GPR43) recognizes short chain fatty acids and is implicated in obesity, colitis, asthma and arthritis. Here, we present the first full characterization of the GPR43 promoter and 5′-UTR. 5′-RACE of the GPR43 transcript identified the transcription start site (TSS) and a 124 bp 5′-UTR followed by a 1335 bp intron upstream of the ATG start codon. The sequence spanning -4560 to +68 bp relative to the GPR43 TSS was found to contain strong promoter activity, increasing luciferase reporter expression by >100-fold in U937 monocytes. Stepwise deletions further narrowed the putative GPR43 promoter (−451 to +68). Site-directed mutagenesis identified XBP1 as a core cis element, the mutation of which abrogated transcriptional activity. Mutations of predicted CREB, CHOP, NFAT and STAT5 binding sites, partially reduced promoter activity. ChIP assays confirmed the binding of XBP1 to the endogenous GPR43 promoter. Consistently, GPR43 expression is reduced in monocytes upon siRNA-knockdown of XBP1, while A549 cells overexpressing XBP1 displayed elevated GPR43 levels. Based on its ability to activate XBP1, we predicted and confirmed that TNFα induces GPR43 expression in human monocytes. Altogether, our findings form the basis for strategic modulation of GPR43 expression, with a view to regulate GPR43-associated diseases.G-protein coupled receptor 43 ( FFAR2 or GPR43) as a critical gene in the prevention of obesity, colitis, asthma and arthritis1234567GPR43 is a G protein coupled-receptor that is activated by mid-micromolar concentrations of short-chain fatty acids (SCFAs) - namely acetate, propionate and butyrate. While the liver metabolism of ethanol can generate micromolar concentrations of acetate in the blood, by far the most abundant source of SCFAs in the human body is the colonic lumen, where hundreds of millimolars are continuously being produced during the anaerobic fermentation of dietary fibre by saccarolytic gut bacteria91112GPR43 has already been shown to mediate some of these beneficial effects, with knockout mice studies confirming a role in obesity and inflammation  or treatment with granulocyte-macrophage colony stimulating factor (GM-CSF) raises GPR43 transcript levels in human monocytesGPR43 expression is tightly regulated and may be important for its function. Indeed, the compelling outcomes of Gpr43 knockout  product being many times more abundant than the longer transcript. It is possible that the two transcript sizes described by Senga et al., 2003GPR43 gene and allow the putative core promoter to be identified for subsequent analysis of transcriptional elements.To define the rt codon . Mappingrt codon . Thus, trt codon , Lane 1,Gpr43 gene , suggesting a lack of involvement in mouse Gpr43 transcriptional control. Deletion from positions −451 to −182 resulted in a marked drop  in activity while further deletions downstream of position −182 resulted in significant reductions in activity , suggesting that the region spanning −451 to −58 might contain crucial activator elements. This is consistent with the observed interspecies sequence conservation  from primary human monocyte genomic DNA and analyzed its promoter activity using dual luciferase reporter system. This putative promoter contained aligned sequences with greater than 70% sequence identity to the corresponding mouse sequence , suggestervation . Strikinin silico predictions using the MatInspector programGPR43 expression. Conversely, no significant loss in activity was found for mutations in the IRF4, PU.1 and c-Rel recognition sites. Mutation of the XBP1 recognition site resulted in near abrogation of promoter activity  was performed with antibodies against XBP1. The GPR43 promoter XBP1 binding site was significantly enriched in the resulting immunoprecipitate, consistent with strong association of XBP1 to that site GPR43. Notably, mutations in the XBP1 gene locus of human patients has been associated with increased risk of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, while −/−Xbp1 mice have increased susceptibility to colitis−/−Xbp1 mice also display impaired glucose and insulin tolerance upon high fat diet-induced obesityGPR43, since −/−Gpr43 mice are similarly more susceptible to colitis1237GPR43 in inflammation and obesity, it might be pertinent to further investigate this hypothetical link.The indispensability of XBP1 for GPR43 promoter was also found to be up-regulated by Tax/CREB, CHOP, NFAT and STAT5 . Following the same line of argument, we cannot rule out the potential involvement of other predicted transcription factors, e.g. IRF4, c-Rel (component of NF-κB), and Pu.1 9GPR43 1415GPR43 in the peripheral blood monocytes might thus enhance the potency of SCFAs on the GPR43-mediated inflammation.The ability of inflammatory stimuli such as LPS, TNFα and GM-CSF  to up-reGPR43 expression observed in phosphoinositide-3 kinase (PI3K) inhibition under basal or immune-challenged conditions  would offer a more complete analysis of the effect of this putative enhancer.In addition to characterizing the promoter, we provide the first description of the 5′-UTR of the human GPR43 promoter are mostly obtained from human monocytes in this study, it would be interesting to explore if the promoter elements identified, regulate GPR43 in other cell types. Our findings on A549 cells suggest that this may indeed be the case. In A549 cells, the 519 bp GPR43 promoter construct up-regulates luciferase expression; an up-regulation that is abrogated by XBP1 null mutation . Ficoll-Paque PREMIUM  gradient centrifugation was performed according to the manufacturer's instructions. Briefly, the mononuclear cell layer was isolated and washed with PBS supplemented with 2% FBS  and 1 mM EDTA, to remove platelets. Monocytes were subsequently purified by negative selection using the Human Monocyte Enrichment Kit (StemCell Technologies) according to the manufacturer's instructions.2. For differentiation of U937 into monocytes, 5 × 105 cells/mL of pre-differentiated cells were induced with 30 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 24 h, before changing to fresh media. The cells were cultured for another 48 h to allow for full differentiation and thereafter used for downstream assays.Primary monocytes and U937 cell line were cultured in RPMI (Life Technologies) while A549 cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS and 1% (v/v) penicillin and streptomycin (Life Technologies). The cells were grown at 37°C and 5% COThe primary neonatal human fibroblasts (Life Technologies) were routinely grown in medium 106 (Life Technologies) before RNA extraction.Amplification of 5′ cDNA ends of mature GPR43 was performed with the GeneRacer™ kit  following manufacturer's instructions. Briefly, 5 μg of U937 total RNA was dephosphorylated with Calf Intestinal Phosphatase (CIP) and ligated with a sequence specified RNA oligonucleotide at 37°C for 1 h. Following ligation, the 5′ ends of GPR43 mRNA were reverse transcribed and amplified using nested PCR primers . The PCRTo generate promoter deletion constructs, primary monocyte genomic DNA was extracted from the interphase and phenol layer using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. To obtain 5′ end promoter deletion products, primers  flankingXhoI and HindIII (Thermo Fisher Scientific). T4 DNA ligase was from Roche. For small-scale purification of plasmids, AxyPrep Plasmid Miniprep kit (Axygen Biosciences) was used. Large-scale purification of plasmids intended for transfection was carried out using PureLink HiPure Plasmid Filter Purification kit (Invitrogen). The full-length sequence of each promoter construct was confirmed by sequencing using Big Dye Terminator cycle sequencing kit and ABI Prism 3100 Genetic Analyzer (Life Technologies).The isolated promoter lengths were separately cloned into pGL4.20 vector (Promega) using standard molecular cloning techniques. The restriction enzymes used were http://www.genomatix.de/Transcription factor binding sites (TFBS) along the delineated promoter regions were identified using the MatInspector programme available online at Promoters containing the specific mutated TFBS were synthesized by Integrated DNA technologies. The mutated promoters were cloned into pGL4.20 vector (Promega) and confirmed by sequencing as described above.Renilla luciferase vector (Promega) were transfected in the molar ratio of 10:1.The full-length promoter, deletion and mutant constructs were transiently transfected into differentiated U937 cells at 48 h after removal of PMA, with X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's guidelines. Promoter-pGL4.20 vector constructs and pRL-CMV control Renilla luciferase reporter activities were assessed using the Dual Luciferase Reporter Assay System (Promega) 22 h after transfection. Luminescence was detected using the Glomax 20/20 Luminometer (Promega), and the resulting measurements from the Firefly luciferase were normalized to the Renilla luciferase. Relative light units were calculated using readouts from the pGL4.20-Basic (promoter-less) vector or the wild type promoter as baseline for deletion and mutation constructs, respectively.Escherichia coli 055:B5 LPS and sodium acetate were purchased from Sigma-Aldrich while GM-CSF and TNFα were from Life Technologies. Purified human monocytes were seeded at a density of 4 × 106 cells/mL in 24-well plates and cultured in the presence of either LPS (100 ng/mL), PMA (30 ng/mL), TNFα (10 ng/mL), GM-CSF (100 ng/mL) or sodium acetate (10 mM) for 3 h before RNA extraction with TRIzol reagent (Life Technologies).For stimulation of cells, SB203580, PKC inhibitor Bisindolylmaleimide I (BisI) , PLC inhibitor U73122, adenylate cyclase activator Forskolin (Tocris Biosciences), proteasome inhibitor MG132 and PI3K inhibitor Wortmannin (Sigma) were obtained.For modulation of signalling pathways, p38 inhibitor Purified human monocytes were pre-treated for 1 h in either 20 μM DMSO (Merck) as vehicle control, SB203580 (10 μM), U73122 (5 μM), MG132 (10 μM), Wortmannin (2 μM) or BisI (4 μM). Cells were then challenged in 100 ng/mL LPS for 3 h before mRNA was extracted for analysis.To identify regulatory pathways involved in the basal regulation of gene expression, the same conditions above were applied except that replicate wells of cells treated with MG132 (10 μM) and Forskolin (20 μM) were lysed and analyzed for mRNA expression after 4 h without LPS treatment. For treatment with other inhibitors, monocytes were lysed for RNA extraction and analyzed after 12 h in culture.XBP1 was detected using the Taqman gene expression assay  and Light Cycler Probes Master (Roche). To obtain relative target mRNA folds, cycle thresholds (Ct) were normalized to the Ct of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or Ribosomal protein L27 (RPL27) reference genes and expressed as fold-change by the 2−▵▵Ct methodTotal RNA was isolated using the TRIzol reagent according to the manufacturer's instructions and the procedure was repeated to ensure thorough removal of genomic DNA. cDNA synthesis of the extracted RNA was performed using Superscript III First Strand Synthesis kit (Life Technologies). Quantitative PCR was performed using GoTaq qPCR Master Mix (Promega) and forward and reverse primers  with LigCell lysates were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-rad). The blots were then probed with antibodies against XBP1 , or β-actin  followed by the corresponding secondary antibodies according to respective manufacturer's instructions. Following incubation with WesternBright ECL chemiluminescent substrate (Advansta), chemiluminescent signals was detected with an ImageQuant™ LAS 4000 mini .GPR43 promoter region. Fold enrichment was obtained by normalizing the amount of precipitated DNA to that of the input sample and then expressed relative to the GPR43 negative control coding region.ChIP assay was performed as described previouslyFor stable knockdown of XBP1, siRNA sequences Sense: 5′-AAAGACAGCAAGTGGTAGATTTA-3′; Anti-sense: 5′-AAAATAAATCTACCACTTGCTGT-3′XBP1s overexpression, XBP1s coding sequence (NM_001079539.1) was cloned into pcDNA3.1A (Invitrogen) and transfected into A549 cells using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instructions.For A.Z.W. and E.J.Z. conceived, designed, and performed the experiments and analyzed the data with intellectual input from J.L.D. J.L.D. provided overall coordination and supervision of the study. A.Z.W., E.J.Z. and J.L.D. wrote the manuscript.Supplementary Information


The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein’s antigenic sites.Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of V The cystoviridae family of viruses, of which ϕ6 was the first discovered species, contain three segments of double stranded RNA. Bacteriophage ϕ6 and its relatives are model systems for virus assembly, genome packaging and dsRNA polymerization. The RNA packaging, replication, transcription mechanism, and overall structure resembles that of reoviruses making the species an excellent model system to study these important pathogens. The initial step in cystoviridae replication is the assembly of a closed and unexpanded, dodecahedral-shaped procapsid (PC). The RNA packaging proceeds in a specific order with the small 2948 bp) viral RNA segment packaged first, followed by the middle 4063 bp) and large 6374 bp) segments –3. Step- bp and l8 bp vira4 bp segmDuring genome packaging the PC undergoes significant conformational morphogenesis with the sequential expansion revealing unique binding sites for each of the three viral RNA segments ,12. The in vitro, indicating that P7 may stabilize P1. Study of self-assembly of PC particles shows potential competition between P2 and P7 proteins  for 1 h at 20°C [× 2048 pixel CCD .The transcription reaction was initiated by incubating 200 μg of NC in 200 μl of transcription reaction mixture [pH 8.8; 70 mM of bicine; 1 mM of Mg Acetate; 3 mM of MnCl at 20°C . As a coTwo Mabs with different epitope recognition, 1C10 and 6C3, were chosen for the sandwich-based ELISA of the NC. The capture antibody was 1C10 and the detecting antibody was biotinylated 6C3. ELISA plates were coated with 5 μg/ml of 1C10 (the Mabs were dissolved in 0.2 M of carbonate-bicarbonate buffer at pH 9.4). The plates were then blocked with 1% BSA in PBS. The transcription reaction mixture (50 μl) was applied to the first well and subsequent 2-fold dilutions with PBS were applied to the remaining wells. Two control samples consisted of: (1) NCs in 100 mM Tris buffer and (2) NCs in transcription buffer but lacking rNTPs were employed for comparison. Following the addition of biotinylated 6C3, plates were incubated with a 1:1000 dilution of streptavidin conjugated with alkaline phosphatase. Substrate solution was added and plates were read at 405 nm using a Titertek Multiscan ELISA reader .E. coli in a soluble state. The protein was bound to Ni-NTA beads efficiently eluted with 250 mM of imidazole. Purity was estimated from the coomassie-stained SDS-PAGE and the P7 protein was observed to migrate at a molecular mass of approximately 17 kDa. 50 μg of native form P7 in CFA was used to intraperitoneally inoculate 6-week-old female, BALB/c mice. Two booster inoculations which included P7 emulsified inIFA were administered at days 21 and 51.The SDS-PAGE gel taken at different steps of the P7 purification procedure is shown in κ derived from the same germline gene, Vκ4-59*01 and a VH gene derived from the VH5 family, either the VH5-9-3*01 or VH5-17*02 genes  NC in 100 mM Tris buffer (pH 7.5) and (2) NC in early phase transcription buffer but lacking rNTPs. Following the addition of the biotinylated 6C3 the plates were incubated with a 1:1000 streptavidin conjugated alkaline phosphatase. The substrate solution was added and the degree of NC capture was determined at 405 nm with a Titertek Multiscan ELISA reader. In order to determine if the Mabs were able to bind to the P7 protein We confirmed the partial disassembly of the NC using cryo-EM imaging of early phase transcription reaction mixtures. Cryo-EM projection images of the control  shows a We note that the transcribing NC particles and controls (that are primed for transcription in the standard reaction conditions but lacking rNTPs) showed the increasing amount of available P7 in the ELISA reaction. The NCs maintained and tested in 100 mM Tris-HCl buffer (pH 8.0) did not have any evident immunolabeling.in vitro transcribing conditions- either with or without 4mM rNTPs. The precise position of the transcribing viral RNA within the partially disassembled NC is not evident and its transit path through the portal remains to be determined. Notably the addition of Mabs 6C3 or 1C10 to NC transcribing reactions had no effect on synthesis of viral RNA transcripts.The results of both the projection EM and sandwich assay analysis indicate that the P8 matrix is destabilized and likely completely or partially disassembled under κ and VH genes of the same gene family demonstrating restricted gene utilization, while one, 6C3, which recognizes a conformational epitope, utilizes VH and Vκ genes frequently associated with anti-DNA antibodies; and (3) monoclonal antibodies can be utilized alone or in combination as structural probes for the disassembly or assembly of the ϕ6 nucleocapsid during transcription in the standard early-stage reaction conditions. During NC transcription P7 is in an exposed position or in proximity to the NC surface which is accessible to antibodies.We conclude the following from the assays: (1) monoclonal antibodies can be elicited from mice that recognize unique linear and conformational epitopes of protein P7; (2) Three of the four Mabs derived from distinct B-cell clones utilize identical V


CCR5 is under the control of a complexly organized promoter region upstream of the gene. The transcription factor cAMP-responsive element binding protein 1 (CREB-1) transactivates the CCR5 P1 promoter. The cell-specific expression of CCR5 however is realized by using various epigenetic marks providing a multivalent chromatin state particularly in monocytes. Here we discuss the transcriptional regulation of CCR5 with a focus on the epigenetic peculiarities of CCR5 transcription.The chemokine receptor CCR5 regulates trafficking of immune cells of the lymphoid and the myeloid lineage  and microglia. Because of this, there is an increasing recognition of the important role of CCR5 in the pathology of (neuro-) inflammatory diseases such as atherosclerosis and multiple sclerosis. Expression of  Ccr5 knock-out mice show less neointima formation and an increase in production of the anti-inflammatory IL-10 molecule by vascular smooth muscle cells (vSMCs) .,30.et al exon 2b  [16]. UsBoth P1 and P2 lack the canonical TATA and CCAAT motifs, although in P1 a non-classical TATA-box can be found. Unlike other TATA-less promoters, the CCR5 promoters have a relatively low CpG content.CCR5 promoter, Liu and coworkers suggested that CCR5 transcription could be up-regulated by NF-κB .,56.55,56+ T cells, the CCR5 locus displays such a classical pattern [+ T cells are characterized be relatively low levels of AcH3 and relatively high levels of the repressive mark 3MeK20H4. When compared to activated T cells, naïve T cells also contain high levels of 3MeK27H3. Upon activation the repressive marks in the chromatin of the CCR5 promoter are replaced by activating marks. Chromatin of the CCR5 promoter in activated CD4+ T cells is almost exclusively covered with the activating marks AcH3 and 3MeK4H3. In other cell types however, the chromatin makeup of the CCR5 locus is more complex.When comparing activated and naïve CD4 pattern ,33. NaïvCCR5 promoter region revealed that this promoter is extensively covered by multivalent marks [Bivalent chromatin structures, composed of both 3MeK4H3 and 3MeK27H3, were first found in embryonic stem cells. In these cells, bivalent states marked genes encoding transcription factors that play an essential role in development. It was proposed that these bivalent states would silence developmental genes while keeping them poised for activation. Upon differentiation, this bivalent state would be lost . Later wnt marks .CCR5 promoter is covered with high levels of AcH3, but also with relative high levels of 3MeK9H3 and 3MeK27H3 and an intermediate level of 3MeK20H4. The histone modifications in monocytes are accompanied with relatively high levels of DNA methylation , a lysine methyltransferase inhibitor (DZNep) and a histone deacetylase (HDAC) inhibitor , despite the fact that they act genome-wide and they seem to be well tolerated [In Jurkat cells, DNA of the  1 and 3 ,64,65) tll-lines . Interesll types . Togetheolerated ,68.CCR5 is achieved by a complex interplay of transcription factors and various forms of epigenetic regulation. Cell type-specific transcription of CCR5 is achieved by epigenetic regulation. Epigenetic regulation also allows for rapid transcriptional shutdown or initiation of the CCR5 locus upon differentiation of various cell types. This epigenetic regulation is a perfect entry point for pharmaceutical intervention. Given the importance of CCR5 in numerous inflammatory diseases and as co-receptor for HIV-1 gaining entrance into cells, pharmaceutical intervention in the epigenetic regulation of CCR5 transcription may prove to be valuable in future disease management.Transcription of


Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including:  Molecular mechanisms that are conserved throughout evolution, or arise independently to perform similar tasks are of major interest to biology The roles of non-coding RNA (ncRNA) molecules have proven to be especially elusive. Only recently, high-throughput technologies have revealed that ncRNAs have important functions across diverse biological systems and processes Based on their location, TSSaRNAs have been speculated to play a role in transcription initiation Regardless, TSSaRNA ubiquity across eukaryotes and bacteria suggests that TSSaRNAs are ancient and must have been present in LUCA. Discovery of TSSaRNAs in archaea would lend credibility to this hypothesis and provide clues into why they are evolutionarily conserved across all organisms.H. salinarum, M. maripaludis, S. solfataricus, P. furiosus, N. equitans, M. kandleri, H. volcanii, M. psycrophilus, M. mazei and P. abyssiH. salinarumM. maripaludisS. solfataricusP. furiosushttp://www.ncbi.nlm.nih.gov/geo/), SRA http://www.ncbi.nlm.nih.gov/sra) and UCSC Archaeal Genome Browser http://archaea.ucsc.edu/). Datasets not available in public databases were obtained directly from publications.In the present work, we investigated whether TSSaRNAs do indeed exist in archaea and, thus, ubiquitous across all three domains of life. By mining publicly available data, we gathered evidence for TSSaRNAs in 10 archaeal transcriptomes  and microarray (NimbleGen and Agilent) platforms, and all library construction protocols (strand-specific and non-strand specific) H. salinarum is a model organism for halophilic archaea and has been extensively studied in the last decade. It became a prime model to study aspects of gene expression regulation, especially due to the establishment of predictive quantitative models with high accuracy The consistency of TSSaRNAs discovery across all platforms and organisms justified further experimentation for independent validation. H. salinarum NRC-1, we performed a strand-specific RNA-seq experiment using non-fragmented small RNAs in the 20–230 bp range. Two biological replicates were extracted from cultures under standard growth conditions H. salinarum NRC-1 genome.In order to precisely map TSSaRNAs in , Figure S2). A given genomic location can have two sets of aligned reads starting exactly there: (i) reads from transcripts greater than 151 nt but truncated at any length, up to the maximum sequencing length limit (151 bp) and (ii) identical full-length reads from transcripts smaller than 151 nt. Although both sets map to the same initial position, the former show repeatedly the same start and end genomic coordinates. We used relative enrichment of the aligned start position as a feature to automatically detect TSSaRNAs . It is important to note that dRNA-seq experiments are prone to false negatives To distinguish between processed and primary transcripts, we performed a dRNA-seq experiment H. salinarum ranged from 16 nt to 146 nt with a median size of 27 nt . The distribution and median size of TSSaRNAs was consistent across many organisms: murine  E. coli (range: 33 nt to 40 nt) and M. pneumoniae  Figure S3B) are consistent with previous observations that most transcripts in H. salinarum are leaderless The TSSaRNA sizes in The current understanding is that the production of TSSaRNA transcripts is associated with stalled RNA polymerase during cognate gene transcription in eukaryote and bacteria Figure S4) argue against the cleavage biogenesis hypothesis. Moreover, the observation that TSSaRNA compositional/thermodynamical properties are no different from similar regions in non-cognate gene sequences  argue against the degradative biogenesis hypothesis, following the same rationale put forward by Yus et al.File S2). This model explores only two parameters for RNA polymerase: elapsed time paused at any given genomic location and time between successive transcription initiation events .In archaea, the absence of a set of RNA-seq reads starting just before TSSaRNA reads' ends (File S1). If, on the other hand, only one genomic position stalls a RNA polymerase, then only one type of small molecule associated with the TSS would be created. This case is also observed experimentally for VNG1213C gene, a probable exonuclease: RNA-seq data shows a population of reads concentrated around 72 nt, which maps directly with the single band found in the northern-blot experiment  and one derived from a single pausing site (VNG1213G). VNG0101G encodes a conserved cold shock protein and was selected for further validation since the signal associated with its TSSaRNA was top ranked in tiling array experiments Figure S7). Such differential expression patterns would not be expected if transcription of a TSSaRNA and the full-length transcript of its cognate gene were not regulated by environmental signals, nor could it arise as an experimental artifact of tiling array hybridization and processing. Using pausing sites that can vary their retention time along the growth curve, the RNA polymerase pausing model explains our experimental observation that TSSaRNA can have distinct dynamical behavior relative to their cognate gene. Although counterintuitive, it is possible to generate dynamical profiles such as the ones where TSSaRNA levels remains constant and its cognate gene varies and vice versa, only exploring the two parameters of the model: elapsed time spent paused and time between successive transcripts initiation events (File S2).Remarkably, it was clear from gene expression profiles that dynamical behavior of a TSSaRNA may be distinct from that of its cognate gene. In some cases, the cognate gene level does not change, but expression of the TSSaRNA has distinct dynamics, with up to 16 fold up-regulation or down-regulation to different degrees  to remove adapters; ii) mapping against appropriate reference genomes using Bowtie The expression signal for putative TSSaRNAs locations is a distinct signature characterized by a sharp rise in signal that plateaus over a relatively small distance and then decays precipitously. Tiling array probe intensities and log ratio data for all growth curve time points were obtained from GEO and processed as described in H. salinarum NRC-1 was grown in CM media, in a water bath incubator at 37°C with agitation of 125 r.p.m. Reference samples were cultured under standard growth conditions H. salinarum NRC-1 were prepared for sequencing. Small RNAs at mid-log phase cultures were extracted. For each sample, 10 µg of small RNAs were treated with RNAse-free DnaseI (Fermentas) in a final volume of 30 µL. The reaction was incubated for 45 min at 37°C and the RNA was purified using phenol/chloroform purification. 1 µg of treated small RNA was ligated to RNA 3′ Adapter (RA3) using T4 RNA ligase 2 truncated (BioLabs) for 1 hour at 28°C, in the presence of RNase inhibitor. Once RA3 was ligated, we performed the RNA 5′ Adapter (RA5) ligation using T4 RNA ligase in the presence of 10 mM ATP. cDNA was synthesized using specific oligos for 5′ and 3′ adapters using SuperScript III Reverse Transcriptase, according to Illumina Truseq protocol. cDNA libraries were amplified and samples were separated in a Novex 6% PAGE gel. cDNAs from 20 bp up to 230 bp were isolated from the gel and subjected to quantification and quality analysis.Two small RNA libraries  from The resulting double stranded cDNA was sequenced on Illumina Miseq v2 platform. Biological replicates were sequenced in the same flow-cell using different indexes. Strand-specific sequencing was performed in MiSeq set to 151 cycles per manufacturer's instructions.http://hannonlab.cshl.edu/fastx_toolkit/links.html) to remove adapters. Processed reads were aligned against H. salinarum NRC-1 reference genome  using Bowtie Reads were trimmed using FASTX-toolkit  reads marking the transcription start site (TSS) of the gene itself, truncated at diverse lengths up to 151 bp; and (ii) reads smaller than 151 bp consistently found with the same 3′ end, thus, being full-length reads . Type (ii) reads are generally generated by TSSaRNAs.Since We used relative enrichment of reads' aligned start coordinates as a parameter to automatically detect TSSaRNAs. We looked for the most frequent start coordinate near the start codon of a CDS. The search was performed in a window starting 50 bp upstream of the translation start site and comprising at maximum 20% of CDS length. To make sure that the TSS is reliable, reads must sum up more than 20 counts. This procedure can detect TSSs, but it is still necessary to split TSSaRNA and cognate gene signals. To isolate the TSSaRNA signal, the most abundant read smaller than 151 bp is defined as the TSSaRNA full-length sequence. All other reads starting at the same position are related to the cognate gene. To be conservative, TSSaRNA reads are only retained if they sum up at least 10 counts.Total RNAs were treated with Turbo™ DNase (Ambion) and incubated with Terminator™ 5′-phosphate-dependent exonuclease (Epicentre) (TEX+ sample) or only in buffer reaction (TEX- sample) at 30°C for 60 min, at proportion of 1 U TEX per 1 µg total RNA. Reaction products were purified with RNeasy MinElute Cleanup Kit (QIAGEN) and incubated with 1 U of Tobacco Acid Pyrophosphatase (TAP) (Epicentre) at 37°C for 1 hour in order to generate 5′-mono-phosphates RNAs able to bind to sequencing adapters. Reactions were purified again with RNeasy MinElute Cleanup Kit (QIAGEN).Sequencing libraries were prepared with 1 µg of treated (TEX+) and untreated (TEX-) samples using a similar protocol described above for RNA-seq experiments. To ensure sequencing of a wider range of transcripts we increased the extension time on cDNA amplification step to 1 min and isolated molecules from 20 bp up to ∼480 bp on the gel. Paired-end sequencing was performed on Illumina Miseq v2 platform using 300 cycles kit. Forward reads were trimmed and mapped to the reference genome using Bowtie 32P-labeled oligonucleotides (5′-AGTGTCGTTGAAGAAGTCAACTTCGCCTGTCGCCATTGCAACT-3′ for VNG0101G and 5′-AAAAGTGGCCGTGGGCAGCGGCCACCCGAT-3′ for VNG1213C) using Rapid-hyb buffer . Signals were detected by autoradiography using a M35A X-Omat Processor (Kodak). Genes encoding a conserved cold-shock protein (VNG0101G)  was separated on polyacrylamide gel . RNAs were transferred to Hybond-N+ membranes  and hybridized with DNA sequences of 11 bp around TSSaRNA 3′ ends were analyzed for conserved patterns using MEME with default parameters H. salinarum NRC-1 was computed from a published dataset generated by tiling array hybridization of total RNA from 13 time points over a growth curve Figure S10). We compared the TSSaRNA representative probe intensity with the median intensity of the upstream region and also, with the intensity of the cognate gene. To be considered differentially expressed, this probe must have a substantial difference in relative intensity when compared to the other cognate gene probes and its surrounding . A TSSaRNA probe must show at least 10-fold difference relative to the overall relative intensity of its cognate gene: V = MTSSaRNA – Mcognate≥1, where M = log10(t/tref), tref is taken at the reference time point in t is taken at the growth curve time point when the second most different |V| is seen, Mcognate is the median of all cognate gene probes starting beyond TSSaRNA 3′ end. The same procedure is also required for an upstream region to make sure that TSSaRNA probe is not a merely continuum of adjacent transcript signal. Therefore, a differentially expressed probe must also show at least a 2-fold difference relative to the overall relative intensity of an upstream region. This upstream region is 300 bp long, 120 bp away from TSSaRNA start . If, there is an annotated gene closer than 200 bp from the TSSaRNA start, the aforementioned region is ignored and the whole adjacent CDS region is considered for probe averaging.Differential expression of TSSaRNAs in Figure S6) and implemented in R programing language (File S2). The model attributes a waiting time for each base position along a virtual gene. For simplicity, this waiting time is taken to be 1 arbitrary time unit. A RNA polymerase pausing site is a position where a moving RNA polymerase stalls for more than the default waiting time. This time is called “stalled time” (Δt). There is an “intrinsic transcription initiation time interval” (Δτ), which is the time it takes between two successive RNA polymerases to start their trajectory along the gene from the first base pair to the gene's end. These two time interval parameters are the most important parameters. Other auxiliary parameters are: gene length L, pause position L′ and total simulation elapsed time T. A RNA polymerase is not allowed to keep traveling along the gene if there is another one stalled at the next base pair. In this case it releases its transcript and detaches from DNA, terminating the transcription process. Also, the stalled RNA polymerase that blocked the previous one is not affected and only keep moving forward when its waiting time at pausing site is up.A simple RNA polymerase pausing model was created Click here for additional data file.Figure S2Additional examples of transcriptome mapping using small RNA sequencing. Strand-specific RNA-seq experiment was performed using non-fragmented small RNAs (20–230 pb). Panels A–D show examples of TSSaRNAs and their cognate genes. (A) VNG0725H, forward strand (B) VNG1182H, reverse strand (C) VNG2014H, forward strand. (D) VNG2658G, reverse strand. For each panel, the genome position and CDSs location and orientation (grey arrows) are indicated at the horizontal axis. The uppermost graphic show log2 transformed amount of reads covering a given genomic coordinate. Vertical dashed lines represents the TSSaRNA region. The intermediary graphic represents the amount of aligned reads whose start position maps to a given genomic coordinate, the “start profile”.(PDF)Click here for additional data file.Figure S3Properties of the 179 TSSaRNAs identified by small RNA-seq and dRNA-seq.A – Size distribution. B – Distribution of the distances between TSSaRNA start position and cognate gene CDSs start codon position. C – Distribution of Pearson correlation between each TSSaRNA and its cognate gene.(PDF)Click here for additional data file.Figure S4Schematic illustration of a putative signature if non-degradative processing biogenesis hypotheses would hold. Dark blue points represent RNA-seq reads coverage data. The yellow arrow represents a gene. Green vertical bars represent mapped reads start positions along genome coordinates and their abundances. Light blue highlight represents TSSaRNA sequence region. The prediction illustrated by the figure is not found in H. salinarum sequencing experiments.(PDF)Click here for additional data file.Figure S5Gibbs Free Energy distribution of secondary structure predictions for TSSaRNAs and regular near-TSS sequences. Histogram considers non-cognate genes sequences with similar localization and same size as the TSSaRNA median sizes. Vertical blue bars represent values for actual TSSaRNA sequences.(PDF)Click here for additional data file.Figure S6Schematic illustration of the RNA polymerase pausing computational model. An arbitrary gene of length L bp is considered. Genomic position L′ represents the pausing site. Each moment in time is depicted by successive drawings from the upper left panel downwards until the rightmost lower panel. Time passing is not represented in constant flux and downward vertical arrows illustrate the amount of time passed. Every Δτ units of time a new RNA polymerase arrives at position 1 bp and keep transcribing forward at a constant velocity of v bp/unit of time. Arriving at the pausing point, a RNA polymerase receives v = 0 and waits there for Δt units of time, leaving then again with the same velocity v until it reaches the end of the gene at position L and releasing the full-length transcript. The parameters Δτ and Δt are the most critical for the model and are called “time spent stalled” and “intrinsic transcription initiation interval”, respectively. If an incoming RNA polymerase encounter another RNA polymerase just a base pair downstream, it cannot go further and releases the DNA sequence freeing the transcript synthesized up to that position/moment. The RNA polymerases released due to downstream blocking are shown with their IDs inside their red circle representation and those still active are show with their IDs below. This illustration depicts several moments between the first RNA polymerase (ID #1) start at position 1 bp until it reaches the last position L bp, along with several RNA polymerases  that produced TSSaRNAs due to early transcription termination.(PDF)Click here for additional data file.Figure S7Expression profiles of TSSaRNAs differentially expressed over a typical growth-curve relative to the control growth condition. Horizontal axis represent 13 growth curve points from different phases in a standard laboratory batch culture. Solid lines are TSSaRNA expression profiles and dashed lines their cognate gene expression profile.(PDF)Click here for additional data file.Figure S8Simple RNA polymerase pausing computational model simulation. Simulated expression profiles of TSSaRNAs differentially expressed during a growth curve relative to the control condition. Tiling microarray output simulation for a 35 bp TSSaRNA in a 2 kb cognate gene. Varying the only two model parameters it is possible to generate situations in which the cognate gene expression level remains constant over time and TSSaRNA levels can vary almost arbitrarily. Panels A to D are build mimicking our experimental setup displayed in manuscript's tI) and the amount simulated at reference time-point (refI). Black solid line – TSSaRNA expression profile. Black dashed line – cognate gene expression profile . Panel E shows all kinds of log2 rations that can be obtained for a TSSaRNA probe and its cognate gene when scanning the parameter space: Δτ and Δt, “intrinsic transcription initiation interval” and “time spent stalled”, respectively. This example scans Δτ from 2 to 700 time units, Δt from 3 to 700 time units and simulates a 2 kb gene with a 35 bp TSSaRNA associated. Highlighted points in Panel E are examples of relatively constant TSSaRNA levels with an appropriately 3-fold difference in cognate gene level , and a 32-fold difference in TSSaRNA levels with relatively constant cognate gene levels . Qualitatively, almost any complex dynamical behavior can be obtained if the pausing rhythm and the RNA polymerase arriving rate are jointly regulated by environmental clues.(PDF)Click here for additional data file.Figure S9Illustration of the TSSaRNA identification procedure. The yellow arrow represents a cognate gene's coding sequence region (CDS). Green vertical bars represent mapped reads' start coordinates and their abundances. The grey circle highlights the most frequent start coordinate. The grey box zoom illustrates the set of reads which mapped to this specific coordinate position, composed by two populations: identical reads from TSSaRNAs and other reads that originate from cognate gene transcripts. Black horizontal bars represent regions, relative to translation initiation site position (start codon position), around which the search for the most frequent start coordinate position was performed.(PDF)Click here for additional data file.Figure S10Illustration of the probe selection for TSSaRNA differential expression analysis. Light blue horizontal bars illustrate tiling array probe intensities for the reference condition. The best probe that represent the TSSaRNA is highlighted in red. The yellow arrow represents a cognate gene. Black dashed lines represent the TSSaRNA boundaries defined by RNA-seq. Gray boxes represent the regions used to calculate the neighbourhood expression intensity, which was compared to the TSSaRNA probe.(PDF)Click here for additional data file.Figure S11H. salinarum.Illustration of the method used to define a differentially expressed TSSaRNA in  The three-dimensional tiling microarray data (relative intensities vs growth-curve vs genome loci) is reduced to two dimensions and then to a single representative value. A TSSaRNA expression profile is considered distinct from its cognate gene if there are at least two time-points in which their relative intensities are at least 10-fold apart. Relative intensities are considered between a time-point and the reference growth condition. Relative intensities for TSSaRNAs are provided by the best tiling array probe  to fit a RNA-seq based TSSaRNA boundaries definition .tion see . Relativ(PDF)Click here for additional data file.Table S1Halobacterium salinarum transcriptome.TSSaRNAs identified in (XLS)Click here for additional data file.Table S2Halobacterium salinarum transcriptome differentially regulated relative to their cognate gene.Subset of TSSaRNAs identified in (XLS)Click here for additional data file.Table S3Detailed information on all 10 organism datasets used to detect TSSaRNA in archaea.(XLS)Click here for additional data file.File S1RNApol pausing computational model with multiple pausing sites (R language script).(R)Click here for additional data file.File S2RNApol pausing computational model with varying pausing rhythm (R language script).(R)Click here for additional data file.


Our previous studies established an important role of bromodomain-containing protein-4 (BRD4) in promoting estrogen-regulated transcription and proliferation of ER+ breast cancer cells. Here, we investigated the association between genome-wide occupancy of histone H4 acetylation at lysine 12 (H4K12ac) and BRD4 in the context of estrogen-induced transcription. Similar to BRD4, we observed that H4K12ac occupancy increases near the transcription start sites (TSS) of estrogen-induced genes as well as at distal ERα binding sites in an estrogen-dependent manner. Interestingly, H4K12ac occupancy highly correlates with BRD4 binding and enhancer RNA production on ERα-positive enhancers. Consistent with an importance in estrogen-induced gene transcription, H4K12ac occupancy globally increased in ER-positive cells relative to ER-negative cells and these levels were further increased by estrogen treatment in an ERα-dependent manner. Together, these findings reveal a strong correlation between H4K12ac and BRD4 occupancy with estrogen-dependent gene transcription and further suggest that modulators of H4K12ac and BRD4 may serve as new therapeutic targets for hormone-dependent cancers.Hormone-dependent gene expression requires dynamic and coordinated epigenetic changes. Estrogen receptor-positive (ER The estrogen receptor-alpha (ERα) plays a central role in determining a luminal epithelial phenotype and tumor progression in a large fraction of breast cancers. Notably, interfering with ERα-regulated gene transcription represents a major therapeutic target in breast cancers where anti-estrogen therapies are the primary indicated therapy for patients with ERα-positive tumors. Estrogen-mediated gene induction is tightly and dynamically regulated. Stimulation with estrogen induces the binding of ERα to estrogen response elements (EREs), which act as enhancers in a manner largely dependent upon the pioneer factor, Forkhead protein A-1 (FOXA1) . Upon acBRD4 belongs to the bromo- and extraterminal (BET) domain protein family and serves as a major epigenetic reader of histone acetylation. BRD4 preferentially binds to multiple acetylated lysine residues including K5, K8, K12 and K16 of histone H4 , 13 and A number of studies have uncovered an essential role for BRD4 in various malignancies including Myc-driven cancers , 29, leuIn this study, we examined the association of H4K12ac with BRD4 occupancy genome-wide and analyzed its function in estrogen-regulated transcription. We show that H4K12ac occupies estrogen-responsive gene promoters and EREs in an inducible manner  where itIn order to determine whether H4K12ac may play a role in estrogen-responsive gene expression, we performed genome-wide ChIP-sequencing analyses of H4K12ac with or without estrogen treatment in the ERα-positive MCF7 luminal breast cancer cell line. Aggregate profiles and heatmap analyses showed that estrogen induction increases H4K12ac occupancy near the transcription start sites (TSS) Fig. . InteresGiven the association of BRD4 with enhancers , 32 and To further evaluate the association of H4K12ac with distal enhancer function, distal EREs were grouped into four classes  according to the enrichment of H4K12ac around EREs. Consistent with their potential function as active enhancers, these groups of distal regions also correlated with BRD4 and RNAPII occupancy as well as nascent RNA transcription Fig. . TogetheIn order to investigate a potential association with the function of BRD4, the average intensity of BRD4, H4K12ac, H3K27ac, H3K4me3, H2Bub1 and RNAPII downstream of the TSS (TSS and 3 kb downstream) were calculated on genes whose expression either decreased (“downregulated”) or was unaffected (“independent”) upon BRD4 depletion under vehicle treated conditions from our previously published RNA sequencing data. Interestingly, genes whose expression decreased following BRD4 depletion were lower expressed than non-regulated genes prior to knockdown Fig.  and alsoIn order to further validate the association of H4K12ac with active gene transcription, average signals of the active transcription marks around TSS were compared with the normalized counts of expression for each gene. As shown in scatterplots along with the respective correlation coefficients, H2Bub1, RNAPII, H3K4me3 and H4K12ac significantly correlated with mRNA expression Fig. . TogetheAs H4K12ac occupancy is closely associated with ERα-dependent gene activity, we also compared H4K12ac occupancy in ERα-positive MCF7 breast cancer cells to that in the ER-negative MCF10A normal mammary epithelial cell line both in ChIP and in whole cell protein lysates. Both single gene ChIP-qPCRs as well as genome-wide ChIP-seq analyses displayed induced H4K12ac occupancy in MCF7 comparing to MCF10A around TSS of estrogen-dependent genes Fig. . SurprisSignal-induced transcription requires a highly coordinated and complex interplay between various transcription factors, post-translational histone modifications, epigenetic readers and chromatin remodelers in order to induce gene expression in a proper temporal and spatial manner. Dysregulation of these mechanisms frequently results in disorders such as cancer. Positive association of H4K12ac with estrogen-induced gene transcription reveals a potentially important and specific role of this modification in promoting ERα-induced gene transcription. According to our model, ERα-directed H4K12 acetylation facilitates the recruitment of BRD4 to both enhancers and promoters. Subsequently, BRD4 recruits CDK9 in order to promote eRNA synthesis at enhancers and transcriptional elongation of ERα-dependent mRNAs. The latter occurs, at least in part via phosphorylation of RNAPII at serine-2 and the subsequent monoubiquitination of histone H2B, which may help to open chromatin structure and facilitate the activity of other chromatin-associated elongation factors , 38, 39.Transcriptional activation is tightly coupled with histone acetylation -48. HoweThe transcriptional mechanisms controlling stimuli-induced gene expression is tightly coupled to the rapid association and disassociation of transcriptional factor and cofactor complexes as well as dynamic changes in chromatin remodeling and histone modifications , 56-58. Previous studies have suggested that BRD4 predominantly binds to histone H4 acetylated at lysine residues 5, 8, 12 and 16 , 13. HowInduced occupancy of H4K12ac at both TSS and enhancers in cancer cells support a role for histone acetylation and enhancer activity in ERα-dependent cancer progression. Moreover, we describe a significant correlation between histone acetylation and eRNA production, which can control targeted gene induction in various disease-specific conditions , 24-26. MCF7 cells were kindly provided by K. Effenberger  and were grown in phenol red-free high-glucose Dulbecco's modified Eagle's media  supplemented with 10% fetal bovine serum (Thermo Scientific), 1% sodium pyruvate, and 1% penicillin/streptomycin (Sigma-Aldrich) at 37°C. For estrogen induction, cells were deprived of hormones by treating them with DMEM media supplemented with 5% charcoal-dextran-treated fetal bovine serum , 1% sodium pyruvate, and 1% penicillin/streptomycin after 24 hr of growth. After 48 hr, cells were treated with 10 nM 17 β-estradiol (Sigma-Aldrich) and/or 1 μM ICI-182,780 (Fulvestrant) for 2 hr. MCF10A cells were kindly provided by M. Oren  and were grown in phenol-red free DMEM/F12 medium supplemented with 5% horse serum, 20 ng/mL epidermal growth factor, 0.1 μg/mL Cholera toxin, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 1% penicillin/streptomycin at 37°C.As mentioned previously , 39, whoChromatin immunoprecipitation of H4K12ac was performed as described previously  using anChIP-seq libraries were made using the NEBNext Ultra DNA library preparation kit according to the manufacturer's instructions . The sizChIP-seq reads were mapped to the human reference genome (UCSC HG19) using Bowtie (version 1.0.0) . Sam fil


This promoter is naturally induced under anaerobic conditions and upregulated by nitrite and nitrate.Anaerobic-inducible promoters are alternatives of chemical-inducible promoters for expression of recombinant proteins especially in conditions where chemical induction is not possible or anaerobic conditions are preferable. The nirB promoter that does not respond to chemical inducers (nitrite or nitrate), but instead responds to anaerobic induction. For this purpose, a new plasmid was constructed (pFSnirB78-23LTB), which contains a synthetic nirB promoter. The activity of this plasmid was evaluated in E. coli under both aerobic and anaerobic conditions and in response to chemical inducers, nitrite and nitrate.The current study was carried out to construct a synthetic nirB promoter was firstly cloned into a pKK223 derivative plasmid and then the heat labile toxin B subunit gene (LTB) of entrotoxigenic E. coli was cloned under the control of this promoter. The inducibility of this plasmid in E. coli was measured under anaerobic conditions in the presence or absence of nitrite or nitrate by ganglioside GM1 ELISA.A synthetic Our data showed that this promoter is strongly induced under anaerobic conditions while it showed much lower activity (11%) under aerobic conditions. In contrast to the native promoter, this promoter was not induced by chemical inducers, nitrite or nitrate.nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes. In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins. Based on the results, this synthetic promoter is suitable for use in live delivery vaccines or drug systems and for production of recombinant proteins especially oxygen sensitive proteins.This study showed that the recombinant protein produced under the control of synthetic  Escherichia coli are usually performed in high oxygen tension and controlled by various chemically or physiologically inducible promoters  protein-binding site and mutation in this region results in decreasing promoter induction. FNR protein encoded by the on codon . nirB promoter activity; this means that NarL and NarP are cofactors of FNR in the transcription process. It has been demonstrated mutation in the NarL binding site suppresses nitrite induction whereas it has no effect on nitrate induction , which are binding sites of two other regulatory proteins; NarP and NarL. Mutation in NarP, NarL or both decreases nduction , 9. A sy tension . in vivo, the present study was performed to construct a synthetic nirB promoter, which has no responding regions for nitrite and nitrate inducers and is only able to induce the recombinant protein under low oxygen pressure or anaerobic conditions.In recombinant protein expression systems, removing these inducers from culture medium result in further steps for purification and then more cost. In addition, these chemical inducers may have some side-effects on cell physiology, and since using these inducers is not applicable E. colinirB promoter, we designed a synthetic nirB promoter and constructed a plasmid that it able to produce recombinant proteins under anaerobic induction and not by chemical inducers (nitrite or nitrate) and then the promoter activity was evaluated with regarding to the expression of a heterologous gene; heat-labile toxin B subunit (LTB gene), under various conditions.For this purpose and based on the native sequence of E. coli DH5 was used in all cloning and expression procedures. Restriction enzymes, T4 DNA ligase, Taq DNA polymerase and dNTP were purchased from Fermentas (Lithuania). Ganglioside GMI type III, standard E. coli heat-labile toxin (LT) and anti-mouse horse-raddish peroxidase (HRP) conjugate antibodies were obtained from Sigma (USA). All cloning steps such as transformation, plasmid extraction, PCR amplification procedures and restriction digestion were performed on the basis of standard protocols  and constructed by two oligomers BamnirBSD78; (5′ GATCCaggtaAATTTGATgTACATCAAatggtaccccttgctgaatcgtTAAGGTaggcggtaTaagGAGGaaaaaac) and NcoNirBSD78 (5′CATGGttttttcctccttataccgcctaccttaacgattcagcaaggggtaccatttgatgtacatcaaatttacctg). The two oligomers were mixed by molar ratio at 85 °C and cooled slowly to form a double-stranded fragment with cohesive ends of BamHI and NcoI sites at the 5′ and 3′ ends, respectively. On the BamnirBSD78 oligo diagram, the FNR region has been shown by underlined capital letters, the ribosome-binding site (RBS) as bold letters and -10 region as italic capital letters.The nirB promoter (nirB: BamHI- NcoI fragment) was firstly cloned in a pkk223 derivative plasmid and then the amplified LTB gene (LTB: NcoI-HindIII fragment) was cloned downstream of the promoter. This new construct was confirmed by restriction analysis and named pFSnirB78-23LTB.By two-step cloning, the synthetic E. coli and one colony of E. coli/ pFSnirB78-23LTB was cultured overnight in Luria-Bertani (LB) medium  supplemented with ampicillin (100 ug/mL) at 37 C with vigorous shaking (220 rpm). For evaluation of promoter activity in aerobic conditions, 1/500 of overnight culture was inoculated into 5 mL of fresh medium containing ampicillin and incubated at 37 C with vigorous shaking (220 rpm) until OD600 nm reached 0.4-0.6. Induction was carried out by adding sodium nitrite (2.5 mM) or sodium nitrate (20 mM), while a reaction without chemical induction was also performed; incubation was continued for 4 hours. The cells were then harvested and resuspended in 1 mL of PBS.The plasmid was transformed into competent  9 cfu. Alternatively, for the reaction under anaerobic conditions, 1/30 of the overnight culture was inoculated into a 5 mL screw-cap conical tube completely filled with LB medium containing ampicillin. Sodium nitrite (2.5 mM) or sodium nitrate (20 mM) was added to each tube as an inducer for comparison with the control, which was kept under anaerobic conditions but did not contain the inducer. After 24 hours of incubation at 37 C, the cfu was determined and rLTB measurement was performed as mentioned above.The colony forming unit (cfu) was determined by duplicate culture of 20 μL of each sample, which was serially diluted. The harvested cells were lysed by freezing and thawing three times with vigorous shaking and the crude lysate was analyzed for detection and measurement of the produced LTB by GM1-ELISA. The recombinant LTB concentration was calculated as ng/ 102O2 were added to each well. The reaction was stopped by 2N H2SO4 after 15 minutes and absorbance was read at 450 nm. The amount of rLTB in each sample was determined by interpolation on standard curves generated using the reference LT. Finally, production rate of rLTB was calculated for 10 9 cfu.The rLTB levels were determined and measured by the GM1 ELISA method as previously reported . BrieflynirB promoter in a pKK223 derivative plasmid and then the LTB gene (375 bp) was cloned under the control of this promoter and the final plasmid was named pFSnirB78 -23LTB. The second codon of the native LTB gene was changed from AAT to GAT, which altered Asn to Asp in the second amino acid of the LTB signal peptide. The plasmid was transformed into E. coli DH5 in order to measure promotor activity following restriction analysis of the plasmid.In this study, we constructed a new plasmid by two-step cloning as shown in E. coli/ pFSnirB78-23 produces rLTB when anaerobiosis is solely the inducer. Under anaerobic conditions and also in the absence of any chemical inducer, the amount of rLTB was as high as 1560 ng per 109 cfu  are extensively used for in vitro expression of recombinant proteins in bacteria. Due to the inducer’s stability, additional time and expensive steps to remove the inducers from the final product are required. Also, these promoters are not suitable for anaerobic conditions both in vitro and in vivo where there is not enough oxygen tension for cell growth. It seems that anaerobically inducible promoters such as the nirB promoter are an attractive alternative choice as they have successfully been used by numerous investigators. Various plasmids containing synthetic  E. coli , 15. The strains . nirB promoter has also been used for expression of foreign genes in Salmonella live vaccine strains and has been shown to be an efficient system for immunization  reported by Oxer et al. (14), as shown in Intact native nization . With thpromoter . Secondler  is essential for binding LTB to its receptor (ganglioside GM1) and also for the immunogenicity and adjuvanticity of this molecule.We choose the TAAGGAGG sequence that it is exactly the same as the native promoter in four 3′ nucleotides but with more distance to the ATG codon. During the expression process, our data showed that the synthetic nirB promoter and well tolerated by E. coli (E. coli FNR-dependent transcription is modulated by other regulatory systems (Nar/NarP), which appear to coordinate transcriptional responses with nitrate and nitrite.  of intact native nirB promoter (nirB can anaerobically produce foreign proteins, and removing the chemical inducibility of nirB promoter from its anaerobic activity is possible. In addition, anaerobiosis has no adverse effects on the produced protein. Accordingly, we think the nirB promoter is a suitable system for in vitro expression especially when high oxygen tension might have destructive effects on the recombinant protein. This system is potentially suitable for in vivo delivery of antigens or drugs by live bacterial strains such as salmonella live delivery vaccine strains.In native