The CR3 portion of hAd5 E1A is a potent transcriptional activation module and serves as a paradigm of viral transactivation (5
). CR3 is the most highly conserved of the four conserved regions within E1A, yet there are dramatic differences in the potency of the CR3 domains from six representative E1As with respect to transactivation (1
). We have previously demonstrated that these differences are independent of the conserved coactivators of E1A CR3: MED23, TBP, SUG1, pCAF, and p300/CBP (1
). Moreover, competition experiments suggest that additional cellular factors are involved in E1A CR3 transactivation and may include negative regulators (1
). We report here the identification of an interaction surface in E1A CR3 for the cellular KAT GCN5. Furthermore, our experiments indicate that GCN5 is a novel negative regulator of E1A transactivation that may function by altering the acetylation state of the viral genome assembled with cellular histones into chromatin and the phosphorylation status of RNAPII at the viral E4 promoter.
Although the interaction of GCN5 with the N terminus of E1A had been reported previously, these experiments used hAds in a background in which only the 12S (243R) isoform of E1A was synthesized and which thus did not contain CR3 (23
). We repeated these interaction experiments using viruses harboring the same mutation, i.e., deletion of residues 26 to 35 of E1A, in a background that synthesizes the larger 13S-encoded (289R) isoform of E1A. In this context, the E1A Δ26-35 mutant retained interaction with GCN5 (), suggesting that GCN5 also binds to a second site present within CR3 of E1A. We confirmed that CR3 alone is sufficient for interaction with GCN5 (). The GCN5 interaction site within CR3 was mapped to require at least residues 178 to 188, which excludes the residues required for interaction with SUG1 () (38
). The promoter-targeting domain of E1A CR3 was originally shown to be residues 183 to 188, which does overlap this newly identified GCN5 binding site (17
). However, we have shown using ChIP analysis that E1A Δ178-184 is still recruited to the E4 promoter ( and ). Co-IP of E1A CR3 with GCN5 required residues 178 to 184 of CR3 in reciprocal Co-IP scenarios indicating a robust and specific interaction. Although the Δ178-184 mutant of CR3 appears to be impaired for interaction with MED23 and TBP, we have shown that the GCN5 interaction is independent of interaction with pCAF, MED23, and TBP using key point mutants that disrupt specific interaction with TBP, MED23, and pCAF (V147L, H160Y, and Δ139-147, respectively). Furthermore, the GCN5 interaction is independent of the zinc finger binding subdomain of CR3. Taken together, these data suggest that the Δ178-184 mutant of CR3 may impact the integrity of the zinc finger subdomain, which would explain the defects in MED23 and TBP binding as well as the impaired activation function of this CR3 mutant. Importantly, E1A CR3Δ178-184 retains interaction with the closely related pCAF KAT. Thus, this mutant further differentiates these two closely related KATs, which both interact with E1A yet appear to have opposing roles in E1A transactivation. Perhaps these two KATs are associated with distinct complexes that bind CR3 through distinct interaction surfaces. Despite indications that CR3 is indeed a structured region of E1A, no detailed information on the structure of CR3 is currently available, but the possibility that E1A-CR3 could be recruiting both activating and repressive modules through distinct surfaces is enticing.
The interaction of GCN5 with CR3 was conserved across all six hAd species, suggesting that the functional role of GCN5 is also conserved (). There is a dichotomy of conservation at the primary amino acid level among hAds in the region required for GCN5 interaction (). Residues 178 to 181 of E1A are poorly conserved among the six representative hAds in our panel. However, residues 182 to 188 share a much greater degree of conservation. Residue 182 in species A through D is phenylalanine; however, in species E and F it is cysteine, which may contribute to the poor interaction of GCN5 with hAd4 CR3. Residues 183 through 188 are very highly conserved among E1As. S185 and S188 are phosphorylated in vivo
and are critical for E1A-dependent activation of transcription for the E4 promoter (54
The primary function of the largest E1A proteins is to activate early viral gene expression by recruiting the cellular transcription machinery to the early viral promoters (5
). Thus, GCN5 would be expected to be present at the viral promoter with E1A, and indeed GCN5 is associated with the viral E4 promoter in an E1A-dependent manner ( and ). Interestingly, the recruitment of GCN5 to the E4 promoter requires both GCN5 binding sites within E1A, demonstrating for the first time a physical cooperation between the N terminus of E1A and CR3 to recruit a cellular protein to the viral E4 promoter (). It should be noted that although an E1A mutant lacking residues 26 to 35 (the N-terminal GCN5 binding site) can still coimmunoprecipitate GCN5 (), this N-terminal binding site is also required to efficiently recruit GCN5 to the viral E4 promoter ( and ). Furthermore, we demonstrated that both binding sites are required for GCN5 to exert its repressive effect on E1A transactivation of the E4 promoter from both plasmid and viral genomic templates (, , and ). Despite the large body of evidence that both the N terminus of E1A and CR3 are transactivation domains, there is very little direct evidence demonstrating that the N terminus of E1A and CR3 can cooperate to interact with and recruit cellular proteins to a viral promoter (55
). Several cellular proteins that bind to CR3 also interact with the N-terminal CR1 domain of E1A, including TBP, pCAF, SUG1, and p300/CBP (24
). There is also evidence that the N terminus of E1A and CR3 can cooperate functionally. For example, both regions can activate transcription when fused to a heterologous DBD (5
) and CR3 can synergize with the N terminus to activate the hAd E2 promoter, presumably via recruitment of TBP and sequestration of pRb (55
). In essence, the interaction of GCN5 with two distinct regions of E1A suggests a mechanism of cooperative recruitment to the viral promoters that may also apply to other proteins that interact with multiple regions of E1A. Similarly, mechanisms of cooperative recruitment of factors to the viral promoters may explain why both these regions of E1A are required for efficient activation and regulation of early viral gene expression (47
RNAi depletion of GCN5 resulted in an increase in E1A CR3 transactivation and provided the first indication that GCN5 is a true negative regulator of E1A transactivation (). This role of GCN5 as a negative regulator was conserved among a subset of our panel of representative CR3s of each hAd species, with the exception of hAd4 E1A CR3 (species D), which also had the weakest physical interaction with GCN5 (). Perhaps hAd4 E1A CR3 does not require the same level of negative regulation that the other CR3s display as a consequence of its weaker intrinsic activation function. Depletion of GCN5 resulted in an increase in transcriptional activation (), whereas depletion of the other cellular factors required by CR3 (MED23, p300 SUG1, or pCAF) results in a decrease in transactivation (1
). This gain of function phenotype suggests that GCN5 is part of another layer of transcriptional control that is recruited to promoters by E1A to optimize early gene expression. This is also a rare example where removing a cellular binding partner of CR3 results in enhanced E1A transactivation. The same phenotype has been previously demonstrated by depleting CtBP with siRNA or sequestering CtBP with the C terminus of E1A (10
). In the context of infection, such a paradigm may be required for regulation of the hAd E4 transcription unit, as many of the gene products of the E4 region are toxic and stimulate cellular defenses and/or antagonize survival pathways (46
). Therefore, it appears that E1A utilizes GCN5 to help fine-tune transactivation of the E4 promoter. This pathway could similarly regulate expression of other viral early genes as well.
If GCN5 negatively regulates E1A CR3-dependent transactivation and is available in limited quantities, then ectopic expression of GCN5 should exacerbate the negative effect on CR3 transactivation. This was indeed the case, since overexpression of GCN5 resulted in a decrease in transactivation by CR3 fused to a heterologous DBD, and also by full-length E1A acting on an E4-responsive reporter (). It is somewhat counterintuitive that CR3 alone is still affected by GCN5, given the requirement for both sites to recruit GCN5 to the viral E4 promoter. However, it is possible that the interaction with the CR3 binding region is stronger than the N-terminal interaction (), and that may be why GCN5 affects CR3 transactivation as a Gal4DBD fusion. We cannot exclude that artificial targeting of CR3 to a promoter through the Gal4DBD renders the GCN5 binding site more accessible or indirectly enhances GCN5 recruitment by direct targeting of CR3 to the promoter. However, the effect of GCN5 overexpression again highlights the cooperative nature of the GCN5 interactions with full-length E1A, as mutation of either binding site rendered full-length E1A nonresponsive to the repressive effects of GCN5 on E4 promoter activation ().
GCN5 has both a KAT activity and a deubiquitinating activity in the SAGA complexes of both yeast and mammalian cells (4
). Importantly, we have demonstrated the functional effect of GCN5 on E1A transactivation using multiple experimental systems of increasing sensitivity. Depleting GCN5 by siRNA indicated that GCN5 was a negative regulator of E1A transactivation but could not distinguish which function of GCN5 was responsible, since all of its functions, including its KAT and deubiquitinating activity, were depleted along with GCN5. However, both pharmacological inhibition and targeted genetic ablation of KAT function clearly demonstrated that the repressive effect on E1A transactivation requires the KAT activity of GCN5. Although pharmacological inhibition was not as effective as genetic ablation, we observed that CPTH2 is exquisitely specific to GCN5, as this inhibitor had no effect on E1A transactivation in hat/hat
MEFs (), which retain expression of the closely related KAT pCAF. Both the Gal4DBD-responsive and E4 reporters, as well as direct measurement of the transcriptional output of the E4 promoter via E4orf6/7 mRNA transcript levels during infection, demonstrated that loss of GCN5 or GCN5 KAT activity resulted in an increase in E1A transactivation and that both GCN5 interaction sites in E1A were required to affect transactivation of the E4 promoter (, , and ). Our results indicate that the KAT activity of GCN5 is required to negatively affect E1A transactivation on both plasmid and viral genomic templates ( and ).
The hAd genome is not packaged into the virion in complex with cellular histones but has been shown to associate with cellular histones following infection. Indeed, genome-wide analysis has revealed acetylated histones across the hAd genome (15
). These include H3, H3.1, and H3.3, which are acetylated at K9 and K14, presumably by GCN5, and also at K18, which is presumably acetylated by p300/CBP (15
), yet no direct role for E1A or GCN5 in this process was demonstrated in either report. Consistent with previous reports, we show here that E1A is not required for H3 deposition and K9/K14 acetylation, as cells infected with hAd with deletion of E1A (ΔE1A) still acquire both H3 and K9/K14-acetylated H3 (H3-Ac) on the viral E4 promoter () (22
). In the presence of E1A (cells infected with wt hAd), there is a dramatic increase in the ratio of H3-Ac to total H3, or hyperacetylation of H3 on K9 and K14 at the E4 promoter compared to cells infected with E1A deletion virus (ΔE1A) or cells infected with a virus that cannot recruit GCN5 to the E4 promoter (E1A Δ178-184) (). Furthermore, we demonstrated that GCN5 is recruited to the viral E4 promoter in an E1A-dependent manner (). The consequence of E1A-dependent recruitment of GCN5 is an increased acetylation of histone H3 at the viral E4 promoter. Therefore, our data demonstrate for the first time recruitment by E1A of a chromatin-modifying enzyme that acts on cellular histones bound to the viral genome to influence viral transcription.
Although acetylation of histones is generally associated with increases in gene expression (18
), there are examples where increased protein acetylation is associated with transcriptional repression, including the mouse mammary tumor virus promoter (19
). It is also well established that histone deacetylases can function as coactivators at some genes (33
). Furthermore, genome-wide analysis of gene expression in yeast demonstrated that approximately 80 genes (5.2% of genes analyzed) showed increased expression as a result of GCN5
). One of these genes elevated in the gcn5
deletion background is ARG1
. The KAT activity of Gcn5p is specifically required for repression of ARG1
, and ChIP analysis demonstrated that decreased histone H3 acetylation at the ARG1
promoter is correlated with increased expression (39
). This is the same phenomenon that we observe with the viral E4 promoter during infection. Thus, it appears that hyperacetylation of histone H3 and potentially other proteins by GCN5 can be used as a mechanism for fine-tuning transcription. Of particular note, GCN5 can acetylate lysine 48 in the catalytic core of the CDK9/pTEFb complex. This inhibits its ability to phosphorylate RNAPII CTD and reduces transcriptional elongation (42
). Mechanistically, the recruitment of GCN5 to the viral promoter by E1A could acetylate factors such as CDK9 and reduce phosphorylation of RNAPII CTD, as depicted in . This model is entirely consistent with our experiments using hat/hat
MEFs, in which we conclusively demonstrated that the GCN5 KAT activity was essential for limiting the phosphorylation status of RNAPII and reducing activation by E1A at the E4 transcription unit.
The net effect of eliminating the KAT function of GCN5 on the hAd replication cycle was reduced virus yield (). The deletion or inhibition of GCN5 KAT activity coincides with an increase in E4 promoter activity, suggesting that a balanced level of E1A transactivation is required for optimal virus growth (). This is not unexpected, as increased transcription of the E4 promoter is known to enhance premature killing of infected cells (54
). Thus, the adenoviral E4 promoter may have multiple levels of regulation. Not only does GCN5 appear to limit E1A stimulation of E4 promoter activity, but the E4 transcription unit also encodes the E4orf4 protein, which interacts with cellular PP2A to dephosphorylate S188 of E1A, completing a negative-feedback loop for E1A transactivation (54
). Therefore, the negative effect of GCN5 on E1A-dependent transactivation may help provide the optimal intracellular milieu for virus replication by fine-tuning expression of the viral transcription program needed for the viral replication cycle.