Upon heat stress, monomeric human heat shock transcription factor 1 (hHSF1) is converted to a trimer, acquires DNA-binding ability, is transported to the nucleus, and becomes transcriptionally competent. It was not known previously whether these regulatory changes are caused by a single activation event or whether they occur independently from one another, providing a multilayered control that may prevent inadvertant activation of hHSF1. Comparison of wild-type and mutant hHSF1 expressed in Xenopus oocytes and human HeLa cells suggested that retention of hHSF1 in the monomeric form depends on hydrophobic repeats (LZ1 to LZ3) and a carboxy-terminal sequence element in hHSF1 as well as on the presence of a titratable factor in the cell. Oligomerization of hHSF1 appears to induce DNA-binding activity as well as to uncover an amino-terminally located nuclear localization signal. A mechanism distinct from that controlling oligomerization regulates the transcriptional competence of hHSF1. Components of this mechanism were mapped to a region, including LZ2 and nearby sequences downstream from LZ2, that is clearly separated from the carboxy-terminally located transcription activation domain(s). We propose the existence of a fold-back structure that masks the transcription activation domain in the unstressed cell but is opened up by modification of hHSF1 and/or binding of a factor facilitating hHSF1 unfolding in the stressed cell. Activation of hHSF1 appears to involve at least two independently regulated structural transitions.The transcriptional enhancement of heat shock protein (hsp) genes by heat shock or other conditions that are stressful to cells is dependent on the presence of so-called heat shock element (HSE) sequences in their promoter regions (3,17,22) that consist of arrays of alternatively oriented NGAAN modules (2, 37). HSEs are binding sites for heat shock transcription factor (HSF) (21,23,36), which is inactive in unstressed cells and active in stressed cells (16,36,38). Organisms differ in their number of distinct HSF species. Whereas Saccharomyces cerevisiae and fruit flies express a single HSF species, birds, mammals, and plants express multiple HSF species (reviewed in reference 31). Furthermore, the strategies employed to regulate HSF activity differ drastically in S. cerevisiae and higher eukaryotes (30). While yeast HSF binds DNA constitutively, heat-activable HSF in higher eukaryotes is incapable of DNA binding in the absence of heat stress. Thus, in higher eukaryotes, HSF activity may be regulated mainly or exclusively at the level of DNA-binding ability. However, a number of situations involving mammalian cells, in which HSF DNAbinding ability was induced but a concomitant increase in hsp gene expression did not occur, have been described (6,14,15,24), suggesting that activation of HSF is a multistage process and that induction of DNA-binding ability may be only an early event in a complex activation process.It has been shown previously that stress activation of hsp genes in mammalian cells is ...

Neuronal nicotinic acetylcholine receptor of the a4/non-a (a4/na) type was reconstituted in Xenopus oocytes after nuclear injection of cDNA expression vectors. Functional neuronal receptor was only formed when the two subunits a4 and na were coiniected, neither a4 nor na alone being effective. Responses to bath application of acetylcholine (AcCho) have been measured in voltage clamp. AcCho doses as low as 10 nM induce currents of up to 50 nA. Dose-response studies indicate a Kd of about 0.77 x 10-6 M and a Hill coefficient of 1.5, thus predicting more than one AcCho binding site per receptor molecule. The current-voltage relationship of AcCho-induced currents presents a strong inward rectification. Responses to AcCho were compared to those of three other agonists: L-nicotine, carbachol, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Sensitivities to AcCho, nicotine, and DMPP are quite similar. Sensitivity to carbachol is much lower, but the currents are otherwise indisinguishable from those induced by AcCho. Five AcCho antagonistsneuronal bungarotoxin (K-bungarotoxin), tubocurarine (TC), hexamethonium bromide (Hex), decamethonium bromide (Dec), and mecamylamine (Mec)-have been tested. Neuronal bungarotoxin has no effect on the a4/na channel, whereas 2.5 ,M TC reduces by half the current peak evoked by 1 ,uM AcCho. The block by TC is independent of membrane voltage. By contrast, the block of AcCho-induced currents by Hex or Dec is strongly voltage dependent, suggesting that these substances enter the channel. The block by Mec is detectable at concentrations as low as 100 nM when applied together with 1 ,uM AcCho and is voltage independent. Hex, Dec, and Mec are effective only when AcCho is present. While the effects of all other agents are fully reversible, the Mec block is persistent.The nicotinic acetylcholine (AcCho) receptor (nAcChoR) mediating synaptic transmission at the neuromuscular junction of vertebrates has long been a favored model system in the study of ligand-gated ion channels. Because of the efforts of many, much is now known about its biochemistry, electrophysiology, and molecular biology (1). In contrast, little is known about the different types of neuronal nAcChoRs that recently have been identified in the central and peripheral nervous system by using antibody and cDNA probes derived from muscle receptor (2-8). At least six neuronal genes have been cloned, sequenced, and found to encode proteins homologous to the four related subunits assembled in the mature muscle receptor (2, 4-8). As functional nAcChoRs can be reconstituted in Xenopus oocytes by cytoplasmic injection of complementary RNA (2, 7, 8) or by nuclear injection of cDNA expression vectors (9), it is now possible to characterize the physiology and pharmacology of the numerous receptor types resulting from the assembly of cloned subunits. Such studies have revealed that functional neuronal nAcChoRs assemble from two subunits only (an a subunit and a non-a-subunit in our nomenclature), whereas four different subunits...

A human 70-kDa heat shock protein (hsp7O) gene segment has been isolated. The (4) and Escherichia coli (5) and, most recently, after the material presented here had been assembled, also in Xenopus (6). That the Drosophila hsp7O genes are expressed in a heat-regulated fashion in frog, mouse, and monkey cells (2,3,7,8) implies that not only the structures, but even the mechanism of transcription regulation of these genes have been conserved throughout evolution.To extend our understanding of the evolutionary relationship between heat shock response mechanisms in different organisms, we have isolated and functionally tested a human hsp7O gene. This study also provides the basis for future experiments in which the newly isolated human hsp gene will be used to investigate the mechanism through which transforming genes activate cellular genes: hsp7O genes are induced as a result of the action of the adenoviral EIA gene (9) and even of a c-myc gene (10). MATERIALS AND METHODSScreening of a Human DNA Library. A phage X library of human genomic DNA (11) was kindly provided by Tom Maniatis. This library was screened by plaque hybridization (12) using as probe a 2-kbp Sal 1 fragment from plasmid SalO (13), which includes a complete Drosophila hsp70 RNAcoding region. After hybridization under standard conditions, the filters were washed at 420C in 5 x NaCl/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M Na citrate). About 5 x 10 plaques were screened. As a final test, DNAs were prepared from the positive clones (14) and were hybridized to labeled Drosophila hsp7O gene DNA in dot blot experiments (15).DNA Sequence Analysis. Plasmid DNAs were prepared by CsCl banding from cleared lysates. DNA sequence analysis was carried out according to Maxam and Gilbert (16) except that DNA fragments were end-labeled by DNA polymerase fragment A and (a-32P)dXTP.Construction of Human hsp7O-E. coli fi-Galactosidase Hybrid Genes. To construct plasmid 671SX, an Xho I linker sequence was introduced into the unique Bgl II site of the previously described plasmid 671 (17), which contains, in between the XmaIII and BamHI sites of pSVOd (18), a 3-kbp E. coli 03-galactosidase fragment from pMC1871 (19) and downstream from it, 2.3 kbp of eukaryotic 3' gene flanking sequence. The construction of p173 is described in Fig. 3a. Sticky ends of restriction fragments were filled in by DNA polymerase fragment A. To construct p173P, a 5-kbp Pst I fragment from p173 was isolated that includes 600 bp of 5' nontranscribed and 500 bp of transcribed human hsp7O gene sequence, the 3-galactosidase-coding region and 3' flanking sequences. This fragment was inserted into the Pst I site of pUC8 (20). Plasmid 173S was prepared by digesting p173 with Sph I. Ends were made blunt by T4 DNA polymerase. After digestion with Sma I, the material was religated and used for transformation of E. coli MC1061. Plasmid 173X was made by introducing an Xho I linker into the Sph I site of p173. To construct p1730R, an Xho I/Cla I fragment from p173X containing 2.95 kbp of human hsp...

In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF-κB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by α-amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replicationdependent, possibly passive mechanism.

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