Primary Transcription Start Site Research Articles

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

BackgroundPathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells.ResultsWe performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sR’s), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5′-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Seventy-five coding genes exhibited different TSSs depending on the host environment. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche.ConclusionsIn sum, the findings of this study authenticate the presence of novel Rc_sR’s in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile.

The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5′-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5′ untranslated regions. By following an in silico iterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors of C. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile, we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

BackgroundShigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches.ResultsWe have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new hybrid strategy to prepare gapless, highly accurate genome sequences, which also cover AT-rich tracks or repetitive sequences that are transcribed. Furthermore, we have performed genome-wide analysis of transcriptional start sites (TSS) and determined the length of 5′ untranslated regions (5′-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6723 primary TSS (pTSS) and 7328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam.mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) databases to use their analysis tools in the S. flexneri 5a M90T genome.ConclusionsWe provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. Further, we present a new hybrid strategy to prepare gapless, highly accurate genome sequences. Unlike currently used hybrid strategies combining long- and short-read DNA sequencing technologies to maximize accuracy, our workflow using long-read DNA sequencing and short-read RNA sequencing provides the added value of using non-redundant technologies, which yield distinct, exploitable datasets.

Defining the Transcriptional and Post-transcriptional Landscapes of Mycobacterium smegmatis in Aerobic Growth and Hypoxia.

The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5′-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense, or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

Cap‐seq reveals complicated miRNA transcriptional mechanisms in C. elegans and mouse

BackgroundMicroRNAs (miRNAs) regulate target gene expression at post‐transcriptional level. Intense research has been conducted for miRNA identification and the target finding. However, much less is known about the transcriptional regulation of miRNA genes themselves. Recently, a special group of pre‐miRNAs that are produced directly by transcription without Drosha processing were validated in mouse, indicating the complexity of miRNA biogenesis.MethodsIn this work, we detect clusters of aligned Cap‐seq reads to find the transcription start sites (TSSs) for intergenic miRNAs and study their transcriptional regulation in Caenorhabditis elegans and mouse.ResultsIn both species, we have identified a class of special pre‐miRNAs whose 5′ ends are capped, and are most probably generated directly by transcription. Furthermore, we distinguished another class of special pre‐miRNAs that are 5′‐capped but are also part of longer primary miRNAs, suggesting they may have more than one transcription mechanism. We detected multiple cap reads peaks within miRNA clusters in C. elegans. We surmised that the miRNAs in a cluster may either be transcribed independently or be re‐capped during the microprocessor cleavage process. We also observed that H3K4me3 and Pol II are enriched at those identified miRNA TSSs.ConclusionsThe Cap‐seq datasets enabled us to annotate the primary TSSs for miRNA genes with high resolution. Special class of 5′‐capped pre‐miRNAs have been identified in both C. elegans and mouse. The capping patter of miRNAs in a cluster indicate t hat clustered miRNA transcripts probably undergo a re‐capping procedure during the microprocessor cleavage process.

Nitrogen cost minimization is promoted by structural changes in the transcriptome of N-deprived Prochlorococcus cells

Prochlorococcus is a globally abundant marine cyanobacterium with many adaptations that reduce cellular nutrient requirements, facilitating growth in its nutrient-poor environment. One such genomic adaptation is the preferential utilization of amino acids containing fewer N-atoms, which minimizes cellular nitrogen requirements. We predicted that transcriptional regulation might further reduce cellular N budgets during transient N limitation. To explore this, we compared transcription start sites (TSSs) in Prochlorococcus MED4 under N-deprived and N-replete conditions. Of 64 genes with primary and internal TSSs in both conditions, N-deprived cells initiated transcription downstream of primary TSSs more frequently than N-replete cells. Additionally, 117 genes with only an internal TSS demonstrated increased internal transcription under N-deprivation. These shortened transcripts encode predicted proteins with an average of 21% less N content compared to full-length transcripts. We hypothesized that low translation rates, which afford greater control over protein abundances, would be beneficial to relatively slow-growing organisms like Prochlorococcus. Consistent with this idea, we found that Prochlorococcus exhibits greater usage of glycine–glycine motifs, which causes translational pausing, when compared to faster growing microbes. Our findings indicate that structural changes occur within the Prochlorococcus MED4 transcriptome during N-deprivation, potentially altering the size and structure of proteins expressed under nutrient limitation.

A systemic identification approach for primary transcription start site of Arabidopsis miRNAs from multidimensional omics data.

The 22-nucleotide non-coding microRNAs (miRNAs) are mostly transcribed by RNA polymerase II and are similar to protein-coding genes. Unlike the clear process from stem-loop precursors to mature miRNAs, the primary transcriptional regulation of miRNA, especially in plants, still needs to be further clarified, including the original transcription start site, functional cis-elements and primary transcript structures. Due to several well-characterized transcription signals in the promoter region, we proposed a systemic approach integrating multidimensional "omics" (including genomics, transcriptomics, and epigenomics) data to improve the genome-wide identification of primary miRNA transcripts. Here, we used the model plant Arabidopsis thaliana to improve the ability to identify candidate promoter locations in intergenic miRNAs and to determine rules for identifying primary transcription start sites of miRNAs by integrating high-throughput omics data, such as the DNase I hypersensitive sites, chromatin immunoprecipitation-sequencing of polymerase II and H3K4me3, as well as high throughput transcriptomic data. As a result, 93% of refined primary transcripts could be confirmed by the primer pairs from a previous study. Cis-element and secondary structure analyses also supported the feasibility of our results. This work will contribute to the primary transcriptional regulatory analysis of miRNAs, and the conserved regulatory pattern may be a suitable miRNA characteristic in other plant species.

Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315.

BackgroundBurkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aims to annotate transcription start sites and identify novel transcripts on a whole genome scale.MethodsRNA extracted from B. cenocepacia J2315 biofilms was analysed by differential RNA-sequencing and the resulting dataset compared to data derived from conventional, global RNA-sequencing. Transcription start sites were annotated and further analysed according to their position relative to annotated genes.ResultsFour thousand ten transcription start sites were mapped over the whole B. cenocepacia genome and the primary transcription start site of 2089 genes expressed in B. cenocepacia biofilms were defined. For 64 genes a start codon alternative to the annotated one was proposed. Substantial antisense transcription for 105 genes and two novel protein coding sequences were identified. The distribution of internal transcription start sites can be used to identify genomic islands in B. cenocepacia. A potassium pump strongly induced only under biofilm conditions was found and 15 non-coding small RNAs highly expressed in biofilms were discovered.ConclusionsMapping transcription start sites across the B. cenocepacia genome added relevant information to the J2315 annotation. Genes and novel regulatory RNAs putatively involved in B. cenocepacia biofilm formation were identified. These findings will help in understanding regulation of B. cenocepacia biofilm formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1993-3) contains supplementary material, which is available to authorized users.

Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis.

BackgroundProkaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.ResultsSequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.ConclusionThe results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-684) contains supplementary material, which is available to authorized users.

Transmission of an Oxygen Availability Signal at the Salmonella enterica Serovar Typhimurium fis Promoter

The nucleoid-associated protein FIS is a global regulator of gene expression and chromosome structure in Escherichia coli and Salmonella enterica. Despite the importance of FIS for infection and intracellular invasion, very little is known about the regulation of S. enterica fis expression. Under standard laboratory growth conditions, fis is highly expressed during rapid growth but is then silenced as growth slows. However, if cells are cultured in non-aerated conditions, fis expression is sustained during stationary phase. This led us to test whether the redox-sensing transcription factors ArcA and FNR regulate S. enterica fis. Deletion of FNR had no detectable effect, whereas deletion of ArcA had the unexpected effect of further elevating fis expression in stationary phase. ArcA required RpoS for induction of fis expression, suggesting that ArcA indirectly affects fis expression. Other putative regulators were found to play diverse roles: FIS acted directly as an auto-repressor (as expected), whereas CRP had little direct effect on fis expression. Deleting regions of the fis promoter led to the discovery of a novel anaerobically-induced transcription start site (Pfis-2) upstream of the primary transcription start site (Pfis-1). Promoter truncation also revealed that the shortest functional fis promoter was incapable of sustained expression. Moreover, fis expression was observed to correlate directly with DNA supercoiling in non-aerated conditions. Thus, the full-length S. enterica fis promoter region may act as a topological switch that is sensitive to stress-induced duplex destabilisation and up-regulates expression in non-aerated conditions.

Genome-wide Mapping of Transcriptional Start Sites Defines an Extensive Leaderless Transcriptome in Mycobacterium tuberculosis

SummaryDeciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades. We have combined RNA sequencing (RNA-seq) of 5′ triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5′ UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.

Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence

The bacterium Legionella pneumophila is found ubiquitously in aquatic environments and can cause a severe pneumonia in humans called Legionnaires' disease. How this bacterium switches from intracellular to extracellular life and adapts to different hosts and environmental conditions is only partly understood. Here we used RNA deep sequencing from exponentially (replicative) and post exponentially (virulent) grown L. pneumophila to analyze the transcriptional landscape of its entire genome. We established the complete operon map and defined 2561 primary transcriptional start sites (TSS). Interestingly, 187 of the 1805 TSS of protein-coding genes contained tandem promoters of which 93 show alternative usage dependent on the growth phase. Similarly, over 60% of 713 here identified ncRNAs are phase dependently regulated. Analysis of their conservation among the seven L. pneumophila genomes sequenced revealed many strain specific differences suggesting that L. pneumophila contains a highly dynamic pool of ncRNAs. Analysis of six ncRNAs exhibiting the same expression pattern as virulence genes showed that two, Lppnc0584 and Lppnc0405 are indeed involved in intracellular growth of L. pneumophila in A. castellanii. Furthermore, L. pneumophila encodes a small RNA named RsmX that functions together with RsmY and RsmZ in the LetA-CsrA regulatory pathway, crucial for the switch to the virulent phenotype. Together our data provide new insight into the transcriptional organization of the L. pneumophila genome, identified many new ncRNAs and will provide a framework for the understanding of virulence and adaptation properties of L. pneumophila.

Abstract 4085: Bidirectional noncoding RNAs mediate rRNA processing and cell death in human lung adenocarcinoma cells

Abstract Increase in ribosome biogenesis is a characteristic of proliferating cells and may be causative in tumorigenesis. Significant increases in basal levels of several primary precursor transcripts during ribosomal RNA (rRNA) processing and of a cis antisense noncoding rRNA (AS-ncrRNA) near the main transcription start site were detected in human lung adenocarcinoma cells (H441 and A549) compared with non-transformed peripheral lung epithelial cells (HPL). A cis sense noncoding rRNA (S-ncrRNA) was also discovered but its levels showed cell-specific changes. It was hypothesized that a high level of the AS-ncrRNA is required to maintain the tumor phenotype. The functions of the AS-ncrRNA and S-ncrRNA were assessed using complementary oligonucleotides, termed −86S and −84AS (negative numbers correspond to the target locations in reference to the primary transcription start site) and intended for inhibiting the above targets, respectively. The levels of precursor rRNAs and both ncrRNAs in cells transfected with the −86S and −84AS oligonucleotides were measured using reverse transcription and quantitative polymerase chain reaction. Cell phenotypes and apoptotic cell death were also examined. A two-hit treatment with −86S 24 hrs apart induced cell detachment predominantly in H441 and A549 cancer cells. Detached cancer cells showed several hundred- to a thousand-fold upregulation of S-ncrRNA over the vehicle-treated controls while the S-nrcRNA in rare floating HPL cells was only increased by about 20 fold. Precursor rRNAs and AS-ncrRNA were also upregulated but to a lesser extent after −86S treatment in both adherent and detached cells. For the −84AS treatment, detached cells were fewer than after the −86S treatment; however those few floating cancer cells also exhibited several hundred-fold increase of the S-ncrRNA. Although the mechanism of upregulation is unclear, the S-ncrRNA may possibly act as a cell death regulator. Flow cytometric assays to detect fragmented DNA in cells after 24-hr and 48-hr treatments showed an apparent increase in apoptosis (2- to 4-fold) for the −86S treatment compared to −84AS and scrambled control. Further morphological analysis by transmission electron microscopy identified large and abundant autophagosomes and vacuoles preferentially in cancer cells, suggesting another possible cell death mechanism mediated by the oligonucleotide targeting AS-ncrRNA. In sum, rRNA processing and cis bidirectional noncoding rRNAs may form an interconnected network to maintain tumor phenotype. Interruption of this network by oligonucleotides targeting AS-ncrRNA activates massive elevation of the S-ncRNA level and sensitizes cancer cells preferentially for apoptotic and autophagic cell death. Targeting of AS-ncrRNA could have potential for human lung cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4085.

Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

Cyclin D1 expression is elevated and Wnt10b is repressed by cAMP during the first few hours of adipogenesis. cAMP-responsive element-binding protein (CREB) is a primary target for cAMP signaling, and we have shown that activation of CREB promotes adipogenesis and adipocyte survival. Here we tested the impact of CREB on expression of cyclin D1 and wingless-related mouse mammary tumor virus integration site 10b (Wnt10b) in 3T3-L1 cells. Forced depletion of CREB blocked Bt(2)cAMP-stimulated cyclin D1 expression and basal Wnt10b gene expression. Two CREB-binding sites were identified in the Wnt10b promoter region. Ablation of either site partially blocked promoter activity, while mutation of both sites completely suppressed promoter activity. These results suggest that CREB activates transcription from both the cyclin D1 and Wnt10b gene promoters. What accounts for the differential regulation of cyclin D1 and Wnt10b genes by cAMP? Chromatin immunoprecipitation revealed CREB bound to the Wnt10b promoter in untreated preadipocytes but not following treatment with Bt(2)cAMP. CREB binding to the cyclin D1 promoter was detected in untreated cells and post-Bt(2)cAMP. Differences between CREB binding to the two genes correlated with increasing methylation of the Wnt10b promoter following Bt(2)cAMP treatment, whereas no methylation of the cyclin D1 promoter was observed. Treatment of cells with the methylase inhibitor 5-azacytidine restored CREB binding to the Wnt10b gene promoter and prevented the inhibition of Wnt10b RNA expression by Bt(2)cAMP. We conclude that cAMP stimulates phosphorylation and binding of CREB to the cyclin D1 gene promoter. Simultaneously, hypermethylation of the Wnt10b gene promoter suppresses binding of CREB, allowing adipogenesis to proceed.

The proximal promoter governs germ cell‐specific expression of the mouse glutathione transferase mGstm5 gene

To explain the tissue-selective expression patterns of a distinct subclass of glutathione S-transferase (GST), transgenic mice expressing EGFP under control of a 2 kb promoter sequence in the 5'-flanking region of the mGstm5 gene were produced. The intent of the study was to establish whether the promoter itself or whether posttranscriptional mechanisms, particularly at the levels of mRNA translation and stability or protein targeting, based on unique properties of mGSTM5, determine the restricted expression pattern. Indeed, the transgene expression was limited to testis as the reporter was not detected in somatic tissues such as brain, kidney or liver, indicating that the mGstm5 proximal promoter is sufficient to target testis-specific expression of the gene. EGFP expression was also more restricted vis-a-vis the natural mGstm5 gene and exclusively found in germ but not in somatic cells. Real-time quantitative PCR (qPCR) data were consistent with alternate transcription start sites in which the promoter region of the natural mGstm5 gene in somatic cells is part of exon 1 of the germ cell transcript. Thus, the primary transcription start site for mGstm5 is upstream of a TATA box in testis and downstream of this motif in somatic cells. The 5' flanking sequence of the mGstm5 gene imparts germ cell-specific transcription.

Primary structure of guinea pig preprodynorphin and preproenkephalin mRNAs: multiple transcription initiation sites for preprodynorphin

Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A (1–17) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5′ end. This sequence has high homology to the 5′ flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160–190 nt 5′ with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.

PTAR-encoded proteins in plasmid partitioning.

Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465-478, 1987). However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host Escherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

Isolation, characterization, and differential expression of the murine Sox-2 promoter.

Sox proteins are expressed at many stages of development and in numerous tissues. The transcription factor Sox-2 is first expressed throughout the inner cell mass and subsequently becomes localized to the primitive ectoderm, developing central nervous system, and the lens. Sox-2 is also highly expressed in F9 embryonal carcinoma cells, but becomes undetectable following differentiation of these cells. In this study, we have isolated, sequenced, and performed the first characterization of the Sox-2 promoter of any species. Approximately 2kb of the Sox-2 5'-flanking region has been sequenced and the primary transcription start site mapped by primer extension analysis. Additionally, two positive regulatory regions within the promoter region have been identified. We also show that expression of Sox-2 promoter/reporter gene constructs is reduced in differentiated EC cells as compared to their undifferentiated counterparts. Furthermore, we have identified a consensus inverted CCAAT box motif present in the Sox-2 promoter. Mutagenesis of this site significantly reduces the expression of Sox-2 promoter/reporter constructs. We also demonstrate that this CCAAT box motif can bind the trimeric transcription factor NF-Y.

Promoter characterization and constitutive expression of the Escherichia coli gcvR gene.

The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the gcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the bcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.