n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

Glucocorticoid receptor (GR)-associated SMRT binding to C/EBPbeta TAD and Nrf2 Neh4/5: role of SMRT recruited to GR in GSTA2 gene repression

The expression of the glutathione S-transferase gene (GST), whose induction accounts for cancer chemoprevention, is regulated by activation of CCAAT/enhancer binding protein beta (C/EBPbeta) and NF-E2-related factor 2 (Nrf2). The present study investigated the repressing effects of activating glucocorticoid receptor (GR) on C/EBPbeta- and Nrf2-mediated GSTA2 gene induction and the mechanism. Dexamethasone that activates GR inhibited constitutive and oltipraz- or tert-butylhydroquinone (t-BHQ)-inducible GSTA2 expression in H4IIE cells. Also, dexamethasone repressed GSTA2 promoter-luciferase gene activity. Dexamethasone-GR activation did not inhibit nuclear translocation of C/EBPbeta or Nrf2 nor their DNA binding activities induced by oltipraz or t-BHQ. Deletion of the glucocorticoid response element (GRE) in the GSTA2 promoter abolished dexamethasone inhibition of the gene induction. Immunoprecipitation-immunoblotting, chromatin immunoprecipitation, and GST pull-down assays revealed that silencing mediator for retinoid and thyroid hormone receptors (SMRT), a corepressor recruited to steroid-GR complex for histone deacetylation, bound to TAD domain of C/EBPbeta and Neh4/5 domain of Nrf2. The GSTA2 promoter-luciferase activities were decreased by SMRT but not by truncated SMRTs. The small interference RNA (siRNA) against SMRT abolished SMRT repression of the gene induction by C/EBPbeta or Nrf2. The plasmid transfection and siRNA experiments directly evidenced the functional role of SMRT in GSTA2 repression. In conclusion, dexamethasone antagonizes C/EBPbeta- and Nrf2-mediated GSTA2 gene induction via ligand-GR binding to the GRE, and steroid-mediated GSTA2 repression involves inactivation of C/EBPbeta and Nrf2 by SMRT recruited to steroid-GR complex.     

A novel minisatellite repeat expansion identified at FRA16B in a Japanese carrier

Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.     

Pliable DNA conformation of response elements bound to transcription factor p63

We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element. The conformation of a 22-bp canonical response element containing an AT spacer between the two half-sites is unaltered compared with that containing a TA spacer, exhibiting superhelical trajectory. In contrast, a GC spacers abolishes the DNA superhelical trajectory and exhibits less bent DNA, suggesting that increased GC content accompanies increased double helix rigidity. A 19-bp DNA, representing an AT-rich response element with overlapping half-sites, maintains superhelical trajectory and reveals two interacting p63DBD dimers crossing one another at 120°. p63DBD binding assays to response elements of increasing length complement the structural studies. We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters.     

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.     

H-NS Can Facilitate Specific DNA-binding by RNA Polymerase in AT-rich Gene Regulatory Regions

Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter −10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter −10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells.     

Instability in tyrR Strains of Plasmids Carrying the Tyrosine Operon: Isolation and Characterization of Plasmid Derivatives with Insertions or Deletions

The transformation of tyrR strains of Escherichia coli with multicopy plasmids which carry the tyrosine operon gave rise to modified plasmids with either insertions or deletions. The effect of each of these insertions or deletions was to decrease the level of expression of this operon. It is proposed that plasmid instability arose as a direct consequence of the metabolic effects of an overproduction of the enzymes coded for by the tyrosine operon. The results have significant implications for the cloning of genes that are repressed by the product of a regulatory gene. Since the predominant plasmid modification observed was the insertion of an IS1 element near the regulatory region of the tyrosine operon, the results also suggest a role for IS1 elements in the regulation of gene expression.     

Regulation of I-transposon activity in Drosophila: evidence for cosuppression of nonhomologous transgenes and possible role of ancestral I-related pericentromeric elements

We have previously shown that the activity of functional I retrotransposons (I factors) introduced into Drosophila devoid of such elements can be repressed by transgenes containing an internal fragment of the I factor itself and that this repressing effect presents the characteristic features of homology-dependent gene silencing or cosuppression. Here we show that the same transgenes can induce silencing of a nonhomologous reporter gene containing as the sole I-factor sequence its 100-bp promoter fragment. Silencing of the nonhomologous reporter gene shows strong similarities to I-factor cosuppression: It does not require any translation product from the regulating transgenes, sense and antisense constructs are equally potent, and the silencing effect is only maternally transmitted and fully reversible. A search for genomic I-like sequences containing domains with similarities to those of both the regulating and the reporter transgenes led to the identification of four such elements, which therefore could act as intermediates-or relays-in the cosuppression machinery. These results strongly suggest that ancestral transposition-defective I-related elements, which are naturally present in the Drosophila genome, may participate per se in the natural conditions of I-factor silencing.