In this study, we have analyzed an ORF from Corynebacterium glutamicum, which codes for a homologue of the Streptomyces coelicolor WhiB-family of proteins known to be involved in sporulation. This ORF encoded a putative protein which harbors a helix-turn-helix DNA-binding motif and a probable redox-sensing motif, and has been designated whcE. We constructed a whcE mutant strain and analyzed the strain under a variety of growth conditions. This mutant strain exhibited a prolonged lag phase and earlier death within the stationary phase, suggesting that the relevant gene may play a role in both growth adaptation and stress responses. Further analysis determined that the mutant strain was not only sensitive with regard to survival under heat stress, but was also markedly susceptible to thiol-specific oxidant diamide and redox cycling compounds, including menadione and plumbagin. The mutant strain also exhibited reductions in thioredoxin reductase activity, which indicates that the trxB gene encoding thioredoxin reductase is under the control of WhcE. Expression of whcE was stimulated during the stationary phase of cell growth and could be modulated by diamide. We also delineated the relationship between whcE and the sigH gene, which is located downstream of whcE, and has been shown to be involved in heat stress responses, via the encoding of an ECF sigma factor. In a sigH mutant strain, the whcE gene was no longer expressed, thereby suggesting that the sigmaH sigma factor is involved in whcE expression. Our results suggest that WhcE functions as a transcription factor which can activate the trxB gene, as well as other genes, possibly by sensing redox changes during the metabolic downshifting of cells from exponential growth to the stationary phase, whereas sigmaH appears to function as the sigma factor for these genes, including whcE.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage- and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.

Several bHLH proteins are involved in vertebrate neurogenesis, but those controlling early stages of neuronal determination have not yet been identified. Here we describe a novel, NeuroD-related bHLH protein, NEUROGENIN, whose expression precedes that of NeuroD in both mouse and Xenopus. Expression of Xenopus NEUROGENIN-related-1 (X-NGNR-1) defines the three prospective territories of primary neurogenesis. Overexpression of X-NGNR-1 (or NEUROGENIN) induces ectopic neurogenesis and ectopic expression of XNeuroD mRNA. Endogenous X-ngnr-1 expression becomes restricted to subsets of cells by lateral inhibition, mediated by X-Delta-1 and X-Notch. The properties of X-NGNR-1 are thus analogous to those of the Drosophila proneural genes, suggesting that it functions as a vertebrate neuronal determination factor.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3' and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.

Sterol regulatory element 1 (SRE-1), a decamer (5'-ATC-ACCCCAC-3') flanking the low density lipoprotein (LDL) receptor gene, activates transcription in sterol-depleted cells and is silenced by sterols. We report the cDNA cloning of human SREBP-1, a protein that binds SRE-1, activates transcription, and thereby mediates the final regulatory step in LDL metabolism. SREBP-1 contains a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) motif, but it differs from other bHLH-ZIP proteins in its larger size (1147 amino acids) and target sequence. Instead of an inverted repeat (CANNTG), the target for all known bHLH-ZIP proteins, SRE-1 contains a direct repeat of CAC. Overexpression of SREBP-1 activates transcription of reporter genes containing SRE-1 in the absence (15-fold) and presence (90-fold) of sterols, abolishing sterol regulation. We suggest that SREBP-1 is regulated by an unknown factor that is overwhelmed when SREBP-1 is overexpressed. Understanding the regulation of SREBP-1 may be crucial for understanding the control of plasma cholesterol in humans.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates essential homeostatic responses to reduced O2 availability in mammals. Recent studies have provided insights into the O2-dependent regulation of HIF-1 expression, target genes regulated by HIF-1, and the effects of HIF-1 deficiency on cellular physiology and embryonic development.

Exposure of rats to hypoxia (7% O2) markedly increased the level of heme oxygenase-1 (HO-1) mRNA in several tissues. Accumulation of HO-1 transcripts was also observed after exposure of rat aortic vascular smooth muscle (VSM) cells to 1% O2, and this induction was dependent on gene transcription. Activation of the mouse HO-1 gene by all agents thus far tested is mediated by two 5'-enhancer sequences, SX2 and AB1, but neither fragment was responsive to hypoxia in VSM cells. Hypoxia-dependent induction of the chloramphenicol acetyltransferase (CAT) reporter gene was mediated by a 163-bp fragment located approximately 9.5 kilobases upstream of the transcription start site. This fragment contains two potential binding sites for hypoxia-inducible factor 1 (HIF-1). A role for HIF-1 in HO-1 gene regulation was established by the following observations: 1) HIF-1 specifically bound to an oligonucleotide spanning these sequences, 2) mutation of these sequences abolished HIF-1 binding and hypoxia-dependent gene activation in VSM cells, 3) hypoxia increased HIF-1alpha and HIF-1beta protein levels in VSM cells, and 4) hypoxia-dependent HO-1 mRNA accumulation was not observed in mutant hepatoma cells lacking HIF-1 DNA-binding activity. Taken together, these data demonstrate that hypoxia induces HO-1 expression in animal tissues and cell cultures and implicate HIF-1 in this response.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes including the human erythropoietin (EPO) gene. In this study, we report structural features of the HIF-1alpha subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1alpha and HIF-1beta (ARNT; aryl hydrocarbon receptor nuclear translocator) subunits were coimmunoprecipitated from nuclear extracts, indicating that these proteins heterodimerize in the absence of DNA. In vitro-translated HIF-1alpha and HIF-1beta generated a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 present in nuclear extracts from hypoxic cells. Compared to 826-amino acid, full-length HIF-1alpha, amino acids 1-166 mediated heterodimerization with HIF-1beta (ARNT), but amino acids 1-390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1alpha eliminated DNA binding without affecting heterodimerization. In cotransfection assays, forced expression of recombinant HIF-1alpha and HIF-1beta (ARNT) activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1alpha (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription. Overexpression of a HIF-1alpha construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.

Certain morphological, physiological and molecular characteristics are shared by all neurons. However, despite these similarities, neurons constitute the most diverse cell population of any organism. Recently, considerable attention has been focused on identifying the molecular mechanisms that underlie this cellular diversity. Parallel studies in Drosophila and vertebrates have revealed that proneural genes are key regulators of neurogenesis, coordinating the acquisition of a generic neuronal fate and of specific subtype identities that are appropriate for the location and time of neuronal generation. These studies reveal that, in spite of differences between invertebrate and vertebrate neural lineages, Drosophila and vertebrate proneural genes have remarkably similar roles.

Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.