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How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

E1A is an adenoviral protein which is expressed at the early phase after viral infection and contains four conserved regions (CR1, CR2, CR3 and CR4). Our previous work suggests that E1A facilitates the formation of cyclin A-CDK2 complex and thereby enhances CDK2 activity. However, the molecular function of E1A in CDK2 activation has been unclear. Here, we studied the mechanism of enhancement of CDK2 activity by E1A, using the E1A variant forms which selectively contain CR domains. We isolated four E1A variant forms, i.e. 13S (containing CR1, CR2, CR3, CR4), 12S (CR1, CR2, CR4), 10S (CR2, CR4) and 9S (CR4), derived from HEK293 cells which express E1A. 13S promoted G2/M-phase arrest, upon CDK2 hyper-activation by co-expressing a stabilized cyclin A mutant, most strongly among those E1A variant forms. Concomitantly, the specific activity of the 13S-associated CDK2 was highest among them. 10S exhibited lower affinity for CDK2 than the 13S while the affinity for CDK2 was comparable between 13S and 12S. Nonetheless, 12S did not enhance the CDK2 specific activity. On the other hand, a mutation in CR2 domain, which is essential for binding to p107, suppressed both the binding and activation of CDK2. These results suggest that CR1 domain, in addition to CR2 domain via p107 interaction, is important for binding to CycA-CDK2 complex while CR3 domain facilitates CDK2 activation. Since the function of CR3 in cell cycle regulation has been relatively unknown, we propose the enhancement of CDK2 activity as a novel function of CR3 domain. • Adenovirus E1A enhances cyclin A-CDK2 activity. • CR1 and CR2 domains of E1A play a key role in E1A-CDK2 binding via p107. • CR3 domain of E1A is involved in the enhancement of CDK2 activity. [ABSTRACT FROM AUTHOR]

Cyclin A-cyclin dependent kinase (CDK) activity is regulated by cyclin A proteolysis and CDK inhibitors (CKIs) during M and G1 phases. Our previous work has shown that constitutive activation of cyclin A-CDK in mouse somatic cells, by ectopic expression of stabilized human cyclin A2 (lacking the destruction box: CycAΔ80) in triple CKI (p21, p27, and p107)-knocked-out mouse embryonic fibroblasts, induces rapid tetraploidization. However, effects of such cyclin A-CDK hyperactivation in human cells have been unknown. Here, we show hyperactivity of cyclin A-CDK induces G2/M-phase arrest in human cell lines with relatively low expression of p21 and p27. Moreover, adenovirus E1A protein promoted CycAΔ80-derived G2/M-phase arrest by increasing the amount of cyclin A and cyclin A-CDK2 complex. This response was suppressed by an addition of ATR or Chk1 inhibitor. The amount of repressive phosphorylation of CDK1 at tyrosine 15 (Y15) was decreased by Chk1 inhibitor treatment. Moreover, we observed that co-expressing CDK1AF mutant, which is resistant to the repressive phosphorylation at threonine 14 and Y15, or cdc25A, which dephosphorylates CDK1 at Y15, suppressed the G2/M-phase arrest by CycAΔ80 with E1A. These results suggest that G2/M-phase arrest in human cells by hyperactivity of cyclin A-CDK2 is caused by repression of CDK1 via the cell cycle checkpoint ATR-Chk1 pathway. [ABSTRACT FROM AUTHOR]

Highlights: [•] Adenovirus E1A interacts with a novel nucleolar protein, RaRF. [•] RaRF promotes the nucleolar translocation of E1A from the nucleoplasm. [•] E1A disrupts the interaction of RAR with RaRF, as well as RAR translocation to the nucleolus. [•] Nucleolar sequestration of E1A by RaRF could be a novel mechanism of RAR regulation. [ABSTRACT FROM AUTHOR]

Abstract: Proteasomes represent the major non-lysosomal mechanism responsible for the degradation of proteins. Following interferon γ treatment 3 proteasome subunits are replaced producing immunoproteasomes. Adenovirus E1A interacts with components of the 20S and 26S proteasome and can affect presentation of peptides. In light of these observations we investigated the relationship of AdE1A to the immunoproteasome. AdE1A interacts with the immunoproteasome subunit, MECL1. In contrast, AdE1A binds poorly to the proteasome β2 subunit which is replaced by MECL1 in the conversion of proteasomes to immunoproteasomes. Binding sites on E1A for MECL1 correspond to the N-terminal region and conserved region 3. Furthermore, AdE1A causes down-regulation of MECL1 expression, as well as LMP2 and LMP7, induced by interferon γ treatment during Ad infections or following transient transfection. Consistent with previous reports AdE1A reduced IFNγ-stimulated STAT1 phosphorylation which appeared to be responsible for its ability to reduce expression of immunoproteasome subunits. [Copyright &y& Elsevier]

Abstract: Tumor suppressor p53 induces apoptosis by transcriptional induction of Noxa and Puma, which encode the proapoptotic BH3-only member of the Bcl-2 family proteins. In the p53-mediated tumor surveillance system, p53 induces apoptosis or replicative senescence in oncogene-expressing cells, resulting in elimination of such cells. In this context, we previously found that Noxa and Puma synergistically induce apoptosis. Here, we found the adenovirus oncogene E1A to induce p53-dependently expression of Puma, but not Noxa. The induced Puma associates with antiapoptotic Bcl-2 protein Mcl-1, accompanied by accumulated Mcl-1 protein on mitochondria. Moreover, E1A also reduces expression of the antiapoptotic Bcl-2 protein Bcl-XL. In contrast, the DNA-damaging agent adriamycin induces Noxa expression in E1A-expressing cells. Interestingly, Mcl-1 knockdown itself induced apoptosis in E1A-expressing MEFs. Furthermore, Noxa displaced Puma’s association with Mcl-1, accompanied by Mcl-1 degradation and apoptosis induction by activating mitochondrial apoptotic executers Bax and Bak. These results suggest that p53-induced apoptosis in oncogene-expressing cells is regulated by differential induction and sequential activation of Noxa and Puma. Accumulated Puma by oncogene enhances susceptibility to apoptosis through “catch” in mitochondria by Mcl-1. Subsequently, in response to DNA-damage, Noxa efficiently induces apoptosis by “release” of Puma from Mcl-1. [Copyright &y& Elsevier]

Abstract: FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27. [Copyright &y& Elsevier]

C-terminal-binding protein interacting protein (CtIP) was first isolated as a binding partner of C-terminal-binding protein (CtBP). It is considered to contribute to the transcriptional repression and cell cycle regulatory properties of the retinoblastoma (Rb) family of proteins and to have a role in the cellular response to DNA damage. Here, we have shown that CtIP is a novel target for the adenovirus oncoprotein early region 1A (AdE1A). AdE1A associates with CtIP in both Ad5E1-transformed cells and Ad5-infected cells and binds directly in glutathione-S-transferase pull-down assays. Two binding sites have been mapped on Ad5E1A – the N-terminal α-helical region (residues 1–30) and conserved region 3 (CR3) – the transcriptional activation domain. CtIP can bind AdE1A and CtBP independently, raising the possibility that ternary complexes exist in Ad-transformed and -infected cells. Significantly, reduction of CtIP expression with small interfering RNAs results in reduction of the ability of a Gal4 DNA-binding domain-CR3 construct to transactivate a Gal 4-responsive luciferase reporter and this effect is reversed by reduction of CtBP expression. Therefore, in this model, CtIP acts as a transcriptional co-activator of AdE1A when dissociated from CtBP, through the action of AdE1A. These data are consistent with observations that CtIP expression is induced by AdE1A during viral infection and that reduction of CtIP expression with RNA interference can retard virus replication. In addition, AdE1A causes disruption of the CtIP/Rb complex during viral infection by its interaction with CtIP, possibly contributing to transcriptional derepression.Oncogene (2007) 26, 7467–7479; doi:10.1038/sj.onc.1210551; published online 4 June 2007 [ABSTRACT FROM AUTHOR]

Background: An imbalance between neutrophil protease and surrounding antiprotease levels has been shown to be important in the pathogenesis of chronic obstructive pulmonary disease (COPD). Adenoviral E1A DNA and protein are frequently detected in the lungs of COPD patients. As secretory leukoprotease inhibitor (SLPI) and elafin/skin-derived antileukoproteinase (SKALP) are locally produced in the lung and inhibit neutrophil elastase activity, we hypothesized that adenoviral E1A might affect the production of these antiproteases. Objectives: To examine the effect of E1A on SLPI and elafin/SKALP secretion in A549 (alveolar epithelial) cells and primary human bronchial epithelial (HBE) cells. Methods: SLPI and elafin/SKALP were quantitated from cell culture supernatants using an ELISA. SLPI mRNA expression was examined by Northern blotting, and SLPI promoter activity was measured using a reporter gene assay. Results: E1A significantly suppressed SLPI and elafin/SKALP secretion by A549 cells upon interleukin (IL)-1β stimulation. E1A also suppressed SLPI and elafin/SKALP secretion by HBE cells. SLPI mRNA expression in A549 cells was suppressed by E1A regardless of IL-1β stimulation. IL-1β-induced SLPI promoter activity was suppressed by E1A gene transfection into A549 cells. Conclusions: Our findings of adenoviral E1A-mediated suppression of SLPI and elafin/SKALP secretion suggest that E1A may be involved in the enhancement of alveolar damage and play a role in the COPD process. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Adenovirus 2 and 12 early region 1A (Ad2 and Ad12 E1A) proteins were cleaved during cisplatin-induced apoptosis of Ad-transformed rat and human cells. Cleavage was inhibited in the presence of caspase inhibitors such as Z-VAD-FMK. In Ad12 transformants both 13S and 12S E1A proteins were cleaved at a similar rate. In Ad2 transformants the E1A 13S component was appreciably less stable than the 12S component. In in vitro studies Ad2 and Ad12 E1A 13S and Ad2 12S proteins were rapidly cleaved by caspase 3 whereas Ad12 12S E1A and Ad12 13S E1A were rapidly degraded by caspase 7. Cleavage sites in Ad12 13S proteins for caspase 3 have been determined. Initial cleavage occurred at D24 and D150; this was followed by cleavage at D204 and D242. Caspase-3-mediated cleavage of Ad12 13S E1A destroyed its ability to bind to CBP and TBP but interaction between C terminal E1A polypeptides and CtBP was observed. During viral infection Ad5 and Ad12 E1A 12S proteins were markedly more stable than 13S proteins but no difference was observed in Ad E1A levels in the absence or presence of the caspase inhibitors Z-VAD-FMK or Z-D(OMe)-E(OMe)-V-D(OMe)-CH2F. Limited caspase 3 and 10 activation occurred during infection with the E1B 19K− virus Ad2 pm1722 but little or no activation of caspase 3 was observed during wt virus infection. Examination of protein cleavage during viral infection of A549 cells showed proteolysis of lamin B and PARP in response to Ad5 wt and Ad2 pm1722. Protein degradation in response to both viruses was partially inhibited by Z-VAD-FMK. Following infection of human skin fibroblasts lamin B was degraded, although only limited changes in PARP levels were observed. We have concluded that Ad E1A is cleaved by caspases during apoptosis but not during viral infection. However, some of the processes commonly associated with apoptosis occur during viral infection, particularly with E1B 19K− mutants, although apoptosis per se is not evident. [Copyright &y& Elsevier]

We have identified the N-terminus of adenovirus early region 1A (AdE1A) as a region that can regulate the 26S proteasome. Specifically, in vitro and in vivo co-precipitation studies have revealed that the 19S regulatory components of the proteasome, Sug1 (S8) and S4, bind through amino acids (aa) 4–25 of Ad5 E1A. In vivo expression of wild-type (wt) AdE1A, in contrast to the N-terminal AdE1A mutant that does not bind the proteasome, reduces ATPase activity associated with anti-S4 immunoprecipitates relative to mock-infected cells. This reduction in ATPase activity correlates positively with the ability of wt AdE1A, but not the N-terminal deletion mutant, to significantly reduce the ability of HPV16 E6 to target p53 for ubiquitin-mediated proteasomal degradation. AdE1A/ proteasomal complexes are present in both the cytoplasm and the nucleus, suggesting that AdE1A interferes with both nuclear and cytoplasmic proteasomal degradation. We have also demonstrated that wt AdE1A and the N-terminal AdE1A deletion mutant are substrates for proteasomal-mediated degradation. AdE1A degradation is not, however, mediated through ubiquitylation, but is regulated through phosphorylation of residues within a C-terminal PEST region (aa 224–238). [ABSTRACT FROM AUTHOR]

The activity of transcription factor NFB is regulated by its subcellular localization. In most cell types, NF ΚB is sequestered in the cytoplasm due to binding of the inhibitory protein I ΚBα. Stimulation of cells with a wide variety of agents results in degradation of I ΚBα, which allows translocation of NFB to the nucleus. Degradation of I ΚBα is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen­activated 90 kDa ribosomal S6 kinase (p90rsk1) is an I ΚBα kinase. p90rsk1 phosphorylates I ΚBα at Ser32 and it physically associates with I ΚBα in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, I ΚBα degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an kinase required for phosphorylation and subsequent degradation of I ΚBα in response to mitogens. [ABSTRACT FROM AUTHOR]

In accordance with previous reports by others, the E7 gene of human papillomavirus (HPV) 16, considered to be etiologically associated with cervical cancer, transactivated the E2 promoter of adenovirus. This promoter, however, was equally stimulated by the E7 gene of HPV1, a skin type HPV never associated with human malignancy. A variety of promoters were tested to see the effect of the E7 genes of low-risk and high-risk type HPVs. The result indicated that there was no obvious relationship in the levels of transactivation or transrepression by the E7 gene between low-risk and high-risk types. It has been suggested that the binding of the E7 protein to the product of retinoblastoma susceptibility gene (pRb) is the underlying mechanism by which the E7 protein transactivates the E2 promoter. Therefore, the association of the E7 protein and pRb alone did not seem to fully explain the mechanism by which this protein participates in the activation of transcription and induction of human malignancies. [ABSTRACT FROM AUTHOR]

Adenovirus transformed cells have a dedifferentiated phenotype. Eliminating E1A in transformed human embryonic kidney cells derepressed ∼2600 genes, generating a gene expression profile closely resembling mesenchymal stem cells (MSCs). This was associated with a dramatic change in cell morphology from one with scant cytoplasm and a globular nucleus to one with increased cytoplasm, extensive actin stress fibers, and actomyosin-dependent flattening against the substratum. E1A-induced hypoacetylation at histone H3 Lys27 and Lys18 (H3K27/18) was reversed. Most of the increase in H3K27/18ac was in enhancers near TEAD transcription factors bound by Hippo signaling-regulated coactivators YAP and TAZ. E1A causes YAP/TAZ cytoplasmic sequestration. After eliminating E1A, YAP/TAZ were transported into nuclei, where they associated with poised enhancers with DNA-bound TEAD4 and H3K4me1. This activation of YAP/TAZ required RHO family GTPase signaling and caused histone acetylation by p300/CBP, chromatin remodeling, and cohesin loading to establish MSC-associated enhancers and then superenhancers. Consistent results were also observed in primary rat embryo kidney cells, human fibroblasts, and human respiratory tract epithelial cells. These results together with earlier studies suggest that YAP/TAZ function in a developmental checkpoint controlled by signaling from the actin cytoskeleton that prevents differentiation of a progenitor cell until it is in the correct cellular and tissue environment., (© 2019 Zemke et al.; Published by Cold Spring Harbor Laboratory Press.)

The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein. [ABSTRACT FROM AUTHOR]

The expression of the Adenovirus serotype 2 or serotype 5 (Ad2/5) E1A gene in tumor cells upregulates ligands that are recognized by the NKG2D activating receptor, which is expressed on NK cells and T cells, and reduces their tumorigenicity, a process dependent on NK cells and T cells. In some model systems, the forced overexpression of NKG2D ligands on tumor cells induced antigen-specific CD8+ T cells that mediated anti-tumor immunity. We wanted to determine if the interaction of NKG2D ligands on tumor cells that express E1A with NKG2D on immune cells contributed to the ability of E1A to induce a CD8+ T cell anti-tumor response or reduce tumorigenicity. To address these questions, we used the MCA-205 tumor cell line or MCA-205 cells that expressed Ad5 E1A (MCA-205-E1A cells), a fusion protein of E1A and ovalbumin (MCA-205-E1A-OVA) or OVA (MCA-205-OVA). We found that the expression of E1A or E1A-OVA, but not OVA, upregulated the expression of the NKG2D ligand RAE-1 on the surface of MCA-205 cells. Additionally, MCA-205-E1A cells and MCA-205-E1A-OVA cells were more sensitive to NK cell lysis than MCA-205 or MCA-205-OVA cells in WT B6 mice, but not NKG2D deficient B6 mice. Next, we adoptively transferred WT or NKG2D deficient OT-1 T cells (CD8 T cells that recognize OVA residues 257-264) into WT B6 mice or B6 mice that were deficient in NKG2D respectively and measured the expansion of OT-1 cells following immunization with MCA-205-E1A-OVA or MCA-205-OVA cells. We found that the expansion of OT-1 cells following immunization of either OVA-expressing MCA-205 cell lines was not affected by the presence or absence of NKG2D in B6 mice. Finally, we found that the capacity of E1A to reduce the tumorigenicity of MCA-205 cells was not impaired in B6-NKG2D deficient mice as compared to WT B6 mice. Our results suggest that the ability of E1A to reduce the tumorigenicity of MCA-205 cells, or induce an antigen-specific CD8+ T cell response, is independent of the interaction of NKG2D ligands with the NKG2D receptor.