Multiple pathways for gene activation in rodent cells by the smaller adenovirus 5 E1A protein and their relevance to growth and transformation

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, three highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,8,33,42,(52)(53)(54)61). Protein VII is the major protein component of the core, with an estimated 1,070 copies present per virion (20). Along with , it is bound noncovalently to the DNA in a sequence-independent manner (2, 6, 36, 55). Protein V contacts the DNA as well and also acts as a bridge between protein VII and the outer capsid (19,55). Protein IVa2 makes sequence-specific contacts with the viral DNA packaging sequence and is thought to play a role in DNA packaging (64). Salt-extracted preparations of the core contain only DNA and protein VII, suggesting that this protein is the most tightly DNA bound of all core proteins (60). Similarly, Sarkosyl preparations of the core contain predominantly DNA and protein VII (8).Structural features of the DNA-protein complex within the adenovirus capsid and during infection remain largely unknown. DNA within the capsid is in a highly compact configuration, and electron microscopy studies of purified viral core reveal structures reminiscent of beads on a string, or higherorder chromatin compaction, depending on the method of preparation (8,18,45,49,50,60,63). Nuclease digestion of core preparations results in discrete populations of prote...

Section: Figmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Adenovirus Protein VII Condenses DNA, Represses Transcription, and Associates with Transcriptional Activator E1A

Johnson

Osheim

Xue

et al. 2004

“…In quiescent cells, binding of E1A to p300 is essential for the induction of DNA synthesis and cell transformation (25,27,33). For the past several years, we have been investigating the role of p300/CBP in quiescent cells and the cell cycle G 1 /S transition and the consequences of binding of E1A to p300 in the induction of S phase.…”

mentioning

confidence: 99%

Adenovirus Transforming Protein E1A Induces c-Myc in Quiescent Cells by a Novel Mechanism

Kadeppagari

Natesan

Thimmapaya

2009

Previously we showed that the E1A binding proteins p300 and CBP negatively regulate c-Myc in quiescent cells and that binding of E1A to p300 results in the induction of c-Myc and thereby induction of S phase. We demonstrated that p300 and HDAC3 cooperate with the transcription factor YY1 at an upstream YY1 binding site and repress the Myc promoter. Here we show that the small E1A protein induces c-Myc by interfering with the protein-protein interaction between p300, YY1, and HDAC3. Wild-type E1A but not the E1A mutants that do not bind to p300 interfered in recruitment of YY1, p300, and HDAC3 to the YY1 binding site. As E1A started to accumulate after infection, it transiently associated with promoter-bound p300. Subsequently, YY1, p300, and HDAC3 began to dissociate from the promoter. Later in infection, E1A dissociated from the promoter as well as p300, YY1, and HDAC3. Removal of HDAC3 from the promoter correlated with increased acetylation of Myc chromatin and induction. In vivo E1A stably associated with p300 and dissociated YY1 and HDAC3 from the trimolecular complex. In vitro protein-protein interaction studies indicated that E1A initially binds to the p300-YY1-HDAC3 complex, briefly associates with it, and then dissociates the complex, recapitulating somewhat the in vivo situation. Thus, E1A binding to the C-terminal region of p300 disrupts the important corepressor function provided by p300 in repressing c-Myc. Our results reveal a novel mechanism by which a viral oncoprotein activates c-Myc in quiescent cells and raise the possibility that the oncoproteins encoded by the small-DNA tumor viruses may use this mechanism to induce c-Myc, which may be critical for cell transformation.

“…nontransformed breast epithelial cell line; see reference 34). The induction of DNA synthesis by the transforming E1A protein is a basic property of E1A, and it occurs in many cell types, including those of human and rodent origin (35,36). To determine which of the cell cycle and DNA replication genes are induced in MCF10A cells, we carried out a microarray analysis of RNA samples prepared from quiescent cells infected with an Ad variant that expresses only the transforming E1A gene (dl1500; see reference 27) (referred to as AdE1A in this report).…”

Section: E1amentioning

confidence: 99%

Adenovirus E1A Oncogene Induces Rereplication of Cellular DNA and Alters DNA Replication Dynamics

Singhal

Leo

Setty

et al. 2013

The oncogenic property of the adenovirus (Ad) transforming E1A protein is linked to its capacity to induce cellular DNA synthesis which occurs as a result of its interaction with several host proteins, including pRb and p300/CBP. While the proteins that contribute to the forced induction of cellular DNA synthesis have been intensively studied, the nature of the cellular DNA replication that is induced by E1A in quiescent cells is not well understood. Here we show that E1A expression in quiescent cells leads to massive cellular DNA rereplication in late S phase. Using a single-molecule DNA fiber assay, we studied the cellular DNA replication dynamics in E1A-expressing cells. Our studies show that the DNA replication pattern is dramatically altered in E1A-expressing cells, with increased replicon length, fork velocity, and interorigin distance. The interorigin distance increased by about 3-fold, suggesting that fewer DNA replication origins are used in E1A-expressing cells. These aberrant replication events led to replication stress, as evidenced by the activation of the DNA damage response. In earlier studies, we showed that E1A induces c-Myc as a result of E1A binding to p300. Using an antisense c-Myc to block c-Myc expression, our results indicate that induction of c-Myc in E1A-expressing cells contributes to the induction of host DNA replication. Together, our results suggest that the E1A oncogene-induced cellular DNA replication stress is due to dramatically altered cellular replication events and that E1A-induced c-Myc may contribute to these events. The adenovirus (Ad) transforming E1A protein [a 243-aminoacid E1A protein, also referred to as small E1A protein [1,2]) has the capacity to induce S phase in quiescent cells, and in the presence of activated ras or virus-encoded E1B19K or 55K proteins, E1A can transform rodent cells in culture (1, 2). The S-phase induction and cell transformation activities of the small E1A protein are genetically linked and are dependent on the N-terminal region of E1A binding to cellular protein complexes, including TRRAP/p400/GCN5, histone acetyltransferase p300/CBP, and the Rb family tumor suppressor proteins (1-4). E1A-Rb interactions result in the release of the progrowth E2F family transcription factors from the Rb-histone deacetylase (HDAC) repressor complexes and the induction of the S phase (1, 5). However, studies have shown that in order for E1A to induce S phase efficiently, it must bind to p300/CBP and Rb family proteins simultaneously, suggesting that E1A must also alter the functions of p300/CBP (3, 6).Although a large number of studies have focused on the cellular proteins that contribute to the forced induction of host DNA synthesis in E1A-expressing cells, the nature of the cellular DNA that replicates in these cells is not well understood. Previous studies have shown that the E1A-expressing cells fail to undergo proper mitosis and that such cells accumulate in the S and G 2 /M phases (7-10). Mammalian cells contain a large number of DNA replication origins, a...