Cellular GCN5 is a novel regulator of human adenovirus E1A-conserved region 3 transactivation

Abstract

The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (hat/hat) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.

Fig 1

Identification of a second interaction site for GCN5 within CR3 of E1A. (A) The largest E1A isoform contains a novel interaction site for GCN5. HeLa cells were infected with the indicated viruses at an MOI of 10. At 24 h postinfection, cell lysates were prepared and E1A was immunoprecipitated with M73 antibody, separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and subsequently probed with anti-GCN5, anti-pRb, anti-MED23, and anti-E1A antibodies. (B) The antibody raised against GCN5 does not cross-react with the closely related pCAF protein. Cell extract (20 μg) prepared from HT1080 cells transfected with vectors expressing FLAG-tagged mGCN5 or pCAF was resolved by SDS-PAGE, transferred to a PVDF membrane, and subsequently probed with anti-GCN5 antibody or anti-FLAG antibody. (C) The interaction of E1A-CR3 with mGCN5 maps to residues 178 to 188 of hAd5 E1A. Human HT1080 cells were transfected as described above, immunoprecipitated with anti-FLAG antibody (for GCN5), separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-myc antibody (for CR3). (D) Interaction of E1A Δ178-184 with cellular targets. Immunoprecipitations were performed as described for panel C, using anti-EGFP antibody, and blots were probed with the indicated antibodies.

Fig 2

GCN5 is recruited to the adenoviral E4 promoter in an E1A-dependent manner during infection. (A) Schematic of the right end of the hAd5 genome, showing primer binding sites for ChIP PCR. ITR, inverted terminal repeat. (B) Human A549 cells were infected at an MOI of 5 with the indicated viruses (wt E1A, ΔE1A, E1A Δ26-35, and E1A Δ178-184). Cells were fixed, ChIP assays were performed with the indicated antibodies, and the E4 promoter was detected by PCR with a set of primers specific for a 320-bp region of the adenoviral E4 promoter region as described previously (36, 50). (C) Fold enrichment on the E4 promoter of E1A and GCN5 in cells infected with ΔE1A virus, wt hAd, and E1A Δ178-184 virus with the same primers as for panel B. Fold enrichment was calculated by setting the percent recovery of IgG to 1.0, and mean fold enrichments were compared by Student's t test. *, significant difference from ΔE1A virus (P < 0.05).

Fig 3

The interaction between GCN5 and E1A-CR3 is conserved, and depletion of GCN5 results in an increase in E1A-CR3 transactivation. (A) All six representative E1A-CR3s coimmunoprecipitate mGCN5. Human HT1080 cells were cotransfected with expression vectors for myc-EGFP fusions to the indicated E1A-CR3s and an expression vector for FLAG-tagged mGCN5. CR3s were immunoprecipitated with anti-myc antibody and probed for GCN5 with anti-FLAG antibody. (B) Sequence alignment of residues 178 to 188 of hAd5 E1A and corresponding regions of each indicated hAd species. Greater sequence conservation at each position is indicated by darker shading. (C) HeLa cells were transfected with 20 nM siRNA specific for GCN5 or control siRNA. At 48 h posttransfection, cells were split and cotransfected with equal ratios of a (Gal4)₆-Luc reporter and an expression vector for the indicated Gal4DBD-CR3 fusion. “Vector” denotes cells transfected with Gal4DBD alone, and “N-Term” denotes cells transfected with an expression vector for Gal4DBD fused to residues 1 to 82 of hAd5 E1A. At 48 h post-DNA transfection, cells were harvested and assayed for luciferase activity in duplicate. Changes in activation (fold activation) in control versus GCN5 siRNA-treated cells were compared by Student's t test. *, P < 0.05; ns, not significant (P > 0.05). (Inset) HeLa cells were transfected with increasing amounts (5 nM, 10 nM, or 20 nM) of GCN5-specific siRNA or control siRNA, and the levels of GCN5 and actin were determined at 72 h posttransfection by Western blotting with the indicated antibodies.

Fig 4

Overexpression of GCN5 results in a decrease in E1A-CR3 transactivation. (A) Human HT1080 cells were cotransfected with a Gal4-responsive luciferase reporter, and expression vectors for a Gal4DBD fusion and increasing amounts of FLAG-tagged mGCN5. The fold activation by Gal4-E1A-CR3 over Gal4 alone, when cotransfected with empty pCMX-FLAG, was set to 100%. (Inset) Levels of FLAG-tagged mGCN5 and endogenous actin upon cotransfection of increasing amounts of pMCX-FLAG-mGCN5 as determined by WB with the indicated antibodies. (B) Human HT1080 cells were cotransfected with equal amounts of an adenoviral E4-responsive luciferase reporter, an expression vector for E1A, and either empty pCMX-FLAG or pCMX FLAG-mGCN5. The fold activation of the E4 reporter in cells transfected with wt 13S 289R E1A over empty vector, when cotransfected with empty pCMX-FLAG, was set to 100%. Percentages of activation in pCMX-FLAG versus pCMX FLAG-mGCN5-treated cells were compared by Student's t test; *, P < 0.0; ns, P > 0.05.

Fig 5

Pharmacological inhibition of GCN5 KAT activity recapitulates depletion of GCN5. (A) Human HT1080 cells were cotransfected with a Gal4-responsive luciferase reporter and an expression vector for Gal4-hAd5-CR3 and treated with either DMSO or increasing concentrations of the GCN5-specific KAT inhibitor CPTH2. The fold activation by Gal4-E1A-CR3 over Gal4DBD alone when treated with DMSO (vehicle) was set to 100%. (B) Human HT1080 cells were cotransfected with an adenoviral E4-responsive luciferase reporter and with the indicated expression vector for E1A and treated with either DMSO or 50 μM CPTH2. The fold activation of the E4 reporter in cells transfected with wt 13S 289R hAd5 E1A over empty vector when treated with DMSO (vehicle) was set to 100%. Percentages of activation in DMSO versus CPTH2-treated cells were compared by Student's t test; *, P < 0.05; ns, P > 0.05.

Fig 6

The KAT activity of GCN5 modulates E1A-dependent transactivation of the viral E4 promoter during infection. (A) hat/hat or wt littermate control MEFs were infected with either wt hAd, E1A Δ26-35 hAd, or E1A Δ178-184 hAd at an MOI of 2. At 16 h postinfection, total RNA was isolated and the level of E4orf6/7 mRNA relative to GAPDH mRNA was determined by qRT-PCR. (B) hat/hat or wt littermate control MEFs were infected with wt hAd as for panel A, but after 1 h of absorption of virus, cells were treated with either 50 μM CPTH2 or an equivalent volume of DMSO (vehicle). At 16 h postinfection the relative expression level of E4orf6/7 was determined as described for panel A. Mean relative E4orf6/7 expression levels between hat/hat and wt littermate control MEFs with or without CPTH2 treatment were compared by Student's t test; *, P < 0.05; ns, P > 0.05.

Fig 7

The KAT activity of GCN5 is required for optimal hAd replication. Human A549 cells were infected with wt hAd5 at an MOI of 5 and subsequently treated with either 50 μM CPTH2 or an equal volume of DMSO (vehicle) after 1 h of virus absorption. At 96 h postinfection, cells were harvested and virus yield was determined by plaque assay on HEK293 cells. Mean virus yields in PFU/ml were compared by Student's t test; *, P < 0.05.

Fig 8

The KAT activity of GCN5 alters chromatinization of the hAd E4 promoter during infection. Human A549 cells were infected at an MOI of 5 with the indicated viruses (wt E1A, ΔE1A, or E1A Δ178-184). Cells were fixed, and chromatin was purified, sheared, and immunoprecipitated with anti-H3 or anti-K9/K14-acetylated-H3 antibody. After washing and de-cross-linking, the percentage of input E4 promoter DNA recovered by ChIP was determined by qPCR with a set of primers specific for a 320-bp region of the adenoviral E4 promoter region as described previously (36, 50). Fold enrichment was calculated by setting the percent recovery of IgG to 1.0, and the ratio of K9/K14-acetylated-H3 (H3-Ac) to total H3 was calculated by dividing fold enrichment for H3-Ac by the fold enrichment of total H3. The mean relative H3-Ac/H3 recruitment levels were compared by one-way ANOVA and Tukey's post hoc test. There was a significant difference in the ratio of H3-Ac/total H3 in cells infected with wt hAd- and ΔE1A- or E1A Δ178-184 hAd-infected cells (*, P < 0.05). There was no significant difference in the mean ratios of H3-Ac to total H3 of ΔE1A- or E1A Δ178-184-infected cells (ns, P > 0.05).

Fig 9

GCN5 KAT activity reduces occupancy by phospho-CTD RNAPII at a viral gene, and a mechanism of GCN5-mediated regulation of transactivation is proposed. (A) MEF (hat/hat or wt littermate control) cells were infected at an MOI of 5 with wt hAd. Cells were fixed, and chromatin was purified, sheared, and immunoprecipitated for total RNAPII or for phospho-CTD RNAPII. After washing and de-cross-linking, the percentage of input E4 promoter DNA recovered by ChIP was determined by qPCR with a set of primers specific for a 320-bp region of the adenoviral E4 promoter region as described previously (36, 50). Fold enrichment was calculated by setting the percent recovery of IgG to 1.0, and the ratio of phospho-CTD RNAPII to total RNAPII was calculated by dividing the fold enrichment for phospho-CTD RNAPII by the fold enrichment of total RNAPII. The mean relative phospho-CTD RNAPII/RNAPII recruitment levels were compared by Student's t test; *, P < 0.05. (B) Same as for panel A, except that ChIP was performed with anti-E1A (M73) and anti-GCN5 antibodies, and the relative recruitment of GCN5 to E1A was determined by dividing the fold enrichment of GCN5 by the fold enrichment of E1A. The mean relative GCN5/E1A recruitment levels were compared by Student's t test; ns, P > 0.05. (C) Model of GCN5 regulation of E1A transactivation. GCN5 is recruited to the viral E4 promoter by E1A through binding sites in both the N terminus and CR3 of E1A. The KAT activity of GCN5 then hyperacetylates H3 K9/K14 associated with the E4 promoter and also inhibits phosphorylation of the RNAPII CTD, resulting in an overall repression of E1A-dependent transactivation.