CtBP-dependent activities of the short-range Giant repressor in the Drosophila embryo.

There are at least three short-range gap repressors in the precellular Drosophila embryo: Krüppel, Knirps, and Giant. Krüppel and Knirps contain related repression motifs, PxDLSxH and PxDLSxK, respectively, which mediate interactions with the dCtBP corepressor protein. Here, we present evidence that Giant might also interact with dCtBP. The misexpression of Giant in ventral regions of transgenic embryos results in the selective repression of eve stripe 5. A stripe5-lacZ transgene exhibits an abnormal staining pattern in dCtBP mutants that is consistent with attenuated repression by Giant. The analysis of Gal4-Giant fusion proteins identified a minimal repression domain that contains a sequence motif, VLDLS, which is conserved in at least two other sequence-specific repressors. Removal of this sequence from the native Giant protein does not impair its repression activity in transgenic embryos. We propose that Giant-dCtBP interactions might be indirect and mediated by an unknown bZIP subunit that forms a heteromeric complex with Giant. We also suggest that the VLDLS motif recruits an as yet unidentified corepressor protein.

Local action of long-range repressors in the Drosophila embryo.

Previous studies have identified two corepressors in the early Drosophila embryo: Groucho and dCTBP: Both proteins are recruited to the DNA template by interacting with short peptide motifs conserved in a variety of sequence-specific transcriptional repressors. Once bound to DNA, Groucho appears to mediate long-range repression, while dCtBP directs short-range repression. The short-range Krüppel repressor was converted into a long-range repressor by replacing the dCtBP interaction motif (PxDLSxH) with a Groucho motif (WRPW). The resulting chimeric repressor causes a different mutant phenotype from that of the native Krüppel protein when misexpressed in transgenic embryos. The different patterning activities can be explained on the basis of long-range silencing within the hairy 5' regulatory region. The analysis of a variety of synthetic transgenes provides evidence that Groucho-dependent long-range repressors do not always cause the dominant silencing of linked enhancers within a complex cis-regulatory region. We suggest a "hot chromatin" model, whereby repressors require activators to bind DNA.

Regulated expression of human angiotensinogen gene by hepatocyte nuclear factor 4 and chicken ovalbumin upstream promoter-transcription factor.

We previously identified various upstream and downstream regulatory elements and factors important for hepatic expression of the human angiotensinogen (ANG) gene, the precursor of vasoactive octapeptide angiotensin II. In the present study, to further investigate the molecular mechanism of human ANG transcriptional regulation, we generated transgenic mice carrying the fusion gene composed of the 1. 3-kilobase promoter of the human ANG gene, its downstream enhancer, and the chloramphenicol acetyltransferase reporter gene. Because expression of the chloramphenicol acetyltransferase gene was observed strongly in the liver and weakly in the kidney, we suspected that hepatocyte nuclear factor (HNF) 4 with a tissue expression pattern similar to that of the reporter gene would regulate ANG transcription. In vitro assays indicated that HNF4 bound to the promoter elements and strongly activated the ANG transcription, but that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a transcriptional repressor, dramatically repressed human ANG transcription through the promoter elements and the downstream enhancer core elements. Furthermore, COUP-TF dramatically decreased the human ANG transcription in the mouse liver by the Helios Gene Gun system in vivo. These results suggest that an interplay between HNF4 and COUP-TF could be important in hepatic human ANG transcription.

Transcriptional coregulators in development.

Small differences in the levels of an extracellular signaling molecule can specify cell fate during development. Threshold responses are often determined at the level of transcription. Cell-specific and spatially localized patterns of gene expression depend on combinations of sequence-specific activators and repressors that bind to extensive cis-regulatory regions. Different mechanisms for integrating this complex regulatory information are discussed, particularly the role of coregulatory proteins, which are recruited to the DNA template by sequence-specific transcription factors. Recent studies suggest that a growing set of coactivators and corepressors mediate communication between diverse upstream regulatory proteins and the core RNA polymerase II transcription complex.

dCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo.

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co-repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression.

Solid-Liquid Phase Behavior of Binary Mixture of Tetraethylene Glycol Decyl Ether and Water.

Solid-liquid phase behavior of binary mixture of nonionic surfactant, tetraethylene glycol decyl ether (C10E4), and water was examined by means of differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), polarized optical microscopy (POM), and visual observation in the temperature range -40-70 degreesC. The DSC experiments allowed us to determine many phase boundaries among various phases appearing in this mixture system, including mesomorphic phases. The T-X phase diagram for fluid phases of C10E4/H2O mixture constructed from the results of DSC and visual observation was in good agreement with that previously reported by Lang and Morgan for the same mixture system except for the occurrence of a dilute lamellar phase (Lang, J. C. and Morgan, R. D., J. Chem. Phys., 73, 5849 (1980). The FT-IR spectra obtained for the mixture in a solid state demonstrated that the phase compound expressed by C10E4 . 6H2O is formed in the solid phase, which indicates that there is a strong interaction in the solid state between water and the hydrophilic polyoxyethylene chain of the surfactant. The FT-IR results also suggested that the strength of the hydrogen bond between water and the polyoxyethylene chain of C10E4 differs among the phase states of the mixture, and increases in the order L2 < L1 < Lalpha approximately H1 < solid, where L2 refers to the inverted micellar phase, L1 the normal micellar phase, Lalpha the lamellar phase, and H1 the normal hexagonal phase. Copyright 1998 Academic Press.

Phase Behavior of Aqueous Mixtures of Some Polyethylene Glycol Decyl Ethers Revealed by DSC and FT-IR Measurements.

Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) were used to investigate the phase behavior of aqueous binary mixtures of three polyoxyethylene type nonionic surfactants, penta-, hexa-, and octaethylene glycol decyl ethers (C10E5, C10E6, and C10E8, respectively) in the temperature range -40-70 degreesC. Many thermotropic transitions among various phases assumed by these mixture systems, including mesomorphic phases, were detected in DSC thermograms, from which the T-X phase diagrams were constructed with the aid of polarized optical microscopic observation to characterize the mesomorphic phases. It was revealed by FT-IR measurements that the phase compounds are formed in a solid phase between these surfactants and water molecules; the compositions of the compounds for the three surfactant species are expressed as C10E5 . 10H2O, C10E6 . 12H2O, and C10E8 . 16H2O, respectively. The stoichiometry of the phase compounds clearly demonstrates that just two water molecules are bound per oxyethylene unit of polyoxyethylene (POE) chain of the surfactants in the solid phase, probably due to the hydrogen bonding. It was also found that the phase state of the mixture is reflected in the wavenumber of the absorption maximum associated with the OH stretching vibration, nuOH, although no significant difference was appreciable in nuOH among mesomorphic phases. The nuOH decreased in the order L (liquid phase) > H1 (normal hexagonal phase) approximately V1 (normal cubic phase) approximately Lalpha (lamellar phase) > solid phase, which indicates that the hydrogen bonding between water and the POE chain of the surfactants becomes stronger in the sequence liquid < mesophase < solid. Copyright 1998 Academic Press.

Interaction of short-range repressors with Drosophila CtBP in the embryo.

Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. The E1A sequence motif that interacts with CtBP, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K), is present in the repression domains of two unrelated short-range repressors in Drosophila, Knirps and Snail, and is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element-induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps, which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals.

Phase Behavior of Binary Mixture of Heptaethylene Glycol Decyl Ether and Water: Formation of Phase Compound in Solid Phase

Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) were used to construct and characterize the phase diagram for a binary mixture of heptaethylene glycol decyl ether (C10 E7 ) and water in the temperature range from -60 to 80°C. Plots of the endothermic peak temperatures obtained by DSC measurements against compositions provided eutectic solid-liquid phase boundaries with a eutectic composition of 34 wt% of H2 O. On the other hand, heat of fusion per unit weight of the mixture changed discretely at the composition corresponding to the "eutectic" composition. Furthermore, the IR spectra obtained for the mixture in the solid phase were well reproduced as a superposition of those for the mixture of 34 wt% H2 O and pure components but were not reproduced by superimposing the spectra obtained for the solid surfactant and ice. These observations indicate that a solid phase compound is formed between C10 E7 and water with a stoichiometry of 1:14 and that the compound and pure components exist as separate phases, rather than the phases separating into surfactant and ice, which would be expected if the C10 E7 /water mixture formed a true eutectic mixture system. It is estimated from the composition corresponding to the phase compounds that two molecules of water per oxyethylene unit are bound to hydrophilic polyoxyethylene chain of C10 E7 to form a hydrated compound.

Human activin betaA gene. Identification of novel 5' exon, functional promoter, and enhancers.

On the basis of cDNA cloning, primer extension, and transfection experiments, we identified a novel 5' exon of the human activin betaA subunit gene, and found its enhancer and promoter regions as well as multiple transcription start sites. A series of deletion and mutation analyses of the enhancer sequences defined the 45-base pair core region (DR-1 core) containing two short elements with similarity to AP-1 (12-O-tetradecanoylphorbol-13-acetate response element; TRE) and CREB/ATF (cyclic AMP response element; CRE) binding sites, both of which were necessary for full enhancer activity. Gel shift and antibody supershift assays using DR-1 core region revealed the formation of two specific DNA-protein complexes, one of which could be partially dissociated by a competing oligonucleotide containing a single copy of the consensus TRE, but the other of which contained neither CREB/ATF nor AP-1 as major components. Although 12-O-tetradecanoylphorbol-13-acetate and cAMP induced the activin enhancer/promoter-driven CAT activity, such drug induction was obscured when either the TRE- or CRE-like elements were mutated in the native promoter context. Our results demonstrate that the promoter and enhancer regions identified here are essential for maintaining the efficient promoter activity of the human activin betaA subunit gene.

A cis-acting DNA element located between TATA box and transcription initiation site is critical in response to regulatory sequences in human angiotensinogen gene.

The promoter of the human angiotensinogen (hAG) gene functioned in its own core promoter context but not when replaced with simian virus 40 (SV40) core promoter, suggesting the presence of a transcriptionally important cis-acting sequence. Electrophoretic mobility shift assays demonstrated that a ubiquitously expressed nuclear factor, AGCF1, bound to AGCE1 (hAG core promoter element 1; positions -25 to -1) located between the TATA box and transcription initiation site. Substitution mutation in AGCE1 which disrupted AGCF1 binding affected the promoter activity more severely than a nonsense mutation of the hAG TATA sequences did. When AGCE1 was placed at the downstream of SV40 core promoter, the responsiveness to hAG upstream region was significantly restored. Furthermore, mutation and in vivo competition experiments suggested that AGCF1 acts as a critical regulator of hAG transcription by mediating the activity of the hAG upstream and downstream enhancer elements. DNase I footprinting and UV cross-linking analyses showed that AGCF1 with apparent molecular masses of 31, 33, and 43 kDa as the components protected the region from -26 to -9 which partially overlapped with the TATA box consensus sequences. These findings indicate that AGCE1 in addition to the TATA box plays a key role in mediating the hAG regulatory elements.

Effect of headgroup type on the miscibility of homologous phospholipids with different acyl chain lengths in hydrated bilayer.

The miscibility of homologous phosphatidylcholines with different acyl chain lengths in hydrated bilayer was examined through the binary phase diagram constructed by differential scanning calorimetry. By analyzing the phase diagram according to a thermodynamic model based on the Bragg-Williams approximation to evaluate the excess free energy of mixing, the non-ideality parameter of mixing, rho(0), was estimated, which allows one to interpret the mixing behavior of the two lipid components in terms of the difference in the pair-interaction energies between like-pairs and mixed-pairs formed in the mixture. By summarizing the rho(0) values obtained previously for other classes of phospholipids, it was found that rho(0) increases in the order of phosphatidylglycerol (PG) approximately phosphatidylcholine (PC) < phosphatidylethanolamine (PE) < phosphatidic acid (PA). Since the difference in the pair-interaction energies is considered to be determined by the relative contribution of inter-headgroup interaction to the overall intermolecular interaction, this sequence of rho(0) value suggests that the headgroup interaction in hydrated bilayer increases in the order of PA < PE < PC approximately PG.

Cloning of the rat angiotensin II type 2 receptor gene and identification of its functional promoter region.

We have cloned the rat angiotensin II receptor type 2 (AT2) gene, whose physiological function remains unclear. Sequence analysis indicated that exons 1 and 2 exist in the 5'-untranslated region and the initiation codon ATG is located in exon 3. The 1.6-kb genomic fragment at positions -1567 to +26 relative to the putative transcription start site was found to contain a functional promoter region using transient chloramphenicol acetyltransferase assay. This is the first report demonstrating the nucleotide sequence of the promoter region of this gene.

Purification and characterization of extracellular alginate lyase from Enterobacter cloacae M-1.

An alginate lyase from the culture supernatant of Enterobacter cloacae M-1 was purified by ammonium sulfate precipitation, cation-exchange chromatography (SP-Toyopearl), and gel filtration (Ultrogel AcA44). The final preparation thus obtained showed a single band on SDS-PAGE. The purified enzyme had the molecular weight of 38,000 and 32,000 by SDS-PAGE and gel filtration, respectively. The pI of the enzyme was 8.9. The optimum pH and temperature for the enzyme reaction were around 7.8 and 30 degrees C, respectively. The enzyme was unstable on heating. EDTA completely inhibited the enzyme activity, but the activity was completely restored by the treatment with CaCl2. The enzyme was specific for poly-guluronate and produced several kinds of unsaturated oligomers from the gluluronate. This suggested that the enzyme could be classified as an endo poly-guluronate lyase.

A cell type-dependent enhancer core element is located in exon 5 of the human angiotensinogen gene.

We have recently characterized a cell type-dependent downstream enhancer that is contained within an 832-bp B2 sequences spanning the exon 5 and 3'-flanking region of the human angiotensinogen gene and have localized one of core elements to a 24-bp region in the 3'-flanking region. In the present study, we functionally dissected the 5'-half of B2 region and identified an additional cell type-dependent enhancer core element composed of the 80-bp sequences (+1399 to +1478) in the exon 5. Electrophoretic mobility shift assays demonstrated that the element interacts with two HepG2 cell-specific and several ubiquitous nuclear factors. These results, together with our previous results, suggest that the 80-bp exon 5 enhancer core element as well as 24-bp region plays an important role in the downstream enhancer effect.

Identification of cell type-dependent enhancer core element located in the 3'-downstream region of the human angiotensinogen gene.

We previously demonstrated that the 3.8-kilobase DNA fragment containing exons 4 and 5 of the human angiotensinogen (hAG) gene enhances the expression of chloramphenicol acetyltransferase gene, under control of the hAG promoter, in human hepatoma HepG2 cells. In the present study, to define regulatory elements of the hAG gene, we have functionally dissected this downstream region and localized a cell type-dependent enhancer to the 832-base pair sequence containing the exon 5 and 3'-flanking region. Further deletion analyses revealed that the 24-base pair core DNA fragment present in the 3'-flanking region was responsible for this enhancement. Electrophoretic mobility shift assay demonstrated that the 3'-enhancer core element interacts specifically with two nuclear factors from the HepG2 cells, one of which is an uncharacterized factor (human angiotensinogen enhancer factor-1: hAEF-1), the other is an AP-3-related factor. Mutation analyses indicated that the disruption of hAEF-1 binding alone completely impaired the enhancer activity of the core element. These results suggested that the downstream enhancer core element interacting with hAEF-1 plays an important role in activating the angiotensinogen promoter in a cell type-dependent manner.