The transcription factor E2F-1 is a downstream target of RB action.

Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular transcription factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated transcriptional repression.

Association of p300 and CBP with simian virus 40 large T antigen.

p300 and the CREB-binding protein CBP are two large nuclear phosphoproteins that are structurally highly related. Both function, in part, as transcriptional adapters and are targeted by the adenovirus E1A oncoprotein. We show here that p300 and CBP interact with another transforming protein, the simian virus 40 large T antigen (T). This interaction depends on the integrity of a region of T which is critical for its transforming and mitogenic properties and includes its LXCXE Rb-binding motif. T interferes with normal p300 and CBP function on at least two different levels. The presence of T alters the phosphorylation states of both proteins and inhibits their transcriptional activities on certain promoters. Although E1A and T show little sequence similarity, they interact with the same domain of p300 and CBP, suggesting that this region exhibits considerable flexibility in accommodating diverse protein ligands.

Mutations in topA interfere with the inducible expression of DNA damage response loci in Salmonella typhimurium.

Strains of Salmonella typhimurium deficient in topoisomerase I activity (topA mutants) are UV sensitive and non-mutable (Overbye and Margolin: J Bacteriol 146:170-178, 1981). Using lac-operon fusions to DNA damage inducible (din) loci we investigated whether these observations could be explained by an inability of topA strains to efficiently induce DNA damage responses. Mitomycin C (MMC)-induced expression of lac-operon fusions to uvrB and to a second SOS locus, din-9, was largely eliminated in topA bacteria. The inducible expression of several other din-fusions was also diminished. This inducibility defect was mimicked by growth of din-9 topA+ bacteria in media of high osmolarity, a condition that leads to increased DNA supercoiling. Inhibitors of DNA gyrase efficiently induced din-9 in topA bacteria. Together, these results suggest that the topA effect on din expression may be mediated at the level of DNA supercoiling. The sensitivities of a number of din-fusions to topA paralleled the degree to which they were repressed by excess LexA, suggesting that mutations in topA might influence LexA-operator interactions and/or increase lexA expression.

Partitioning apoptosis: a novel form of the execution phase of apoptosis.

Apoptosis is characterized by chromatin condensation, DNA cleavage, redistribution of phosphatidylserine, and apoptotic body formation via an actin-dependent process. We describe a novel form of the execution phase of apoptosis in human multiple myeloma cells that is morphologically and mechanistically distinct from classical apoptosis, but is caspase-dependent and inhibited by IL-6 and overexpression of Bcl-2. Electron microscopic analysis of these cells demonstrated chromatin condensation without nuclear fragmentation, and 'partitioning' of cell constituents into two components: a single, large bleb containing soluble protein and free ribosomes, and a region containing the nucleus, organelles, and RER. In some cases, the bleb separated, becoming a free vesicle exhibiting random kinetic motion. These morphologic features occurred despite inhibition of the actin and tubulin cytoskeletal systems. This novel form of apoptosis, called partitioning apoptosis, was observed in a variety of tumor cell types and in primary cells. The execution phase of apoptosis can occur in a manner that is morphologically and mechanistically distinct from classical apoptosis.

DNA damage-inducible loci in Salmonella typhimurium.

lac operon fusions to DNA damage-inducible (din) loci were generated in Salmonella typhimurium LT2. Many of these din fusions were efficiently repressed by cloned Escherichia coli LexA, while others were not; all required RecA for induction. Several din fusions exhibited strong inducibility and will be useful in developing an SOS induction assay in S. typhimurium to detect genotoxins.

A family of transcriptional adaptor proteins targeted by the E1A oncoprotein.

The cellular protein p300 is a target of the adenoviral E1A oncoprotein and is thought to participate in preventing the G0/G1 transition in the cell cycle, activating certain enhancers and stimulating differentiation pathways. CBP is a protein that is associated with and coactivates the transcription factor CREB, mediating the induction by cyclic AMP of certain responsive promoters. The sequences of p300 and CBP are highly related. We show here that p300, like CBP2, can stimulate transcription. This activity is directly and specifically inhibited by E1A. We also find that CBP exists in a DNA-bound complex containing a member of the CREB family and that E1A and CBP interact with one another in vivo. In keeping with the idea that E1A functionally targets CBP, cAMP-dependent transcription is repressed by E1A. Thus, p300 and CBP define a family of transcriptional adaptor proteins that are specifically targeted by the E1A oncoprotein.

Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1alpha protein. Both HIF-1alpha and HIF-1beta mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1alpha protein was barely detectable in normoxic cells, even when HIF-1alpha was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1beta protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1alpha protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1alpha protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1alpha protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1alpha protein.

Phosphorylation of the adenovirus E1A-associated 300 kDa protein in response to retinoic acid and E1A during the differentiation of F9 cells.

Transcription of the c-jun gene is up-regulated by either retinoic acid (RA) or adenovirus E1A during the differentiation of F9 cells. We show here that RA and E1A induce phosphorylation of the E1A-associated 300 kDa protein (p300) during the differentiation of F9 cells. The region of E1A that is required for interaction with cellular protein p300 overlaps with the region of E1A required for E1A to induce expression of the c-jun gene. Treatment of F9 cells with RA or infection of the cells by adenovirus led to a decrease in the electrophoretic mobility of p300. Phosphatase treatment of p300 from RA-treated or adenovirus-infected F9 cells reversed the changes in migration of p300, indicating that RA- and E1A-mediated changes in the mobility of p300 were due to phosphorylation. We also found factors, designated DRF1 and DRF2, that bound specifically to a sequence element that is necessary and sufficient for RA- and E1A-mediated up-regulation of the c-jun gene. The mobility of DRF complexes was changed by E1A or RA and the complexes were supershifted by addition of a polyclonal p300 antiserum. Moreover, overexpression of p300 resulted in an increase in the level of DRF1 complex. p300 fused to the DNA binding domain of the E2 protein of papilloma virus stimulated E2-dependent reporter activity in response to RA or E1A in F9 cells. Our results suggest that p300 is part of the DRF complexes, that it is differentially phosphorylated in undifferentiated versus differentiated cells and that it is likely involved in regulating transcription of the c-jun gene during F9 cell differentiation.

Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase.

Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting transcription factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.e., RB during G1 and cyclin A-kinase during S/G2) may contribute to the periodicity of expression of certain E2F-1-responsive genes at the G1/S transition.

Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1.

Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1alpha. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1alpha/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhanced endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1alpha to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP.

Cooperation of Stat2 and p300/CBP in signalling induced by interferon-alpha.

The transcription factor ISGF3 transduces interferon (IFN)-alpha signals and activates the transcription of cellular antiviral defence genes. Adenovirus E1A blocks the IFN-alpha response, allowing unhindered viral replication. ISGF3 consists of Stat1, Stat2 and p48. Here we show that p300 and/or CBP (CREB-binding protein), which are transcription adaptors targeted by E1A, interact specifically with Stat2. Binding occurs between the first cysteine-histidine-rich region of p300/CBP and the carboxy-terminal segment of Stat2, a domain essential for ISGF3 function. We find that this domain of Stat2 has transactivation potential, which correlates with its binding to p300/CBP. Moreover, E1A represses Stat2 transactivation and IFN-alpha-activated transcription by inhibiting p300/CBP function. This provides a new mechanism for inhibition of the IFN-alpha-activated antiviral response by E1A, and supports the view that E1A binding to p300/CBP has functional significance for adenovirus replication in its natural host.

An essential role for p300/CBP in the cellular response to hypoxia.

p300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. They participate in numerous biological processes, including cell cycle arrest, differentiation, and transcription activation. p300 and/or CBP (p300/CBP) also coactivate CREB. How they participate in these processes is not yet known. In a search for specific p300 binding proteins, we have cloned the intact cDNA for HIF-1 alpha. This transcription factor mediates hypoxic induction of genes encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular endothelial growth factor. Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1 alpha and p300/CBP. Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP. Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by E1A. Hence, p300/CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including one (vascular endothelial growth factor) that plays a major role in tumor angiogenesis. Paradoxically, these data, to our knowledge for the first time, suggest that p300/ CBP are active in both transformation suppression and tumor development.

Erythropoietin gene regulation depends on heme-dependent oxygen sensing and assembly of interacting transcription factors.

Studies on erythropoietin (Epo) gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia. Both in vivo and in Hep3B cells. Epo production is induced not only by hypoxia but also by certain transition metal (cobalt and nickel) and by iron chelation. When Hep3B cells were incubated in an iron deficient medium, Epo mRNA expression was enhanced fourfold compared to Hep3B cells in iron enriched medium. Epo induction by cobalt was inversely related to iron concentration in the medium, indicating competition between the two metals. Under hyperbaric oxygen, cobalt induction of erythropoietin mRNA was modestly suppressed while nickel induction was markedly enhanced. These recent observations support the proposal that the oxygen sensor is a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. The up-regulation of Epo gene transcription by hypoxia depends on at least two known DNA binding transcription factors, HIF-1 and HNF-4, which bind to cognate response elements in a critical approximately 50 bp 3' enhancer. Hypoxia induces HIF-1 binding. HNF-4, an orphan nuclear receptor constitutively expressed in kidney and liver, binds downstream of HIF-1 and cooperates with HIF-1, contributing importantly to high level and perhaps tissue specific expression. The C-terminal activation domain of HNF-4 binds to the beta subunit of HIF-1. The C-terminal portion of the alpha subunit of HIF-1 binds specifically to p300, a general transcriptional activator. Hypoxic induction of the endogenous Epo gene in Hep3B cells as well as an Epo-reporter gene was fully inhibited by E1A, an adenovirus protein that binds to and inactivates p300, but only slightly by a mutant E1A that fails to bind to p300. Moreover, overexpression of p300 enhanced hypoxic induction. Thus, it is likely that in hypoxic cells, p300 or a related family member plays a critical role in forming a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3' enhancer to the apparatus on the promoter responsible for the initiation of transcription.