Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis.
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The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at -954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1(-/-)) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1(+/+) fibroblasts but increased apoptosis in cyclin D1(-/-) cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G(1) analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties. (+info)
The adenovirus type 5 early region 1A gene (E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras and E1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of the Her-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the epidermal growth factor receptor, Her-2/Neu, Src, and Axl, are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (D. Robinson, F. Lee, T. Pretlow, and H.-J. Kung, Proc. Natl. Acad. Sci. USA 93:5958-5962, 1996) to identify potential tyrosine kinases regulated by E1A. Reverse transcription (RT)-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified by analyzing the AluI-digested RT-PCR products. We isolated the DNA fragment of interest and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfected axl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl. The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in ip1-E1A control cells and protected the Axl-expressing cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis and thereby contributes to E1A's antitumor activities. (+info)
Biochemical analysis of distinct activation functions in p300 that enhance transcription initiation with chromatin templates.
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To investigate the mechanisms of transcriptional enhancement by the p300 coactivator, we analyzed wild-type and mutant versions of p300 with a chromatin transcription system in vitro. Estrogen receptor, NF-kappaB p65 plus Sp1, and Gal4-VP16 were used as different sequence-specific activators. The CH3 domain (or E1A-binding region) was found to be essential for the function of each of the activators tested. The bromodomain was also observed to be generally important for p300 coactivator activity, though to a lesser extent than the CH3 domain/E1A-binding region. The acetyltransferase activity and the C-terminal region (containing the steroid receptor coactivator/p160-binding region and the glutamine-rich region) were each found to be important for activation by estrogen receptor but not for that by Gal4-VP16. The N-terminal region of p300, which had been previously found to interact with nuclear hormone receptors, was not seen to be required for any of the activators, including estrogen receptor. Single-round transcription experiments revealed that the functionally important subregions of p300 contribute to its ability to promote the assembly of transcription initiation complexes. In addition, the acetyltransferase activity of p300 was observed to be distinct from the broadly essential activation function of the CH3 domain/E1A-binding region. These results indicate that specific regions of p300 possess distinct activation functions that are differentially required to enhance the assembly of transcription initiation complexes. Interestingly, with the estrogen receptor, four distinct regions of p300 each have an essential role in the transcription activation process. These data exemplify a situation in which a network of multiple activation functions is required to achieve gene transcription. (+info)
Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas.
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Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development. (+info)
Adenovirus E1A blocks oxidant-dependent ferritin induction and sensitizes cells to pro-oxidant cytotoxicity.
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Ferritin is a protein that oxidizes and sequesters intracellular iron in a mineral core. We have reported that the E1A oncogene selectively represses ferritin H transcription, resulting in reduced levels of the ferritin H protein. Here we demonstrate that cells respond to pro-oxidant challenge by inducing ferritin mRNA and protein, and that this response is completely blocked by E1A. Concordantly, E1A sensitized cells to the cytotoxic effects of oxidative stress and enhanced the accumulation of reactive oxygen species in response to pro-oxidant challenge. These results demonstrate that expression of E1A impedes the cellular response to oxidative stress, including the induction of ferritin. (+info)
Transcriptional down-regulation of poly(ADP-ribose) polymerase gene expression by E1A binding to pRb proteins protects murine keratinocytes from radiation-induced apoptosis.
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Adenovirus E1A confers enhanced cell sensitivity to radiation and drug-induced DNA damage by a mechanism involving the binding to cellular proteins. Mutant analysis in E1A-transfected murine keratinocytes demonstrates that increased sensitivity to DNA damage requires at least E1A binding to the p300/CREB-binding protein (CBP) transcriptional coactivators and to pRb family members, indicating that this biological activity of E1A is the result of the concomitant perturbation of different cell pathways. Here we show that in the same cells E1A binding to members of the retinoblastoma protein family induces transcriptional down-regulation of the poly(ADP-ribose) polymerase (PARP) gene, coding for a NAD-dependent enzyme stimulated by DNA breaks. Inhibition of PARP expression is accompanied by a decrement of gamma-irradiation-induced apoptosis, which is overridden by reconstitution of wild type levels of PARP. Hence, E1A effects on PARP transcription are central determinant of the apoptotic sensitivity of E1A-expressing keratinocytes. Conversely, E1A binding to only p300/CBP results in an increase in PARP enzyme activity and consequently in cell death susceptibility to irradiation, which is effectively counteracted by the PARP chemical inhibitor 3-aminobenzamide. Therefore, our results identify in the E1A-mediated effects on PARP expression and activity a key molecular event involved in E1A-induced cell sensitization to genotoxic stress. (+info)
Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue.
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The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dcl3, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not pRB inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle arrest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130. (+info)
Activity of the Nurr1 carboxyl-terminal domain depends on cell type and integrity of the activation function 2.
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Nurr1, a member of the nuclear hormone receptor superfamily, was recently demonstrated to be of critical importance in the developing central nervous system, where it is required for the generation of midbrain dopamine cells. Nuclear receptors encompass a transcriptional activation function (activation function 2; AF2) within their carboxyl-terminal domains important for ligand-induced transcriptional activation. Since a Nurr1 ligand remains to be identified, the role of the Nurr1 AF2 region in transcriptional activation is unclear. However, here we show that the Nurr1 AF2 contributes to constitutive activation independent of exogenously added ligands in human embryo kidney 293 cells and in neural cell lines. Extensive mutagenesis indicated a crucial role of the AF2 core region for transactivation but also identified unique features differing from previously characterized receptors. In addition, Nurr1 did not appear to interact with, and was not stimulated by, several previously identified coactivators such as the steroid receptor coactivator 1. In contrast, adenovirus protein E1A, stably expressed in 293 cells, was shown to contribute to AF2-dependent activation. Finally, while the AF2 core of RXR is required for ligand-induced transcriptional activation by Nurr1-RXR heterodimers, the functional integrity of Nurr1 AF2 core is not critical. These results establish that the ligand binding domain of Nurr1 has intrinsic capacity for transcriptional activation depending on cell type and mode of DNA binding. Furthermore, these results are consistent with the possibility that gene expression in the central nervous system can be modulated by an as yet unidentified ligand interacting with the ligand binding domain of Nurr1. (+info)