Analysis of G-quadruplexes upstream of herpesvirus miRNAs: evidence of G-quadruplex mediated regulation of KSHV miR-K12–1-9,11 cluster and HCMV miR-US33

Background G-quadruplexes regulate gene expression, recombination, packaging and latency in herpesviruses. Herpesvirus-encoded miRNAs have been linked to important biological functions. The presence and the biological role of G-quadruplexes have not been studied in the regulatory regions of virus miRNA. We hypothesized that herpesvirus-encoded miRNAs are regulated by G-quadruplexes in their promoters. Results We analyzed the 1 kb regulatory regions of all herpesvirus-encoded miRNAs for the presence of putative quadruplex-forming sequences (PQS). Over two-third (67%) of the regulatory regions of herpesvirus miRNAs had atleast 1 PQS. The 200 bp region of the promoter proximal to herpesvirus miRNA is particularly enriched for PQS. We chose to study the G-quadruplex motifs in the promoters of miR-K12 cluster in Kaposi's sarcoma-associated Herpesvirus (KSHV miR-K12–1-9,11) and the miR-US33 encoded by Human Cytomegalovirus (HCMV miR-US33). Biophysical characterization indicates that the G-quadruplex motifs in the promoters of the KSHV miR-K12 cluster and the HCMV miR-US33 form stable intramolecular G-quadruplexes in vitro. Mutations disrupting the G-quadruplex motif in the promoter of the KSHV miR-K12 cluster significantly inhibits promoter activity, while those disrupting the motif in the promoter of HCMV miR-US33 significantly enhance the promoter activity as compared to that of the respective wild-type promoter. Similarly, the addition of G-quadruplex binding ligands resulted in the modulation of promoter activity of the wild-type promoters (with intact G-quadruplex) but not the mutant promoters (containing quadruplex-disrupting mutations). Conclusion Our findings highlight previously unknown mechanisms of regulation of virus-encoded miRNA and also shed light on new roles for G-quadruplexes in herpesvirus biology.

Role of TEM-1 β-Lactamase in the Predominance of Ampicillin-Sulbactam-NonsusceptibleEscherichia coliin Japan

ABSTRACTWe investigated the epidemiology and resistance mechanisms of ampicillin-sulbactam-nonsusceptibleEscherichia coli, focusing on the role of the TEM-1 β-lactamase. We collected all nonduplicateE. coliclinical isolates at 10 Japanese hospitals during December 2014 and examined their antimicrobial susceptibility, β-lactamases, TEM-1 transferability, TEM-1 β-lactamase activity, outer membrane protein profile, membrane permeability, and clonal genotypes. Among the 329 isolates collected, 95 were ampicillin-sulbactam nonsusceptible. Of these ampicillin-sulbactam-nonsusceptible isolates, β-lactamases conferring resistance to sulbactam, such as AmpC, were present in 33%. Hyperproduction of sulbactam-susceptible β-lactamases, TEMs with a strong promoter, were rare (5%). The remaining 59 isolates (62%) had only sulbactam-susceptible β-lactamases, including TEM-1 with a wild-type promoter (n= 28), CTX-Ms (n= 13), or both (n= 17). All 45 transconjugants from 96 donors with TEM-1 had higher ampicillin-sulbactam MICs (4 to 96 mg/liter) than the recipient (2 mg/liter). In donors with only TEM-1, TEM-1 activity correlated with the 50% inhibitory concentration of sulbactam and ampicillin-sulbactam MICs. The decreased membrane permeation of sulbactam was associated with an increased ampicillin-sulbactam MIC. The reduced permeation was partly attributable to deficient outer membrane proteins, which were observed in 57% of the ampicillin-sulbactam-nonsusceptible isolates with only TEM-1 and a wild-type promoter. Sequence type 131 (ST131) was the most common clonal type (52%). TEM-1 with a wild-type promoter primarily contributed to ampicillin-sulbactam nonsusceptibility inE. coli, with the partial support of other mechanisms, such as reduced permeation. Conjugative TEM-1 and the clonal spread of ST131 may contribute to the prevalence of Japanese ampicillin-sulbactam-nonsusceptible isolates.

Identification of a TFIID Binding Sequence That Increases Expression from the Ankyrin-1 Promoter: Application to Globin Gene Therapy

Effective Gene Therapy of the hemoglobin β-chain disorders β-thalassemia or Sickle Cell Disease (SCD), requires that viral vectors deliver β-like globin gene to hematopoietic stem cells (HSC) and express β-globin at levels >20% of that of endogenous α-globin. However, the β-globin gene is poorly expressed without sequences from the Locus Control Region (LCR). The LCR contains sequences that contribute to inefficient production and low titers of the recombinant virus, and pose a significant risk of insertional activation of leukemia genes. We have taken an alternative approach using enhancer independent promoters from genes expressed in erythrocytes to express sufficient β-like globin. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the four different tissue specific promoters used to express the ANK-1 gene. We have shown that a double copy of Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. We have also demonstrated that in mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein were expressed at a uniform level of 7.5% of α-globin per vector copy. To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide in the transcribed region (+4 or +32 relative to the proximal or distal mRNA start sites) of the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We hypothesized that introducing sequences with higher TFIID binding affinity into the region of the ANK-1E promoter would result in increased level of ANK-1E/γ-globin transcription needed for an effective therapy for β-thalassemia and SCD. An ANK-1E promoter library was generated using degenerate sequence in the TFIID binding region while preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained: wild type (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones ((G/T)(G/C)(G/C)GGTGAG) was found at two other locations in the ANK-1E promoter (−5 and −54 relative to the distal mRNA start site) as well as in 25% of 5′UTR across the human genome (p=2.2e−16). 64% of GC rich promoters contain the consensus sequence, which is enriched at +50 and −70 relative to the mRNA start site. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase than the wild type promoter (p=0.001; 0.005 respectively). Electrophoretic mobility shift assays demonstrated that the two more active promoters bind more TFIID than the wild type promoter. We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

The glnAntrBC operon of Herbaspirillum seropedicae is transcribed by two oppositely regulated promoters upstream of glnA

Herbaspirillum seropedicae is an endophytic bacterium that fixes nitrogen under microaerophilic conditions. The putative promoter sequences glnAp1 (σ70-dependent) and glnAp2 (σ54), and two NtrC-binding sites were identified upstream from the glnA, ntrB and ntrC genes of this microorganism. To study their transcriptional regulation, we used lacZ fusions to the H. seropedicae glnA gene, and the glnA-ntrB and ntrB-ntrC intergenic regions. Expression of glnA was up-regulated under low ammonium, but no transcription activity was detected from the intergenic regions under any condition tested, suggesting that glnA, ntrB and ntrC are co-transcribed from the promoters upstream of glnA. Ammonium regulation was lost in the ntrC mutant strain. A point mutation was introduced in the conserved –25/–24 dinucleotide (GG→TT) of the putative σ54-dependent promoter (glnAp2). Contrary to the wild-type promoter, glnA expression with the mutant glnAp2 promoter was repressed in the wild-type strain under low ammonium levels, but this repression was abolished in an ntrC background. Together our results indicate that the H. seropedicae glnAntrBC operon is regulated from two functional promoters upstream from glnA, which are oppositely regulated by the NtrC protein.Key words: Herbaspirillum seropedicae, nitrogen assimilation, glnAntrBC operon, transcriptional regulation.

Reversal of Glucocorticoids-Dependent Proopiomelanocortin Gene Inhibition by Leukemia Inhibitory Factor

We previously have described molecular mechanisms converging at the Nur response element-signal transducer and activator of transcription (STAT) composite site responsible for synergistic activation of the proopiomelanocortin (POMC) gene promoter by leukemia inhibitory factor (LIF) and CRH. In this study, we asked how glucocorticoids (GC), the physiological negative regulators of POMC gene expression, modulate this synergism. In the corticotroph cell line AtT-20, the response of the wild-type promoter to LIF+CRH was barely inhibited by GC, whereas a distal promoter subregion (−414/−293) encompassing the Nur response element-STAT site and devoid of the negative GC-responsive element located in the proximal domain, displayed a cooperative response to LIF + dexamethasone (DEX) and LIF+CRH+DEX treatments. LIF+CRH-stimulated ACTH secretion was also inefficiently inhibited by DEX in the same cell line. This study was focused thereafter on LIF+DEX cooperativity, which may be responsible, on the wild-type promoter, for lack of negative regulation by DEX of the LIF+CRH synergy. The STAT1–3 low-affinity site, in the context of the (−414/−293) subregion of the POMC promoter, was found necessary and sufficient for transcriptional synergism between activated GC receptor (GR) and STAT1–3. Moreover the activities of reporters specific for STAT1–3 or GR were reciprocally enhanced by DEX or LIF. Single and sequential chromatin immunoprecipitations revealed 1) a STAT-dependent corecruitment of coactivators after LIF and LIF+DEX stimulation and 2) a more lasting recruitment of both STAT3 and GR in the same enhanceosome on the endogenous POMC promoter after LIF+DEX joint stimulation than after the single one. Such events may be responsible for a lack of repressive property of GR unmasked on the whole POMC promoter during LIF+CRH stimulation and may contribute to the tonicity of the hypothalamic-pituitary-adrenal axis during inflammatory-infectious diseases.

Evidence that the Promoter Can Influence Assembly of Antitermination Complexes at Downstream RNA Sites

ABSTRACT The N protein of phage λ acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the λ early P R promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P R, at positions −14 and −45 (yielding P R-GA), reduces effectiveness of N modification, while an additional mutation at position −30 (yielding P R-GCA) suppresses this effect. In vivo, the level of P R-GA-directed transcription was twice as great as the wild-type level, while transcription directed by P R-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P R-GA in vitro was roughly one-third the rate for wild-type P R. We ascribe this apparent discrepancy to an effect of the mutations in P R-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.

Genetic Basis for Conversion of Rough-to-Smooth Colony Morphology in Actinobacillus actinomycetemcomitans

ABSTRACT The basis of the rough-to-smooth conversion of Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained mutations at the flp promoter region. Replacing the mutated flp promoter with the wild-type promoter restored the rough phenotype. The expression level of the flp promoter was ∼100-fold lower in smooth than in rough strains. Mutations of the flp promoter are a cause of the rough-to-smooth conversion.

Genetic analysis of the Bacillus subtilis sigG promoter, which controls the sporulation-specific transcription factor σ G

At the onset of sporulation in Bacillus subtilis, an asymmetric cell division gives rise to two unequal-sized compartments with distinct developmental fates. The smaller compartment, or prespore, becomes the spore, whilst the larger compartment, or mother cell, eventually lyses after contributing to spore maturation. The fate of each compartment is determined by differential gene expression, controlled by the activation of four compartment-specific σ-factors. The expression and activity of all four σ-factors are tightly regulated to ensure the correct sequence of morphological events. Prespore-specific genes are transcribed by two σ-factors, σ F followed by σ G. The gene encoding σ G (sigG) is transcribed by σ F, but also requires the activity of one of the mother-cell-specific σ-factors, σ E, for its expression. The minimal promoter required for dependence on σ E was found to stretch to just upstream of the −35 site. Analysis of mutant sigG promoters generated by site-directed mutagenesis and sigG promoters from other species suggests the presence of a binding site for a transcriptional repressor within the sigG promoter region. Replacement of the wild-type promoter with σ E-independent promoters resulted in impairment of sporulation. These data support the idea that σ E activity is required for the transcription of sigG.

5′-Flanking region polymorphisms of CYP2C9 and their relationship to S-warfarin metabolism in white and Japanese patients

White and Japanese patients require different warfarin dosages to achieve therapeutic anticoagulation, but this can be only partly explained by genetic variability in the coding region of CYP2C9—a critical enzyme in the drug's metabolism. Accordingly, analysis of the -2.1-kb 5′-flanking region of CYP2C9 was undertaken in 22 white and 38 Japanese patients whose unbound oral clearance of S-warfarin had been previously determined. Thirteen single nucleotide polymorphisms (SNPs) were identified, some of which were in linkage disequilibrium with functionally defective coding region variants. Those 5′-flanking patterns linked with at least one CYP2C9*3 allele or CYP2C9*2/*3 were associated with reduced CYP2C9 activity and warfarin dose. Japanese patients possessing the wild-type promoter and coding sequences had significantly (P < .01) greater CYP2C9 activity than white patients with the corresponding genotype. In conclusion, either unidentified polymorphisms further upstream in the promoter region or environmental factor(s) account for the differences in the warfarin doses between whites and Japanese. (Blood. 2004;103: 3055-3057)

Regulation of rRNA Transcription Correlates with Nucleoside Triphosphate Sensing

ABSTRACT We have previously shown that the activity of theEscherichia coli rRNA promoter rrnB P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 differentrrnB P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The sevenrrnB P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the fis gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the rrnB P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which rrnB P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).