Repression of p53-mediated Transcription by Adenovirus E1B 55-kDa Does Not Require Corepressor mSin3A and Histone Deacetylases

Abstract

The Ad E1B 55-kDa protein (E1B) is a potent transcriptional repressor. In vitro biochemical studies revealed that direct p53-E1B interaction is essential for E1B to block p53-activated transcription and a corepressor may be involved. To understand how E1B represses p53-mediated transcription in vivo, we expressed E1B in several tumor cell lines that express wild type p53. Here we show that E1B strongly suppresses the expression of p53 target genes such as p21 and Puma-␣ in normal growth conditions or after cells were treated with p53-activating chemotherapeutic agents, suggesting that E1B-mediated gene repression is dominant and cannot be reversed via p53 activation. Interestingly, we found that E1B binds to corepressor mSin3A. Mutagenesis analysis indicated that the sequence motif "LHLLA" near the NH 2 terminus of E1B is responsible for mSin3A binding, and this motif is conserved among E1B proteins from different Ad serotypes. The conserved paired amphipathic helix domain 1 of mSin3A is critical for mSin3A-E1B interaction. Surprisingly, E1B mutants that cannot bind to mSin3A can still repress p53 target genes, indicating that it is not the corepressor required for E1B-mediated gene repression. In support of this notion, repression of p53 target genes by E1B is insensitive to HDAC inhibitor trichostatin A. We further show that both the NH 2 -and COOH-terminal domains of E1B are required for the repression function. Therefore, E1B employs a unique repression mechanism to block p53-mediated transcription.The p53 and pRb tumor suppressor pathways are inactivated in virtually all human cancers regardless of their etiology (1, 2). Understanding the precise molecular mechanisms of these pathways is at the center stage of current research efforts in cancer biology. Small DNA tumor viruses such as adenoviruses (Ad), 3 human papillomaviruses, and SV40 can transform cells and cause cancer (3, 4). Each of these viruses produces several proteins that disable both p53 and pRb tumor suppressor pathways in infected cells. Ad E1A proteins physically associate with pRb and release it from E2F transcription factors that activate expression of genes required for DNA replication and cell cycle progression. The Ad 1B region encodes two major proteins in two overlapping ORFs: E1B 55-and 19-kDa, both of which are required for efficient cell transformation. The E1B 19-kDa functions as an inhibitor of apoptosis by binding to proapoptotic proteins Bax and Bak (5), and E1B 55-kDa (hereafter called E1B) participates in transformation by inactivating the p53 pathway (6).Several functions of E1B contribute to the inhibition of p53. Apart from sequestration of p53 in the cytoplasm that blocks p53-mediated apoptosis (7), E1B can inhibit acetylation of p53 and disrupt the interaction between p53 and coactivator p300/ CBP-associated protein (8). Early studies clearly showed that the transcriptional repression function of E1B, but not p53-E1B interaction per se, is critical for Ad-mediated cell transformation (9, 10). These studies demonstrated t...

The Repression Domain of the E1B 55-Kilodalton Protein Participates in Countering Interferon-Induced Inhibition of Adenovirus Replication

“…However, such mutations neither induced increased expression of E1B 55-kDa protein-repressed genes in infected HFFs (44) nor increased the sensitivity of viral replication (Table 1) or DNA synthesis (data not shown) to IFN. The E1B protein alone is sufficient to repress p53-dependent transcription in cells transiently or stably synthesizing the viral protein (35,37,38,(75)(76)(77)(78). The E1B 55-kDa protein fully competent to rescue the defects in genome replication of the null mutant AdEasyE1⌬2347 when stably produced in HFFs (Fig.…”

“…Substitutions of the C-terminal sites of phosphorylation of the Ad5 or Ad12 E1B protein reduced its ability to inhibit p53-dependent transcription or to act as a repressor when fused to a heterologous DNA-binding domain (37,75,76). However, such mutations neither induced increased expression of E1B 55-kDa protein-repressed genes in infected HFFs (44) nor increased the sensitivity of viral replication (Table 1) or DNA synthesis (data not shown) to IFN.…”

Adenovirus E1B 55-Kilodalton Protein: Multiple Roles in Viral Infection and Cell Transformation

“…The E1B-55K protein has also been shown to be a strong repressor of basal transcription (157), but interestingly, this activity requires a cellular corepressor which has not yet been identified (103). The E1B-55K protein interacts with a Sin3A/histone deacetylase 1 corepressor complex, but this is apparently not required for the transcriptional repression activity of the E1B-55K protein (118,160). It is therefore tempting to speculate that the identity of this elusive corepressor is E1B-AP5, and it will be interesting to determine if this is the case.…”

Inhibition of p53 by Adenovirus Type 12 E1B-55K Deregulates Cell Cycle Control and Sensitizes Tumor Cells to Genotoxic Agents

“…E1B-55K forms a stable complex with DNAbound p53 and increases p53's binding affinity to its cognate DNA-binding site in vitro (30). Chromatin immunoprecipitation experiments revealed that E1B-55K also associates with the promoters of endogenous p53 target genes such as the p21 gene (57). Association of E1B-55K with p53 represses p53-mediated transcription in vitro and in cells (31,52,57).…”

“…Chromatin immunoprecipitation experiments revealed that E1B-55K also associates with the promoters of endogenous p53 target genes such as the p21 gene (57). Association of E1B-55K with p53 represses p53-mediated transcription in vitro and in cells (31,52,57). It has been suggested that a cellular corepressor is required for E1B-55K to inhibit p53-activated transcription (31).…”