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BBA - Gene Structure and Expression (v.1759, #1-2)
Deciphering B-ZIP transcription factor interactions in vitro and in vivo by Charles Vinson; Asha Acharya; Elizabeth J. Taparowsky (pp. 4-12).
Over the last 15 years, numerous studies have addressed the structural rules that regulate dimerization stability and dimerization specificity of the leucine zipper, a dimeric parallel coiled-coil domain that can either homodimerize or heterodimerize. Initially, these studies were performed with a limited set of B-ZIP proteins, sequence-specific DNA binding proteins that dimerize using the leucine zipper domain to bind DNA. A global analysis of B-ZIP leucine zipper dimerization properties can be rationalized using a limited number of structural rules [J.R. Newman, A.E. Keating, Comprehensive identification of human bZIP interactions with coiled-coil arrays, Science 300 (2003) 2097–2101]. Today, however, access to the genomic sequences of many different organisms has made possible the annotation of all B-ZIP proteins from several species and has generated a bank of data that can be used to refine, and potentially expand, these rules. Already, a comparative analysis of the B-ZIP proteins from Arabidopsis thaliana and Homo sapiens has revealed that the same amino acids are used in different patterns to generate diverse B-ZIP dimerization patterns [C.D. Deppmann, A. Acharya, V. Rishi, B. Wobbes, S. Smeekens, E.J. Taparowsky, C. Vinson, Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: a comparison to Homo sapiens B-ZIP motifs, Nucleic Acids Res. 32 (2004) 3435–3445]. The challenge ahead is to investigate the biological significance of different B-ZIP protein–protein interactions. Gaining insight at this level will rely on ongoing investigations to (a) define the role of target DNA on modulating B-ZIP dimerization partners, (b) characterize the B-ZIP transcriptome in various cells and tissues through mRNA microarray analysis, (c) identify the genomic localization of B-ZIP binding at a genomic level using the chromatin immunoprecipitation assay, and (d) develop more sophisticated imaging technologies to visualize dimer dynamics in single cells and whole organisms. Studies of B-ZIP family leucine zipper dimerization and the regulatory mechanisms that control their biological activities could serve as a paradigm for deciphering the biophysical and biological parameters governing other well-characterized protein–protein interaction motifs. This review will focus on the dimerization specificity of coiled-coil proteins, particularly the human B-ZIP transcription family that consists of 53 proteins that use the leucine zipper coiled-coil as a dimerization motif.
Keywords: B-ZIP; Dimerization specificity; Repeat protein; Leucine zipper; Coiled coil; Double mutant thermodynamic cycle
eEF1B: At the dawn of the 21st century by Frédéric Le Sourd; Sandrine Boulben; Ronan Le Bouffant; Patrick Cormier; Julia Morales; Robert Belle; Odile Mulner-Lorillon (pp. 13-31).
Translational regulation of gene expression in eukaryotes can rapidly and accurately control cell activity in response to stimuli or when rapidly dividing. There is increasing evidence for a key role of the elongation step in this process. Elongation factor-1 (eEF1), which is responsible for aminoacyl-tRNA transfer on the ribosome, is comprised of two entities: a G-protein named eEF1A and a nucleotide exchange factor, eEF1B. The multifunctional nature of eEF1A, as well as its oncogenic potential, is currently the subject of a number of studies. Until recently, less work has been done on eEF1B. This review describes the macromolecular complexity of eEF1B, its multiple phosphorylation sites and numerous cellular partners, which lead us to suggest an essential role for the factor in the control of gene expression, particularly during the cell cycle.
Keywords: Elongation factor; Gene expression regulation; Protein translation; Cancer; Virus
Identification and regulation of novel PPAR-γ splice variants in human THP-1 macrophages by Ye Chen; Anna R. Jimenez; Jheem D. Medh (pp. 32-43).
We have previously identified four novel isoforms of PPAR-γ transcripts in monkey macrophages (J. Zhou, K.M. Wilson, J.D. Medh, Genetic analysis of four novel peroxisome proliferator receptor-γ splice variants in monkey macrophages. Biochem. Biophys. Res. Commun., 293 (2002) 274-283). The purpose of this study was to ascertain that these isoforms are also present in humans. Specific primers were designed to amplify individual isoform transcripts. The presence of PPAR-γ4, PPAR-γ5, and PPAR-γ7 transcripts in human THP-1 macrophages was confirmed by RT-PCR and sequencing. A transcript corresponding to PPAR-γ6 was not detected. The presence of novel full-length transcripts and protein was also ascertained by Northern and Western blot analysis. Treatment of THP-1 cells with 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) resulted in more than 20% induction in the expression of PPAR-γ5 and PPAR-γ7 transcripts by both Northern blot analysis and RT-PCR. Another PPAR-γ ligand, troglitazone, induced expression of only PPAR-γ5. Both ligands inhibited the expression of PPAR-γ1 and PPAR-γ2. Additionally, 15d-PGJ2 and troglitazone increased the level of apolipoprotein E transcript by 60% but decreased lipoprotein lipase expression by 15% in THP-1 cells. The differential regulation of PPAR-γ transcripts suggests that each transcript isoform may contribute to macrophage function.
Keywords: PPAR-γ isoforms; THP-1 macrophages; 15-deoxy-Δ12,14-prostaglandin J; 2; Troglitazone; Lipoprotein lipase; Apolipoprotein E
Activin A-induced expression of PAX4 in AR42J-B13 cells involves the increase in transactivation of E47/E12 by Rei Kanno; Takeshi Ogihara; Yasuhiro Igarashi; Yasushi Tanaka; Stuart B. Smith; Itaru Kojima; Michael S. German; Ryuzo Kawamori; Hirotaka Watada (pp. 44-50).
Pax4 is a paired-homeodomain containing transcriptional factor that controls the differentiation of pancreatic β cells. The aim of this study was to investigate the mechanism of PAX4 expression by activin A. By reporter gene analysis using AR42J-B13 cells, in which treatment with activin A induced PAX4 mRNA expression, we identified that a short sequence located ∼1930 bp upstream of the transcriptional start site is essential for activin A induced PAX4 promoter activation. This region contains an E box and binding sites for hepatocyte nuclear factor (HNF)-1α. Mutation introduced in each binding site markedly reduced activin A responsiveness. It has been reported that HNF-1α synergizes with basic helix–loop–helix (bHLH) proteins in activating the PAX4 promoter, and we demonstrated that activin A strongly enhanced the functional activity of E47/E12 without the increase in its binding ability. In addition, suppression of E47/E12 expression in AR42J-B13 cells using siRNA oligonucleotides results in the significant decrease in the intrinsic activin A-induced PAX4 expression. Our results suggest that activin A enhances PAX4 expression by enhanced transactivation of E47/E12 proteins and might result in a cumulative transactivation of the promoter.
Keywords: bHLH; basic helix–loop–helix; DBD; DNA binding domain; EMSA; electrophoretic mobility shift assay; FCS; fetal calf serum; GAPH; glyceraldehyde-3-phosphate dehydrogenase; HGF; hepatocyte growth factor; HNF; hepatocyte nuclear factor; UAS; upstream activating sequenceInsulin; Pax4; Transcription factor; Gene regulation; beta-cell; Pancreatic development
dps expression in Escherichia coli O157:H7 requires an extended −10 region and is affected by the cAMP receptor protein by Kwang-Cheol Jeong; David J. Baumler; Charles W. Kaspar (pp. 51-59).
The DNA binding protein from starved cells (Dps) is a general stress protein that provides Escherichia coli protection from osmotic, oxidative, and acid stresses. While Dps production and accumulation is primarily associated with stationary phase, during log phase, this protein protects against oxidative stress in an OxyR-dependent manner. In this study, evidence is provided that expands the role of Dps in acid tolerance to both log- and stationary-phase E. coli O157:H7. The transcription of dps occurred in log-phase cells without OxyR or stress and was upregulated during entry into stationary phase. The expression in log and stationary phase involved σ70 and σs, respectively, with both sigma factors recognizing the same promoter region. Site-directed mutagenesis identified an extended −10 region that was essential to both σ70 and σs transcription of dps. cAMP receptor protein (CRP) was found to repress dps expression as a crp mutant had a significant increase in the dps mRNA level. However, a CRP binding site was not found in the dps promoter and upregulation of dps in the crp mutant was absent in a crp rpoS double mutant. The findings from this study demonstrated that dps was expressed at a basal level during growth, both σ70- and σs-driven transcription required an extended −10, and CRP repression is mediated through the alternative sigma factor σs ( rpoS).
Keywords: Abbreviations; Dps; DNA binding protein from starved cells; CRP; cAMP receptor protein; ATR; acid tolerance response; CFU; colony forming units; LB; Luria–Bertani broth; ORF; open reading frame; EDTA; ethylene diamine tetraacetic acid; PCR; polymerase chain reaction; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; DMS; dimethyl sulfateAcid tolerance; E. coli; O157:H7; dps; regulation; CRP; −; 10 region; σ; s
JPO1/CDCA7, a novel transcription factor E2F1-induced protein, possesses intrinsic transcriptional regulator activity by Yuya Goto; Reiko Hayashi; Tomoki Muramatsu; Hitomi Ogawa; Ikumi Eguchi; Yasuhiro Oshida; Kiyoshi Ohtani; Kenichi Yoshida (pp. 60-68).
JPO1/CDCA7 was originally identified as a c-Myc-responsive gene that participates in neoplastic transformation. Here, we report the identification of JPO1/CDCA7 as a direct transcriptional target of transcription factor E2F1. We demonstrated that overexpression of E2F1 by adenoviral-mediated gene transfer upregulated JPO1/CDCA7 mRNA expression in human cells. Analysis of human and mouse JPO1/CDCA7 promoter constructs showed that an E2F-responsive sequence was necessary for E2F1-induced activation of the JPO1/CDCA7 gene transcription. Among the members of the E2F family, E2F1 to E2F4, but not E2F5 or E2F6, activated the JPO1/CDCA7 reporter construct. Chromatin immunoprecipitation analysis demonstrated that E2F1, E2F2, and E2F4 specifically bound to an E2F-responsive sequence of the human JPO1/CDCA7 gene. Like JPO2/R1, which has a homologous transcriptional regulator domain, the C-terminal cysteine-rich region of JPO1/CDCA7 protein induced transcriptional activity in a mammalian one-hybrid assay. Taken together, our results suggest that JPO1/CDCA7 is a unique transcription regulator whose expression is activated by E2F1 as well as c-Myc.
Keywords: JPO1/CDCA7; Promoter; E2F1; c-Myc; Transcriptional regulation
Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1 by Yaopan Mao; Kanchan A. Pavangadkar; Michael F. Thomashow; Steven J. Triezenberg (pp. 69-79).
The Arabidopsis GCN5, ADA2a and ADA2b proteins are homologs of components of several yeast and animal transcriptional coactivator complexes. Previous work has implicated these plant coactivator proteins in the stimulation of cold-regulated gene expression by the transcriptional activator protein CBF1. Surprisingly, protein interaction studies demonstrate that the DNA-binding domain of CBF1 (and of a related protein, TINY), rather than its transcriptional activation domain, can bind directly to the Arabidopsis ADA2 proteins. The ADA2a and ADA2b proteins can also bind directly to GCN5 through their N-terminal regions (comparable to a region previously defined in yeast Ada2) and through previously unmapped regions in the middle of the ADA2 proteins, which bind to the HAT domain of GCN5. The ADA2 proteins enhance the ability of GCN5 to acetylate histones in vitro and enable GCN5 to acetylate nucleosomal histones. Moreover, GCN5 can acetylate the ADA2 proteins at a motif unique to the plant homologs and absent from fungal and animal homologs. We speculate that this modification may represent a novel autoregulatory mechanism for the plant SAGA-like transcriptional coactivator complexes.
Keywords: Chromatin; Histone; Nucleosome; Acetylation; Cold acclimation; SAGA
Expression and cellular localization of dbpC/Contrin in germ cell tumor cell lines by Takeshi Yoshida; Hiroto Izumi; Takeshi Uchiumi; Yasuyuki Sasaguri; Akihide Tanimoto; Tetsuro Matsumoto; Seiji Naito; Kimitoshi Kohno (pp. 80-88).
The transcriptional regulation of the germ cell-specific cold-shock domain protein dbpC/Contrin was investigated, and the promoter region between −272 and −253 relative to the transcription start site was shown to be critical for the manifestation of cell-type specific transcription. In vivo footprint analysis demonstrated that the E-box located between −272 and −253 is protected in the dbpC/Contrin-positive germ cell tumor cell lines NEC8 and TERA1, but not in the dbpC/Contrin-negative bladder cancer cell line T24 or ovarian cancer cell line A2780. The promoter activity of the dbpC/Contrin gene was transactivated by co-transfection with c-Myc and the N-Myc expression plasmid. Western blotting analysis clearly showed that N-Myc is highly expressed in both NEC8 and TERA1 cells, and that c-Myc is expressed in both T24 and A2780 cells. These data demonstrate that cell-type specific dbpC/Contrin expression in germ cells is regulated by N-Myc. In addition, dbpC/Contrin is localized mainly in the cytoplasm of NEC8 and TERA1 cells, but is translocated to the nucleus when its C-terminal region is partially deleted. Our findings also suggest that dbpC/Contrin can be used as a molecular tool for the detection of germ cell tumors.
Keywords: Abbreviations; DAPI; 4,6-diamidino-2-phenylindole; dbpA; DNA-binding protein A; dbpB; DNA-binding protein B; dbpC; DNA-binding protein C; DMS; dimethylsulfate; MSY2; mouse germ cell-specific Y-box protein; YB-1; Y-box binding protein-1Cold-shock domain; dbpC/Contrin; E-box; Germ cell; Myc
Combinatorial regulation modules on GmSBP2 promoter: A distal cis-regulatory domain confines the SBP2 promoter activity to the vascular tissue in vegetative organs by Alessandro J. Waclawovsky; Rejane L. Freitas; Carolina S. Rocha; Luis Antônio S. Contim; Elizabeth P.B. Fontes (pp. 89-98).
The Glycine max sucrose binding protein ( GmSBP2) promoter directs phloem-specific expression of reporter genes in transgenic tobacco. Here, we identified cis-regulatory domains (CRD) that contribute with positive and negative regulation for the tissue-specific pattern of the GmSPB2 promoter. Negative regulatory elements in the distal CRD-A (−2000 to −700) sequences suppressed expression from the GmSBP2 promoter in tissues other than seed tissues and vascular tissues of vegetative organs. Deletion of this region relieved repression resulting in a constitutive promoter highly active in all tissues analyzed. Further deletions from the strong constitutive −700 GmSBP2 promoter delimited several intercalating enhancer-like and repressing domains that function in a context-dependent manner. Histochemical examination revealed that the CRD-C (−445 to −367) harbors both negative and positive elements. This region abolished promoter expression in roots and in all tissues of stems except for the inner phloem. In contrast, it restores root meristem expression when fused to the −132pSBP2-GUS construct, which contains root meristem expression-repressing determinants mapped to the 44-bp CRD-G (−136 to −92). Thus, the GmSBP2 promoter is functionally organized into a proximal region with the combinatorial modular configuration of plant promoters and a distal domain, which restricts gene expression to the vascular tissues in vegetative organs.
Keywords: Abbreviations; GmSBP2; Glycine max; sucrose-binding protein 2; VfSBPL; Vicia faba; sucrose-binding protein-like protein; PCR; Polymerase chain reaction; CRD; Cis-regulatory domain; GUS; β-glucuronidaseSucrose binding protein; Phloem-specific expression; GmSBP; Soybean; Tissue-specificity, promoter activity
Characterization of human Enah gene by Lorena Urbanelli; Carlo Massini; Carla Emiliani; Antonio Orlacchio; Giorgio Bernardi; Aldo Orlacchio (pp. 99-107).
Enabled homolog (Enah) is a mammalian ortholog of Drosophila Enabled (Ena), which is genetically linked to the Drosophila Abl tyrosine phosphorylation signaling cascade and is required for normal neural development. Vertebrates have three Ena-related genes: Enah, VASP (vasodilator-stimulated phosphoprotein) and Ena/VASP like (EVL). These genes play an important role in linking signal transduction pathways to localized remodeling of the actin cytoskeleton. We isolated and sequenced a cDNA encoding human Enah. Comparison of the amino acid sequences of mouse ( Mus musculus) and human ( Homo sapiens) species shows 86.6% identity. The human protein appears longer than the mouse and additional amino acids are concentrated in a region containing repeats of the amino acid sequence LERER. The complete gene is about 157 kb and consists of 14 exons. Analysis of multiple tissue northern blot revealed a major transcript of about 4.8 kb in all tissue examined. Alternatively spliced isoforms were isolated by RT-PCR. The gene is differentially expressed and to gain insight factors affecting its expression we cloned and preliminarily characterized human Enah gene promoter.
Keywords: Abbreviations; Enah; enabled homolog; Mena; mammalian enabled; Ena; enabled; βAPP; amyloid precursor protein; AD; Alzheimer's Disease; VASP; enabled/vasodilator-stimulated phosphoprotein; EVL; Ena/VASP-like protein; EVH; Ena/VASP homology; SH3; Src homology 3; RACE; rapid amplification of cDNA ends; ORF; Open Reading FrameEna/VASP family; Cloning; Cytoskeleton; Alzheimer's disease
Characterization of three Rop GTPase genes of alfalfa ( Medicago sativa L.) by Attila Szűcs; Dulguun Dorjgotov; Krisztina Ötvös; Csilla Fodor; Mónika Domoki; János Györgyey; Péter Kaló; György B. Kiss; Dénes Dudits; Attila Fehér (pp. 108-115).
Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The represented genes could be assigned to various linkage groups by genetic mapping. They were expressed in all investigated plant organs, although at different level. Relative gene expression patterns in response to Sinorhizobium infection of roots as well as during somatic embryogenesis indicated their differential participation in these processes. DNA sequences coding for altogether six different Medicago sp. Rop GTPases could be identified in sequence databases. Based on their homology to each other and to their Arabidopsis counterparts, a unified nomenclature is suggested for Medicago Rop GTPases.
Keywords: Gene expression; Genetic mapping; Medicago truncatula; Nod-factor; Plant Rho GTPase; Somatic embryogenesis; Sinorhizobium meliloti