These cells were then either infected with dl520, mock-infected, or treated with MG132 and incubated for 6 h. CHX chase was used to analyze the levels of GFP, Myc, E1A, and actin.

E1A expression promotes the formation of Myc–p400 complexes. (A) U2OS cells were transiently transfected with HA-tagged Myc and FLAG-tagged p400 for 48 h. Transfected cells were infected for 6 h with β-GAL, dl520, or dl1102 adenovirus. Coimmunoprecitation was performed as described (4) by using anti-Myc (N262) or anti-FLAG (M2) antibodies, and Myc and p400 were detected by WB. (B) U2OS cells were transfected with FLAG-p400 or control DNA for 48 h and then infected with β-GAL, dl520, or dl1102 adenovirus for 6 h. ChIP analysis was performed by using the anti-Myc antibody N262, and coprecipitating DNAs corresponding to the B23 E-box were detected by quantitative PCR. Enrichment is calculated relative to a non-Myc binding sequence. (C) Protein–DNA complexes recovered in B were eluted and subject to a second round of ChIP with anti-FLAG antibodies to recover p400-containing chromatin. Levels of coprecipitating DNA from the B23 E-box were determined by quantitative PCR. To control for nonspecific background, signal in the re-ChIP was normalized to that from an identical, parallel, experiment from cells not expressing FLAG-tagged p400.