We have cloned the ubiquitous form of an octamer-binding, 60-kDa protein (NonO) that appears to be the mammalian equivalent of the Drosophila visual and courtship song behavior protein, no-on-transient A/dissonance (nonAdiss). A region unprecedently rich in aromatic amino acids containing two ribonuclear protein binding motifs is highly conserved between the two proteins. A ubiquitous form of NonO is present in all adult tissues, whereas lymphocytes and retina express unique forms of NonO mRNA. The ubiquitous form contains a potential helix-turn-helix motif followed by a highly charged region but differs from prototypic octamer-binding factors by lacking the POU DNA-binding domain. In addition to its conventional octamer duplex-binding, NonO binds single-stranded DNA and RNA at a site independent of the duplex site.

We previously found that the angiogenic factors TNF␣ and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47 phox and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47 phox and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47phox , was recovered largely in the cytoskeleton/ membrane fraction. Coexpression of p47 phox and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47 phox and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47 phox binding domain of TRAF4 blocked endothelial cell JNK activation by TNF␣ and HIV-1 Tat, suggesting an uncoupling of p47 phox from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNF␣ and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.

NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly into bacteria without a further ligase-dependent reaction. Using this approach, we have constructed recombinant DNA molecules rapidly, efficiently and directionally. This method can effectively replace conventional protocols for PCR cloning, PCR SOEing, DNA subcloning and sitedirected mutagenesis.

Surfactant protein (SP)-B expression is restricted to type II pneumocytes and Clara cells in the lung. Previously, a promoter region of human SP-B gene from -64 to -118 has been identified as critical for the tissue-specific expression of this gene. Two cis-elements for thyroid transcription factor (TTF)-1 and hepatocyte nuclear factor (HNF)-3alpha binding were found within this area. Using an oligonucleotide fragment, we incorporated this region sequence into the promoter of a HIS3 reporter gene in yeast. With this modified yeast a human lung complementary DNA (cDNA) library was screened for DNA-binding proteins, other than TTF-1 and HNF-3alpha, that interacted with this promoter segment. A cDNA clone encoding a novel polypeptide, BR22, was identified that activated the reporter gene expression in yeast. This gene is expressed in many tissues and encodes a protein with bipartite nuclear localization signals. Studies using in vivo yeast two-hybrid analysis, in vitro protein-protein interactions, and coimmunoprecipitation analyses demonstrated that BR22 formed a protein complex with TTF-1. In vivo cotransfection studies further indicated that BR22 could act with TTF-1 to synergistically activate the SP-B promoter in mammalian cells. Our data suggest that BR22 is a TTF-1-associated protein. Through a protein-protein interaction with TTF-1, BR22 can form a complex and activate the human SP-B promoter in vivo.