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BBA - Gene Structure and Expression (v.1759, #3-4)

Editorial Board (pp. ii).

DNA topoisomerase I from parasitic protozoa: A potential target for chemotherapy by R.M. Reguera; C.M. Redondo; R. Gutierrez de Prado; Y. Pérez-Pertejo; R. Balaña-Fouce (pp. 117-131).

The growing occurrence of drug resistant strains of unicellular prokaryotic parasites, along with insecticide-resistant vectors, are the factors contributing to the increased prevalence of tropical diseases in underdeveloped and developing countries, where they are endemic. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity. Due to the fact that effective immunoprophylaxis is not available at present; preventive sanitary measures and pharmacological approaches are the only sources to control the undesirable effects of such diseases. Current anti-parasitic chemotherapy is expensive, has undesirable side effects or, in many patients, is only marginally effective. Under this point of view molecular biology techniques and drug discovery must walk together in order to find new targets for chemotherapy intervention. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted. The present report is an up to date review of the new findings on type IB DNA topoisomerase in unicellular parasites and the role of these enzymes as targets for therapeutic agents.

Keywords: Abbreviations; LdTOPIA; gene encoding the large subunit of; L donovani; topoisomerase I; LdTOPIB; gene encoding the small subunit of; L donovani; topoisomerase; PfTOPI; gene encoding; P. falciparum; topoisomerase I; NLS; nuclear localization signal; kDNA; kinetoplast DNA; Tdp-1; tyrosyl-phosphodiesterase-1; CPT; camptothecin; SUMO; Small Ubiquitin-like Modifiers; QSAR; Quantitative Structure Activity Relationship; DHBA; betulinic acid derivatives; SSB; single-strand break; DSB; double-strand break; RNAi; small interference RNADNA topoisomerase IB; Camptothecin; Indolocarbazole; Parasitic protozoa; Plasmodium; Cryptosporidium; Trypanosomatid

Stability of casein mRNA is ensured by structural interactions between the 3′-untranslated region and poly(A) tail via the HuR and poly(A)-binding protein complex by Kentaro Nagaoka; Toshiyuki Suzuki; Tomomi Kawano; Kazuhiko Imakawa; Senkiti Sakai (pp. 132-140).

The maintenance of mRNA stability has emerged as a mechanism of post-transcriptional control. We demonstrate that β-casein mRNA stability is dictated by the poly(A) tail and sequences in the 3′-UTR. An in vitro mRNA decay assay revealed that β-casein mRNA with a long poly(A) tail had higher stability than that with a short poly(A) tail. The addition of poly(A) homopolymer and 3′-UTR cRNA as competitor induced rapid degradation of β-casein mRNA. The interaction between full-length β-casein mRNA and poly(A) homopolymer was inhibited by the addition of the 3′-UTR cRNA in the lysates, which indicates that one region of the 3′-UTR associates with the poly(A) tail through an RNA–protein interaction. The putative AU-rich element (ARE) is present at nt 897–905; deletion and mutation analysis showed that the ARE site was required for maintaining the stability of the β-casein 3′-UTR. In the immunoprecipitation analysis, the poly(A)-binding protein (PABP) and the RNA-binding protein HuR were pulled down by 3′-UTR cRNA, and the absence of the ARE site reduced the binding of these proteins. These experiments further revealed that the rapid degradation of β-casein mRNA was induced by incubation with HuR- and PABP-depleted RRLs. Collectively, our results suggest that β-casein mRNA is protected from degradation by virtue of the structural interaction between the 3′-UTR and poly(A) tail via a protein complex of HuR and PABP.

Keywords: Stability; Casein; 3′UTR; Poly(A) tail; PABP; HuR

Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression by Hye-Sook Kwon; Boli Huang; Nam Ho Jeoung; Pengfei Wu; Calvin N. Steussy; Robert A. Harris (pp. 141-151).

Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor α (RXRα) and retinoic acid receptor α (RARα) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRα/RARα/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRα/RARα/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

Keywords: Abbreviations; PDK4; pyruvate dehydrogenase kinase 4; RA; retinoic acid; TSA; trichostatin A; HDAC; histone deacetylase; HAT; histone acetyltransferase; RXRα; retinoid X receptor α; RARα; retinoic acid receptor α; CBF; CCAAT box binding factor; RARE; retinoic acid response element; ChIP; chromatin immunoprecipitation assayPyruvate dehydrogenase; Pyruvate dehydrogenase kinase; Retinoic acid; Trichostatin A; Histone deacetylase; Human; Retinoid X receptor; Histone; Retinoic acid receptor; Histone acetylation

β-naphthoflavone represses dystrophin Dp71 expression in Hepa-1 cells by Mario Bermúdez de León; Pablo Gómez; Guillermo Elizondo; Rocío Zatarain-Palacios; Francisco García-Sierra; Bulmaro Cisneros (pp. 152-158).

Dystrophin Dp71 is expressed in hepatic tissue; however, its function in this tissue remains unknown. The Dp71 promoter sequence contains conserved CACGC motifs, which constitute the invariant core sequence of xenobiotic-regulatory elements. These elements function as target sites for the aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (Ahr/ARNT) in genes regulated by this transcription factor. Thus, Dp71 expression in hepatic cells would be regulated by Ahr signaling. In this study, the effect of the xenobiotics β-Naphthoflavone (βNF), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and Benzo[a]Pyrene (BaP) on Dp71 expression was analyzed in Hepa-1 cells. It was demonstrated that βNF, but not BaP or TCDD, represses Dp71 expression at both transcriptional and translational levels. To test directly the involvement of the Ahr signaling in the negative regulation of Dp71, we analyzed the effect of βNF on Dp71 expression in the liver of wild type (Ahr+/+) and AHR-null (Ahr−/−) mice. The Dp71 mRNA repression, caused by the βNF treatment, was also found in the liver tissue of wild type mice; however, such negative effect was reversed in the liver of AHR-null mice, which supports the participation of the Ahr signaling in Dp71 downregulation. Modulation of Dp71 expression by βNF may represent a novel mechanism of Ahr action.

Keywords: Dp71; β-Naphthoflavone; Hepa-1; Hepatic cell; Xenobiotics; AHR-null mice

Cell-surface expression of a new splice variant of the mouse signal peptide peptidase by Jens Urny; Irm Hermans-Borgmeyer; H. Chica Schaller (pp. 159-165).

The signal peptide peptidase (SPP) is an intramembrane-cleaving aspartyl protease that acts on type II transmembrane proteins. SPP substrates include signal peptides after they have been cleaved from a preprotein, hence the name. The known SPP isoform, which we renamed SPPα, contains an endoplasmic reticulum retention signal at the carboxy terminus. We found a new splice variant, SPPβ, with an additional in-frame exon inserted between exons 11 and 12 of SPPα. Insertion of the new exon led to a complete change in the amino-acid sequence of the carboxy tail. A stop codon within this new exon resulted in silencing of exon 12 and eliminated the endoplasmic reticulum retention signal. The new SPP isoform predominantly localised to the cell surface in contrast to the more restricted localisation of SPPα in the endoplasmic reticulum. Differential expression in mouse tissues and in subcellular compartments suggests new functions for SPP in addition to cleaving signal peptides.

Keywords: Signal peptide peptidase; SPP; Cell-surface localisation; ER-retention signal; Intramembrane cleavage; Aspartyl protease

Transcriptional regulation of mouse mast cell protease-7 by TGF-β by Masayuki Funaba; Teruo Ikeda; Masaru Murakami; Kenji Ogawa; Yoshii Nishino; Kunihiro Tsuchida; Hiromu Sugino; Matanobu Abe (pp. 166-170).

Mouse mast cell protease-7 ( mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-β (TGF-β) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-β. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt −126 to −122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-β pathway. In addition, a region from nt −104 to −98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-β signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-β. Treatment with TGF-β in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c- fos but not of c- jun and junB. Expression of c- fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c- jun expression inhibited the transcription. Our results suggest that TGF-β stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c- fos induction, and that the AP-1 components distinctly related with the TGF-β pathway.

Keywords: Smad3; Smad4; c-; fos; c-; jun

Transcription promoter activity of the human S100A7 gene in oral squamous cell carcinoma cell lines by Hideaki Fukuzawa; Tamotsu Kiyoshima; Ieyoshi Kobayashi; Satoru Ozeki; Hidetaka Sakai (pp. 171-176).

The S100A7 (psoriasin) gene has been shown to be markedly over-expressed in squamous cell carcinomas (SCCs) as well as in psoriasis. We herein examined the S100A7 gene promoter activity in human oral SCC cell lines to identify the putative SCC-specific regulatory regions for the S100A7 transcription. Functional deletion assays of 5′-flanking region demonstrated that the segments, (−1513 to −988), (−1954 to −1513) and (−3040 to −2578), play important roles in the transcription activity in the oral SCCs. The internal deletion of the short segments, (−1248 to −1110), (−1109 to −988) and (−1248 to −988), decreased this activity. These segments cloned upstream of the heterologous promoter increased the promoter activity in oral SCC cell line. Electrophoretic mobility shift assays, using the sequence segmental probes, (−1248 to −1110) and (−1109 to −988), showed different DNA–protein complex patterns depending on the types of used cell lines. One of the complexes was only observed in the oral SCCs. These data suggested that the segment from −1513 to −988 contains up-regulatory elements for the transcription activity of the S100A7 gene in oral SCCs.

Keywords: S100A7; Squamous cell carcinoma; Promoter; Transcription; DNA binding protein

Protein–DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera by Bo Hong; Zhi-Fang Zhang; Shun-Ming Tang; Yong-Zhu Yi; Tian-Yi Zhang; Wei-Hua Xu (pp. 177-185).

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH-PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning −467 to −371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from −965 to −534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions −360 to −355 bp, a potential site for interaction with basic helix–loop–helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis-elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression.

Keywords: Abbreviations; SG; suboesophageal ganglion; DH; diapause hormone; PBAN; pheromone biosynthesis activating neuropeptide; DHMBP; DH-modulator-binding protein; EMSA; electrophoretic mobility shift assays; bHLH; basic helix–loop–helix; POU; Pit-Oct-Unc; Har; Helicoverpa armigera; Bom; Bombyx moriDiapause hormone; Pheromone biosynthesis activating neuropeptide; Transcriptional regulation; E-box; Transcription factor; Helicoverpa armigera

A novel member of the NSF family in the corn earworm, Helicoverpa zea: molecular cloning, developmental expression, and tissue distribution by Wei-Hua Xu; Qi-Rui Zhang; David L. Denlinger (pp. 186-190).

N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane transport events. We report the cloning of a cDNA encoding NSF from the noctuid moth, Helicoverpa zea (Hez), a major agricultural pest. The amino acid sequence deduced from the cDNA indicates that Hez-NSF has 88%, 73%, and 70% identities to NSF from Manduca sexta, Aedes aegypti, and Drosophila melanogaster, respectively. Northern hybridization analysis clearly shows a 4.4 kb mRNA, corresponding in size to the cDNA present in the brain–suboesophageal ganglion (SG) complex of pupae. The NSF transcript is present in components of the central nervous system, including the brain, SG, thoracic ganglion and abdominal ganglion, but is not present in nonneural tissues. A developmental profile of gene expression revealed that Hez-NSF mRNA is present throughout the embryonic, larval, and pupal development.

Keywords: Abbreviations; NSF; N-ethylmaleimide-sensitive fusion protein; SNAP; soluble NSF attachment protein; SNARE; SNAP receptor; Br; brain; SG; suboesophageal ganglion; RT-PCR; reverse transcription-polymerase chain reaction; RACE; rapid amplification of cDNA endsN-ethylmaleimide-sensitive fusion protein; cDNA structure; Tissue distribution; Developmental expression; Helicoverpa zea

Molecular characterization of a bHLH transcription factor involved in Arabidopsis abscisic acid-mediated response by Jiyoung Kim; Ho-Young Kim (pp. 191-194).

The suppression subtractive hybridization (SSH) method was used to isolate abscisic acid (ABA)-regulated genes in Arabidopsis. We report that the AtAIG1 gene is up-regulated by ABA and the AtAIG1 encodes a basic/helix–loop–helix (bHLH)-type transcription factor in Arabidopsis. The AtAIG1 was targeted into nucleus and bound to the E-box–DNA sequence, which is found in the promoter regions of many ABA-responsive genes. Functional analyses with the ataig1 knockout mutants revealed that the mutant plants show enhanced sensitivity to ABA. In addition, the ataig1 plants showed reduced expression of ABA-responsive genes, such as RD29A and RD22. Thus, these functional analyses suggest that the AtAIG1 protein plays an important role in ABA-mediated signal transduction pathway.

Keywords: Abscisic acid; Transcription factor; bHLH; Suppression subtractive hybridization; Arabidopsis

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BBA - Gene Structure and Expression (v.1759, #3-4)