Major Transcription Initiation Site Research Articles

ETS-dependent enhancers for endothelial-specific expression of serum/glucocorticoid-regulated kinase 1 during mouse embryo development.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.

GlnR negatively regulates the transcription of the alanine dehydrogenase encoding gene ald in Amycolatopsis mediterranei U32 under nitrogen limited conditions via specific binding to its major transcription initiation site.

Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II) were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.

Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

FOXO1 Up-Regulates Human L-selectin Expression through Binding to a Consensus FOXO1 Motif

L-selectin plays important roles in lymphocyte homing and leukocyte rolling. Mounting evidence shows that it is involved in many disease entities including diabetes, ischemia/reperfusion injuries, inflammatory diseases, and tumor metastasis. Regulation of L-selectin at protein level has been well characterized. However, the regulation of human L-selectin transcription remains largely unknown. To address transcriptional regulation of L-selectin, we cloned 1088 bp 5′ of the start codon ATG. Luciferase analysis of the serial 5′ deletion mutants located the core promoter region at −288/−1. A major transcription initiation site was mapped at −115 by 5′RACE. Transcription factors Sp1, Ets1, Mzf1, Klf2, and Irf1 bind to and transactivate the L-selectin promoter. Significantly, FOXO1 binds to a FOXO1 motif, CCCTTTGG, at −87/−80, and transactivates the L-selectin promoter in a dose-dependent manner. Over-expression of a constitutive-active FOXO1 increased the endogenous L-selectin expression in Jurkat cells. We conclude that FOXO1 regulates L-selectin expression through targeting its promoter.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.

Genomic organization and functional characterization of the promoter for the human suppressor of cytokine signaling 6 gene

In this study, we report the expression and genomic structure of the gene encoding human suppressor of cytokine signaling 6 ( SOCS6), and the characterization of the functional promoter region. The human SOCS6 gene, spanning 40 kb on chromosome 18q22.2, is composed of two exons separated by an intron of 35 kb. Two transcripts are ubiquitously expressed, and both encode the full-length open reading frame of SOCS6. A primer extension assay revealed that the major transcription initiation site is located 469 bp upstream the ATG codon. Luciferase promoter analysis demonstrated that the 5′-flanking region is able to drive transcription, and the CpG-rich sequences near the transcription initiation site are important for the TATA-less SOCS6 promoter activity. Analogous to SOCS1 and SOCS3, which are down-regulated in several human cancers, SOCS6 is expressed at lower levels in carcinomas of stomach and colon. We demonstrated that hypermethylation of the SOCS6 promoter is one of the mechanisms for the epigenetic regulation of SOCS6 expression. Firstly, in vitro methylation of the reporter promoter plasmid significantly suppressed the promoter activity. Secondly, SOCS6 expression in vivo was enhanced by treating cells with a methyltransferase inhibitor. The SOCS6 gene from various species shares significant homology in amino acid sequences, transcription factor binding motifs in promoter regions and the two-exon genomic structure, suggesting that the SOCS6 gene is highly conserved.

5-Lipoxygenase: mechanisms of regulation

5-lipoxygenase (5-LO) catalyzes two steps in biosynthesis of leukotrienes (LTs), a group of lipid mediators of inflammation derived from arachidonic acid (AA). LT antagonists are used in treatment of asthma; more recently a potential role also in atherosclerosis has raised considerable interest. Furthermore, possible effects of 5-LO metabolites in relation to tumorigenesis have emerged. Thus, an understanding of the biochemistry of this lipoxygenase has potential implications for treatment of various diseases.

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins

The human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with Tm of ∼55°C (Q1) and ∼72°C (Q2). DMS-footprinting and CD suggest that Q1 can be a parallel and Q2 a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA–protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS.

Identification and functional characterization of a polymorphic oestrogen response element in the human coagulation factor IX gene promoter

The liver expressed procoagulant factor IX (FIX) shows inter-individual variation in levels, some of which is heritable. Raised levels of FIX are associated with a thrombotic tendency. This study demonstrated that, in females but not males, part of this variation in FIX levels is due to polymorphic genotype at a locus in the factor IX gene (F9) promoter 698 bp upstream of the major transcription initiation site (-698C/T). The -698C allele (associated with higher FIX level) shows closer homology to a canonical ORE sequence and a higher binding affinity for oestrogen receptor alpha than the -698T allele. Reporter gene vectors were constructed with elements spanning residues -738 to +50 of the F9 promoter corresponding to wild type -698C and -698T alleles. A related series of vectors comprising three copies of the F9 ORE driving expression of a minimal synthetic promoter were also created. Transfection into the liver-derived HepG2 and erythroleukaemic K562 cell lines demonstrated increased levels of expression in the presence of oestrogenic factors when compared to those found in their absence; this stimulation was more pronounced in the non-liver derived K562 cell line and from the reporter vectors containing promoter elements corresponding to the -698C allele.

Keratinocyte transcriptional regulation of the human c-Myc promoter occurs via a novel Lef/Tcf binding element distinct from neoplastic cells

The proto-oncogene c-Myc is involved in early neoplastic transformations. Two consensus Lef/Tcf binding elements (TBE) were found to be prerequisite for transcriptional transactivation by the armadillo proteins β-catenin and plakoglobin (PG) together with Tcf4 in human neoplastic cells. In epidermal keratinocytes, c-Myc was reported to be repressed by Lef-1 and PG. Using reporter gene assays, here we demonstrate that deletion of the two consensus TBE fails to abrogate transcriptional regulation by Lef-1/PG in wildtype and β-catenin−/− keratinocytes, while it reduces transcription in pre-neoplastic PG−/− keratinocytes. We identified a TBE sequence variant downstream of the major transcriptional initiation site that binds Lef-1 in vitro and in vivo, and its mutation compromised transcriptional regulation by Lef-1/PG. Collectively, this study demonstrates that the two consensus TBE’s reported in neoplastic cells are dispensable for c-Myc regulation in normal keratinocytes, which instead use a novel TBE sequence variant. This unprecedented finding may have important implications for armadillo target genes involved in carcinogenesis.

A Novel Variable Number of Tandem Repeat of the Natriuretic Peptide Precursor B gene's 5'-Flanking Region is Associated with Essential Hypertension among Japanese Females

Brain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH. The polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants. A novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046). The 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females.

Isolation and Characterization of Human ADAMTS13 Gene Promoter Region - Control of ADAMTS13 Gene Expression by Hepatic Transcription Factors and Inflammatory Cytokines.

ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) controls von Willebrand factor multimer sizes by cleaving the Tyr1605-Met1606 bond in the central A2 domain. Deficiency of plasma ADAMTS13 activity can result in a lethal syndrome, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is primarily synthesized in hepatic stellate cells (HSCs), endothelial cells and megakaryocytes. We determined the transcription initiation site, the core region for promoter activity, the putative transcription factor binding sites as well as the influence of inflammatory cytokines on ADAMTS13 promoter activity. To explore the transcriptional control of ADAMTS13 gene expression, we constructed reporter genes containing 991 base pairs (bp) of the ADAMTS13 5′ untranslated (UT) region. We showed by deletion mutagenesis and luciferase reporter expression that the proximal-most 197 bp region was required for maximal luciferase activity in transfected cells in the human hepatic stellate cell line (LX-2) and in the human hepatocyte-like cell line (HepG2); the major transcription initiation site determined by 5′ - RACE was found at 77 bp upstream from the translation start site (ATG). However, the minimal sequences that were required for the promoter activity varied depending on the cells, with required sequences of approximately 147 and 127 bp in LX-2 and HepG2 cells, respectively. The proximal ADAMTS13 promoter region is evolutionally conserved between humans, mice and rats. This region is rich in GC content (72%) and contains putative binding sites for the transcription factors heat shock factor-2 (HSF2), FOXa2 [also named hepatocyte nuclear factor 3beta (HNF-3b)] and AP-1. A footprint assay demonstrated that the region between −116 and −126, containing the putative FOXa2 binding site, was largely protected by Dnase I digestion. The luciferase reporter activity was suppressed in cells transfected with the plasmid containing the proximal 314 bp human 5′ UT ADAMTS13 sequence in parallel with the inflammatory cytokines found to be elevated in patients with TTP: IL-4, TNF-alpha and INF-gamma. These inflammatory cytokines inhibited the Adamts13 mRNA and protein expression in rat primary HSCs in culture in a dose dependent manner. Approximately 70%, 71% and 80% of Adamts13 mRNA (by real time RT-PCR) and 77%, 78% and 92% of Adamts13 proteolytic activity (by FRETS-VWF73) were suppressed at 48 hours by IL-4 (10 ng/ml), TNF-alpha (10 ng/ml) and INF-gamma (100 ng/ml), respectively. We conclude that under physiological conditions ADAMTS13 synthesis may be strictly maintained at relatively low levels by binding transcription factors, whereas under pathological conditions inflammatory cytokines, released due to systemic inflammation, may further suppress ADAMTS13 gene expression, which may result in thrombotic complications. However, the mechanism regarding how the inflammatory cytokines negatively regulate ADAMTS13 (or Adamts13) synthesis remains to be determined.

Cloning and expression of mouse cytosolic α-mannosidase (Man2c1)

It is known that the neutral/cytosolic α-mannosidase (Man2c1) which can cleave α 1,2-, α 1,3-, and α 1,6-linked mannose residues, is stimulated by cobalt and is inhibited by furanose analogues swainsonine (SW) and 1,4-dideoxy-1,4-imino- d-mannitol (DIM). The enzyme is involved in the degradation of oligomannosides derived from dolichol intermediates and the degradation of newly synthesized glycoproteins. An immunological relationship has been demonstrated between the rat endoplasmic reticulum α-mannosidase and the cytosolic α-mannosidase. In fact antibodies raised against the soluble α-mannosidase recognized the membrane form of the ER α-mannosidase. A cDNA encoding the mouse cytosolic α-mannosidase was obtained by RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of 25 introns within the cytosolic α-mannosidase gene. The gene spans 11.5 kb from the major transcription initiation site to the RNA cleavage site. The transcription initiation site of mouse cytosolic α-mannosidase was mapped to 170 bases upstream of the ATG codon using 5′ RACE. Northern blotting analysis revealed expression of a major transcript of 3.8 kb in all tissues examined. COS cells transfected with the cDNA showed a 20-fold increase in cytosolic α-mannosidase activity. This enzyme activity was stimulated by cobalt and inhibited by DIM and EDTA. Furthermore we demonstrated that the expressed enzyme was active towards the radiolabeled substrate Man9GlcNAc1 giving the final product Man5GlcNAc1 through the formation of Man8GlcNAc1 isomer C as intermediate.

Identification of regulatory sites in the human PXR (NR1I2) promoter region

The human pregnane X receptor (hPXR, NR1I2) is a member of the nuclear receptor superfamily and a key regulator of genes encoding several major cytochrome P450 enzymes and transporters. However, the transcriptional regulation of hPXR itself remains unclear. We recently reported significant diversity in the 5' region of human hepatic PXR transcripts and identified the major transcription initiation site. Here, we investigate the transcriptional regulatory sites in the hPXR 5'-flanking region. Luciferase reporter constructs containing various lengths of 5'-flanking region, up to 10.5 kb upstream of the major transcription initiation site, were assessed for promoter activity in HepG2 cells. We mapped the minimal essential region for promoter activity to a 160 bp region upstream of the transcription initiation site, an area that also showed nuclear protein binding. Constructs with mutations introduced into these protein-binding sites demonstrated reduced promoter activity concomitant with reduced DNA-protein binding activity. hPXR promoter activity was observed in HepG2 cells but not in HeLa cells. Likewise, nuclear protein binding to promoter elements was also observed in HepG2 but not HeLa cells. The present study provides basic information on the transcriptional regulation of hPXR and may help elucidate the regulatory mechanisms of hPXR target genes.

GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site

5-lipoxygenase (5LO) catalyzes formation of leukotrienes, mediators with roles in several inflammatory disorders, including atherosclerosis. The human 5LO gene promoter contain multiple GC-boxes. The relevance of these for expression of 5LO in the human monocytic cell line Mono Mac 6 (MM6) was studied. A downregulating effect of the GC-box binding compound mithramycin indicated that GC-rich sequences in the 5LO gene promoter are important for expression of native 5LO. In DNase I footprinting, mithramycin and Sp1 protected known GC-boxes, but also a novel Sp1 binding site was found, comprising 20 bp upstream of the major transcription initiation site, beside an Initiator-like sequence. Mutation of this site reduced Sp1 binding and expression of reporter genes in MM6 cells, compatible with a function as basal promoter element for the TATA-less 5LO gene. When differentiation was induced by TGFβ and vitamin D 3, 5LO expression became prominent, but expression levels of Sp1/3 and Egr-1 were the same as for control cells. Also, 5LO reporter gene activity in transiently transfected MM6 cells was insensitive to differentiation. Thus, the GC-rich part of the 5LO gene promoter, including a novel Sp1 site, appear important for basal (rather than upregulated) transcription of 5LO in MM6 cells.

Mechanisms and factors in embryonic stem cell self-renewal

Embryonic stem (ES) cells are attracting renewed attention because their capacity to differentiate into cellular derivatives of all three primary germ layers suggests that they could be a source of cells for therapeutic alleviation of disease. This differentiative capacity, termed pluripotency, can be restrained during ES cell culturein vitro with the cells exhibiting an apparently unlimited capacity for symmetrical self-renewing divisions. A thorough understanding of self-renewal is essential if ES cells are to be fully exploited for therapeutic ends. Intrinsic self-renewal determinants include the homeodomain proteins Oct4 and Nanog. Increased Nanog expression alleviates the requirement for BMP and gp130 receptor stimulation and allows factor-independent ES cell self-renewal. Fusions between Nanog and GFP indicate that Nanog is localised to the cell nucleus consistent with a role as a transcriptional regulator. Mapping of the major transcription initiation site of Nanog paves the way towards identification of the factor(s) that direct Nanog gene expression and thus determine the pluripotent state.

The Role of Protein Synthesis and Degradation in the Post-Transcriptional Regulation of Rat Multidrug Resistance-Associated Protein 2 (Mrp2, Abcc2)

Multidrug resistance-associated protein 2 (Mrp2, Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo post-transcriptional regulation. We hypothesized that Mrp2 protein undergoes altered rates of protein synthesis or degradation consistent with different Mrp2 protein expression. We analyzed Mrp2 synthesis, expression, and degradation in control female, 19- and 20-day pregnant, and pregnenolone-16alpha-carbonitrile (PCN)-treated rats using in vivo metabolic-labeling studies with [35S]cysteine/methionine or [14C]NaHCO3, polysomal distribution analyses and ribonuclease protection assays (RPA). Mrp2 protein was significantly increased in rats treated with PCN for 2 days but significantly decreased in 19-day pregnant rats relative to controls; no significant differences were observed in Mrp2 mRNA expression among these groups. The measured half-lives of 14C-labeled Mrp2 in control, pregnant, and PCN-treated rats were 27, 36, and 22 h, respectively, and were not significantly different. The rate of incorporation of 35S into Mrp2 was highest in PCN-treated rats. Polysomal distribution analysis of Mrp2 mRNA was consistent with increased Mrp2 protein synthesis after PCN treatment. The major transcription-initiation site for rat liver determined by RPA was -98 nucleotides (nt), with other start sites observed at -213, -163, -132, and -71 nt; use of transcription sites did not differ among the groups. Differences in the degradation of Mrp2 protein cannot explain the post-transcriptional regulation of Mrp2 in control, pregnant, and PCN-treated rats. Rather, the observed difference in protein synthesis suggests an intrinsic role for the translational regulation of rat Mrp2 protein.

Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

Cell cycle-dependent regulation of the human aurora B promoter

Aurora B is an important regulator of mitosis, and its mRNA and protein levels are tightly regulated during the cell cycle. In this study, we cloned the 5 ′ flanking region of the human aurora B gene and characterized its promoter activity. Two major transcription initiation sites were identified by primer extension. aurora B promoter activity was upregulated during M phase, and its cell cycle-dependent element (CDE) and cell cycle-gene homology region (CHR) upstream of the transcription initiation sites regulated the cell cycle-dependent promoter activity. Several CDE-binding protein complexes were identified using the electrophoretic mobility shift assay. Using the biotin–streptavidin pull-down assay, binding of E2F-1, E2F-4, and DP-2, but not of DP-1, to the CDE was detected. These results demonstrate that aurora B mRNA level is regulated by CDE–CHR and that a subset of E2F/DP family proteins binds to the CDE.