Transcription Initiation Point Research Articles

37LRP induces invasion in hypoxic lung adenocarcinoma cancer cells A549 through the JNK/ERK/c-Jun signaling cascade.

We previously reported that 37-kDa laminin receptor precursor involved in metastasis of lung adenocarcinoma cancer cells. In this study, we further revealed that hypoxia induced 37-kDa laminin receptor precursor expression and activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in lung adenocarcinoma cancer cells. In addition, we further demonstrated that the c-Jun N-terminal kinase inhibitor SP600125 and extracellular signal-regulated protein kinase inhibitor U0126 blocked the c-Jun activity and abolished hypoxia-induced 37-kDa laminin receptor precursor expression and promoter activity in a concentration-dependent manner. However, the p38 mitogen-activated protein kinase inhibitor did not affect 37-kDa laminin receptor precursor expression and c-Jun activity in response to hypoxia. Furthermore, downregulated c-Jun expression by short interfering RNA could also inhibit hypoxia-induced 37-kDa laminin receptor precursor expression and transcriptional activity. The inhibition of 37-kDa laminin receptor precursor expression by SP600125 and U0126 could be rescued by c-Jun overexpression. Studies using luciferase promoter constructs revealed a significant increase in the activity of promoter binding in the cells exposed to hypoxia, which was lost in the cells with mutation of the activator protein 1 binding site. Electrophoresis mobility shift assay and chromatin immunoprecipitation demonstrated a functional activator protein 1 binding site within 37-kDa laminin receptor precursor gene regulatory sequence located at -271 relative to the transcriptional initiation point. Hypoxia-induced invasion of A549 cells was inhibited by the pharmacologic inhibitors of c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated protein kinase (U0126) as well as 37-kDa laminin receptor precursor-specific siRNA or antibody. Our results suggest that hypoxia-elicited c-Jun/activator protein 1 regulates 37-kDa laminin receptor precursor expression, which modulates migration and invasion of lung adenocarcinoma cells.

Мультиспецифичные промоторные островки как участки генома с необычными структурными и функциональными свойствами

The study is aimed at unusual genomic regions of E. coli - mixed promoter islands, which possess high density of potential transcription initiation points. They were predicted by promoter finder PlatPromU, without the consideration of conservative elements, recognized by σ-subunits of RNA polymerase, thus they are supposed to interact with polymerase, which has different σ-factors. We found that the structural and functional properties of mixed promoter islands are very similar to those of promoter islands, previously revealed by the algorithm PlatProm, which was adjusted to the search for σ 70 -specific promoters of E. coli. Thus, both types of islands are preferentially located in the regulatory regions of genes acquired by horizontal transfer. Their double helix is characterized by a higher degree of curvature and twisting, compared to normal promoters. Both types of islands have a higher capacity to form complexes with RNAP, than normal promoters, but lower ability to initiate productive transcription. This suppression can be largely explained by the advanced ability of islands to interact with the histone-like inhibitory protein H-NS. However, RNA synthesis from the promoter island associated with the gene appY, increased upon its transfer into the plasmid pET28b-eGFP and showed dependence not only from the presence of H-NS binding sites, but also from the spatial configuration of the neighboring areas. That means that conformational changes in the genome can increase the transcriptional activity from the islands, delivering the products of adjacent genes for metabolic needs of the bacterial cell.

NMR Study of Hydrogen Exchange in the DNA Decamer Duplexes Containing the -10 galP1 Promoter Sequence

Most Escherichia coli promoters are consists of two 6-bp sequence elements which are centered at ~10 and 35 nucleotides upstream of the transcription initiation point. The conserved region 4.2 and 2.4 of the RNA polymerase σ subunit recognize −10 and −35 promoter elements, respectively. The open complex between σ subunit and promoter elements, in which the promoter DNA is locally melted, is competent to initiate RNA synthesis according to the DNA sequence of template strand. Promoter opening is temperature-dependent; the most −10/−35 promoter complexes display closed forms below 15 C, whereas promoter complexes on the extended −10 galP1 promoter remain open at 5 C. The reason for this unusual behavior of the −10 galP1 promoter is not completely understood. The conserved A·T base pair at the −11 position of the promoters plays very important role in the initiation of E. coli transcription. The in vitro transcription with the −10 galP1 promoter was not initiated when this A base was substituted to other bases. To understand the function of the A-11 base in the transcription initiation process, the imino proton exchange rates were measured for the DNA decamer duplex containing the wild-type −10 galP1 promoter sequence (position: −14 ~ −5) (referred to as wt galP1, Fig. 1). The exchange rate constants of the imino protons for the wt galP1 duplex were compared with those of the modified galP1 promoters at the −11 position (see Fig. 1). This comparison indicates that some differences in the base pair dynamics are correlated with function of the −10 galP1 promoter during transcription initiation.

Cisplatin induced Mitochondrial DNA damage in dorsal root ganglion neurons

Cisplatin is a platinum-based chemotherapeutic agent that induces peripheral neuropathy in 30% of patients. Peripheral neuropathy is the dose limiting side effect, which has no preventative therapy. We have previously shown that cisplatin induces apoptosis in dorsal root ganglion (DRG) sensory neurons by covalently binding to nuclear DNA (nDNA), resulting in DNA damage, subsequent p53 activation and Bax-mediated apoptosis via the mitochondria. We now demonstrate that cisplatin also directly binds to mitochondrial DNA (mtDNA) with the same binding affinity as nDNA. Cisplatin binds 1 platinum molecule per 2166 mtDNA base pairs and 1 platinum molecule per 3800 nDNA base pairs. Furthermore, cisplatin treatment inhibits mtDNA replication as detected by 5-bromo-2′-deoxy-uridine (BrdU) incorporation and inhibits transcription of mitochondrial genes. The relative reduction in mtDNA transcription is directly related to the distance the gene is located from the transcription initiation point, which implies that randomly formed platinum adducts block transcription. Cisplatin treated DRG neurons exhibit mitochondrial vacuolization and degradation in vitro and in vivo. Taken together, this data suggests that direct mtDNA damage may provide a novel, distinct mechanism for cisplatin-induced neurotoxicity separate from the established nDNA damage pathway.

Hypoxia‐mediated up‐regulation of MGr1‐Ag/37LRP in gastric cancers occurs via hypoxia‐inducible‐factor 1‐dependent mechanism and contributes to drug resistance

Our previous study demonstrated hypoxia-inducible factor-1(HIF-1) could prompt multidrug resistance (MDR) phenotype and MGr1-Ag/37LRP, a novel drug-resistance protein was reported by our labortary, associated with multidrug resistance in gastric cancer. Given this association, we hypothesized that MGr1-Ag/37LRP contributed to HIF-1-dependent hypoxia-induced MDR phenotype. Initial experiments revealed that blocking MGr1-Ag/37LRP expression by siRNA in gastric cancer cells effectively reversed multidrug resistance phenotype induced by hypoxia. Subsequent analysis of MGr1-Ag/37LRP mRNA and protein in gastric cancer cells revealed a time-dependent manner increase with hypoxia. While the up-regulation of MGr1-Ag/37LRP was abolished by HIF-1 inhibition with siRNA. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subject to hypoxia and such hypoxia inducibility was lost in cells co-transfected siRNA targeting HIF-1. Analysis of the MGr1-Ag/37LRP promoter revealed several potential binding sites for HIF-1. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated a functional HIF-1 binding site within MGr1-Ag/37LRP gene regulatory sequence located at -16 to -11 relative to the transcriptional initiation point. These observations demonstrate that MGr1-Ag/37LRP is actively engaged by hypoxia and represent a novel HIF-1 target. Such results suggest hypoxia-elicited MGr1-Ag/37LRP expression as a pathway for resistance of gastric cancer to chemotherapeutics.

Alternative transcription initiation of the nitrilase gene (BrNIT2) caused by infection with Plasmodiophora brassicae Woron. in Chinese cabbage (Brassica rapa L.)

In clubroot disease, gall formation is induced by infection with the obligate biotroph Plasmodiophora brassicae, and cell hypertrophy is dependent on increased auxin levels. The enzyme nitrilase is suggested to play an important role in auxin biosynthesis in plants. Here, we investigated the expression of nitrilase genes in clubroot disease in Chinese cabbage (Brassica rapa L.). We isolated four isogenes of nitrilase (BrNIT1, BrNIT2, BrNIT3, and BrNIT4) from Chinese cabbage. When a BrNIT2-specific probe was used for Northern blot hybridization, enhanced accumulation of a 1.4 kb mRNA and additional shorter transcripts (1.1 kb) were only detected in clubbed roots 25 days postinoculation (dpi) onward. The expression of BrNIT1 was not strongly affected by infection with P. brassicae. BrNIT3 expression was detected in the roots at 10 and 20 dpi, and the expression was less in clubbed roots than in healthy roots at 20 dpi. Analysis of the transcription initiation point of the BrNIT2 gene suggests that 1.1 kb transcripts were generated by alternative transcription initiation between the second intron and the third exon. The sequence from the second intron to half of the third exon (+415 to +1037, 623 bp) had promoter activity in Arabidopsis during clubroot formation. Therefore, our results suggest that transcriptional regulation of BrNIT2 might be involved in auxin overproduction during clubroot development.

SIGffRid: A tool to search for sigma factor binding sites in bacterial genomes using comparative approach and biologically driven statistics

BackgroundMany programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (σ) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations.ResultsWe describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data), performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped), allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of Streptomyces coelicolor and Streptomyces avermitilis. Cross-check with the well-defined SFBSs of the SigR regulon in S. coelicolor is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these σ factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. Escherichia coli/Salmonella typhimurium and Bacillus subtilis/Bacillus licheniformis pairs). Motifs of house-keeping σ factors were found as well as other SFBSs such as that of SigW in Bacillus strains.ConclusionWe demonstrate that our approach combining statistical and biological criteria was successful to predict SFBSs. The method versatility autorizes the recognition of other kinds of two-box regulatory sites.

A Case of Prolactin Deficiency with Familial Puerperal Alactogenesis Accompanying Impaired ACTH Secretion

We report here the case of a 34-year-old female with puerperal alactogenesis. Her menstrual cycle was regular and breast development normal. She had delivered a healthy boy but could not breast-feed after parturition. Endocrinological studies disclosed that the cause was a prolactin (PRL) deficiency. In addition, she showed accompanying impaired ACTH secretion that was believed to be triggered by encephalitis, although her plasma levels of GH, TSH, LH and FSH remained intact. Pituitary MRI showed no specific findings and anti-pituitary antibody tests were negative. Interestingly, both her mother and grandmother also reported puerperal alactogenesis. The sequences of all five exons of the PRL gene, including promoter region and transcription initiation point, were surveyed in order to examine for certain genetic disorders, but no mutations were identified. Although it cannot be definitively concluded that this PRL deficiency was not a genomic DNA disorder, in our case at least, her PRL gene was normal and, therefore, was not directly responsible for the patient's impaired PRL secretion. This evidence suggests that familial puerperal alactogenesis and PRL deficiency can be induced by other causes such as via disorders of unknown transcription factors or molecules that contribute to translation of PRL gene.

Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter

It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5' promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

The relationship among nephrin, podocin, CD2AP, and α-actinin might not be a true ‘interaction’ in podocyte

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1 , NPHS2 , CD2AP , and ACTN4 , can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or α -actinin-4 mRNA by using reconstructed RNA interference vector – psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of α -actinin-4, whereas α -actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: I. Effects of pH, temperature, and ligand binding

Transcriptional regulation depends on the appropriate set of positive and negative signals that provide correct gene expression. Studies in eukaryotic gene expression have shown NC2 to be a general repressor of transcription which blocks the interaction between TFIIB and TBP. However, during the last few years NC2 has been found to bind the transcriptionally active promoters and interact with several positive transcription factors. These data suggested a controversial role of NC2 in transcription. Using in vitro transcription on minimal LTR promoter of HIV-1, we show that removal of NC2 from HeLa nuclear extract increases the yield of transcripts as well as unspecific transcription initiation in a template amount dependent manner. Fractions of HeLa nuclear extract containing NC2 can restore basal transcription repression and precise selection of transcription initiation point. This points to a new role for NC2 as a repressor of inaccurate transcription initiation that allows specific transcription to take place.

Molecular Cloning of the m-<I>Golsyn</I> Gene and its Expression in the Mouse Brain

The mouse ortholog of the human GOLSYN gene, termed the m-Golsyn gene, was isolated and mapped to the region on mouse chromosome 15B3.2 syntenic with human chromosome 8q23. Three mRNA species (type la, 1b, and type 2) were produced by use of alternative transcription initiation points and alternative splicing events. The type 1 mRNAs were expressed only in the brain, whereas the type 2 was detected in various tissues. m-Golsyn protein was expressed in various tissues including the brain. Immunohistochemical study of m-Golsyn protein showed its prominent expression in the neuronal cells in various regions of the brain and strong expression in the choroid plexus ependymal cells lining the ventricles. m-Golsyn protein was found to be homologous to syntaphilin, a regulator of synaptic vesicle exocytosis. These results indicate that the m-Golsyn protein may play an important role in intracellular protein transport in neuronal cells of the brain.

Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions -64.5 and -44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position -60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions -81.5 and -42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

Transcriptional Analysis of the Conserved ftsZ Gene Cluster in Mycoplasma genitalium and Mycoplasma pneumoniae

Several experimental approaches were used to construct a detailed transcriptional profile of the phylogenetically conserved ftsZ cell division gene cluster in both Mycoplasma genitalium and its closest relative, Mycoplasma pneumoniae. We determined initiation and termination points for the cluster, as well as an absolute steady-state RNA level for each gene. Transcription of this cluster in both these organisms was shown to be highly strand specific. While the four genes in this cluster are cotranscribed, their transcription unit also includes two genes of close proximity yet disparate function. A transcription initiation point immediately upstream of these two genes was detected in M. genitalium but not M. pneumoniae. In M. pneumoniae, transcription of the six genes terminates at a poly(U)-tailed hairpin. In M. genitalium, this transcription terminates at two closely spaced points by an unknown mechanism. Real-time reverse transcription-PCR analysis of this cluster in M. pneumoniae shows that mRNA levels for all six genes vary at most fivefold and form a gradient of decreasing quantity with increasing distance from the promoter at the beginning of the cluster. mRNA from coding regions was approximately 20- to 100-fold more abundant than that from intergenic regions. We estimated the most abundant mRNA we detected at 0.6 copy per cell. We conclude that groups of functionally related genes in M. genitalium and M. pneumoniae are often preceded by promoters but rarely followed by terminators. This causes functionally unrelated genes to be commonly cotranscribed in these organisms.

Regulation of DNA Methyltransferase 1 by the pRb/E2F1 Pathway

Tumor suppressor gene silencing by DNA hypermethylation contributes to tumorigenesis in many tumor types. This aberrant methylation may be due to increased expression and activity of DNA methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine to cytosines in CpG dinucleotides. Elevated expression of the maintenance DNA methyltransferase, DNA methyltransferase 1 (DNMT-1), has been shown in carcinomas of the colon, lung, liver, and prostate. Based on the nearly ubiquitous alterations of both DNA methylation and the retinoblastoma protein (pRb) pathway found in human cancer, we investigated a potential regulatory pathway linking the two alterations in murine and human prostate epithelial cells. Analysis of DNA methyltransferase levels in Rb-/- murine prostate epithelial cell lines revealed elevated Dnmt-1 levels. Genomic DNA sequence analysis identified conserved E2F consensus binding sites in proximity to the transcription initiation points of murine and human Dnmt-1. Furthermore, the Dnmt-1 promoter was shown to be regulated by the pRb/E2F pathway in murine and human cell lines of epithelial and fibroblast origin. In the absence of pRb, Dnmt-1 transcripts exhibited aberrant cell cycle regulation and Rb-/- cells showed aberrant methylation of the paternally expressed gene 3 (Peg3) tumor suppressor gene. These findings show a link between inactivation of the pRb pathway and induction of DNA hypermethylation of CpG island-containing genes in tumorigenesis.

Characterization of the rolB promoter on mikimopine-type pRi1724 T-DNA

The promoter activity of the rolB gene encoded on mikimopine-type pRi1724 ( 1724rolB) was elucidated. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that 1724rolB mRNA was highly accumulated in roots of 1724rolB-transformed tobacco plants. A β-glucuronidase (GUS) fluorometric assay using a 1724rolB promoter-GUS construct also showed high promoter activity in the roots. A GUS histochemical assay revealed that 1724rolB was expressed in the vicinity of root apical meristematic cells of the vascular cylinder in the main roots, in lateral root primordial and meristematic cells, in veins of cotyledons, and faintly in the shoot apex. Since the product of 1724rolB has a N-terminal stretch 17 amino acid longer than the rolB of agropine-type pRi1855 ( 1855rolB), primer extension analysis was used to show that the transcription initiation point of 1724rolB was located beyond the putative TATA box of 1855rolB, suggesting that the locations of the minimal promoters of the two rolB genes differed from each other. 1724rolB promoter analysis using deletion constructs indicated that the minimal region necessary for this promoter activity was, at most, −215 bp upstream of the translation initiation point. A predominant auxin-responsive sequence was located at position −313 to −216 upstream of the transcription initiation point, containing the Nicotiana tabacum rolB domain B factor 1 (NtBBF1) binding motif ACTTTA.

Newly Discovered Polymorphism in the CD34+ Stem Cell Specific AC133-P1 Promoter Linked to Leukemias.

AC133 (CD133, human Prominin-1) is a cell-surface glycoprotein which is expressed on several types of stem cells, including hematopoietic stem cells and endothelial progenitor cells. AC133 is also expressed on acute and chronic myeloid leukemias, as well as lymphoblastic leukemias. AC133 expression is correlated with a poor prognosis in cases of leukemia; in addition, preliminary observations suggest that AC133 may define a population of leukemic stem cells. We previously identified and characterized the AC133 promoter region, and demonstrated that transcription of AC133 is driven by five alternative promoters in a tissue-dependent manner, resulting in the formation of multiple alternatively spliced isoforms. CD34+ hematopoietic stem cells were found to utilize only one of the five promoters, AC133-P1. Furthermore, preliminary data suggest that hematopoietic stem cells express only one AC133 isoform. We identified four allelic variants in the AC133-P1 promoter, 250 bp upstream of the transcription initiation point, which differ by the number of short tandem repeats as well as by a single nucleotide polymorphism (SNP). One allele, (CAA)3CAG, is found in patients with AML, chloroma, and myeloma. Two different alleles, (CAA)4 and (CAAAA)5, are found in cancer cell lines derived from AML, retinoblastoma, colon cancer, and sarcoma. One allele, (CAA)5, is found only in healthy human subjects. Our preliminary results show that these different alleles may affect the activity of the AC133-P1 promoter. These data might lay a foundation for testing a risk of susceptibility to AC133+ leukemias. Additional investigations are required to establish the role of AC133 in emergence, development and survival of leukemias, as well as to explore the viability of AC133 as a primary therapeutic target in patients with aggressive AC133+ leukemia.

A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II

Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.

Induction of ID2 Expression by Hypoxia-inducible Factor-1: A ROLE IN DEDIFFERENTIATION OF HYPOXIC NEUROBLASTOMA CELLS

ID (inhibitor of differentiation/DNA binding) proteins, frequently deregulated in advanced human malignancies, can participate in multiple fundamental traits of cancer, such as block of differentiation, increased proliferation, tissue invasiveness, and angiogenesis. We have previously demonstrated that hypoxia decreases expression of neuronal marker genes in neuroblastoma, but induces genes expressed in the neural crest, such as ID2. Because of its involvement in normal neural crest development and its ability to inhibit proneuronal bHLH proteins, the hypoxic induction of ID2 was of particular interest. Here we report fast induction kinetics of ID2 expression in hypoxic neuroblastoma cells. The up-regulation of ID2 was abolished by addition of actinomycin D, implicating a hypoxia-driven transcriptional mechanism. Analyzing the ID2 promoter revealed several potential binding sites for hypoxia-inducible factors. Subsequent electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated two functional HIF-1 binding sites within ID2 gene regulatory sequences located at -725 and -1893 relative to the transcriptional initiation point. In transfection assays, DNA constructs of the ID2 promoter, including the functional HIF-1 binding sites, induced luciferase reporter activity in a HIF-1-specific manner. These observations demonstrate that ID2 is actively engaged by hypoxia and represents a novel HIF-1 target. Hypoxia-induced ID2 expression could play a significant role in the previously observed dedifferentiation of hypoxic neuroblastoma cells, which in a clinical setting could lead to less mature and more aggressive tumors.

Localization of matrix-associated elements in the 5'-region of the rat estrogen sulfotransferase gene

Potential matrix-associated elements in the 5'-region of the rat estrogen sulfotransferase gene (Ste1) were searched for and characterized. The DNA fragments corresponding to the regions -800/+1048 and +1049/+2038 relative to the main point of transcription initiation were found to be bound to the nuclear matrix in vitro. A permanent association of the 5'-region of the Ste1 gene with the nuclear matrix in rat hepatocytes was found, the most probable site of attachment being the region -352/-152. No differences were found in the attachment of the 5'-region of the Ste1 gene to the nuclear matrix in the liver of males where the gene is actively transcribed and in that of females where it is inactive.