Bidirectional Promoter Activity Research Articles

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.

Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation.

Targeted selection-based genome-editing approaches have enabled many fundamental discoveries and are used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette in budding yeast led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all mutant phenotypes detected in cells deleted for DBP1. This behavior resembled the 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target mutant phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that the inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). Divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette-induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests it should be considered in the design and interpretation of experiments using integrated expression cassettes in other eukaryotic systems, including human cells.

Effects of activation of the LINE-1 antisense promoter on the growth of cultured cells

Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~ 17% of the human genome. Although ~ 6000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. The L1 5′ untranslated region has bidirectional promoter activity with a sense promoter driving L1 mRNA production and an antisense promoter (ASP) driving the production of L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using CRISPR-Cas9 technology to evaluate its biological impacts. Activation of the L1 ASP upregulated the expression of L1 ASP-driven ORF0 and enhanced cell growth. Furthermore, the exogenous expression of ORF0 also enhanced cell growth. These results indicate that activation of L1 ASP activity fuels cell growth at least through ORF0 expression. To our knowledge, this is the first report demonstrating the role of the L1 ASP in a biological context. Considering that L1 sequences are desilenced in various tumor cells, our results indicate that activation of the L1 ASP may be a cause of tumor growth; therefore, interfering with L1 ASP activity may be a potential strategy to suppress the growth.

Molecular Characterization of a Pathogen-Inducible Bidirectional Promoter from Hot Pepper (Capsicum annuum).

In hot pepper, the sesquiterpene phytoalexin capsidiol is catalyzed by the two final-step enzymes, a sesquiterpene cyclase (EAS) and a hydroxylase (EAH), which are genetically linked and present as head-to-head orientation in the genome. Transcriptomic analysis revealed that a subset of EAS and EAH is highly induced following pathogen infection, suggesting the coregulation of EAS and EAH by a potential bidirectional activity of the promoter (pCaD). A series of the nested deletions of pCaD in both directions verified the bidirectional promoter activity of the pCaD. Promoter deletion analysis revealed that the 226 bp of the adjacent promoter region of EAS and GCC-box in EAH orientation were determined as critical regulatory elements for the induction of each gene. Based on promoter analyses, we generated a set of synthetic promoters to maximize reporter gene expression within the minimal length of the promoter in both directions. We found that the reporter gene expression was remarkably induced upon infection with Phytophthora capsici, Phytophthora infestans, and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 but not with necrotrophic fungi Botrytis cinerea. Our results confirmed the bidirectional activity of the pCaD located between the head-to-head oriented phytoalexin biosynthetic genes in hot pepper. Furthermore, the synthetic promoter modified in pCaD could be a potential tool for pathogen-inducible expression of target genes for developing disease-resistant crops.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional characterization of an inducible bidirectional promoter from Fusarium oxysporum f. sp. cubense

Bidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5′ ends in close proximity. These genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. This intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. Characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.

PRR11 and SKA2 gene pair is overexpressed and regulated by p53 in breast cancer

Our previous study found that two novel cancer-related genes, PRR11 and SKA2, constituted a classic gene pair that was regulated by p53 and NF-Y in lung cancer. However, their role and regulatory mechanism in breast cancer remain elusive. In this study, we found that the expression levels of PRR11 and SKA2 were upregulated and have a negative prognotic value in breast cancer. Loss-of-function experiments showed that RNAi-mediated knockdown of PRR11 and/or SKA2 inhibited proliferation, migration, and invasion of breast cancer cells. Mechanistic experiments revealed that knockdown of PRR11 and/or SKA2 caused dysregulation of several downstream genes, including CDK6, TPM3, and USP12, etc. Luciferase reporter assays demonstrated that wild type p53 significantly repressed the PRR11-SKA2 bidirectional promoter activity, but not NF-Y. Interestingly, NF-Y was only essential for and correlated with the expression of PRR11, but not SKA2. Consistently, adriamycin-induced (ADR) activation of endogenous p53 also caused significant repression of the PRR11 and SKA2 gene pair expression. Notably, breast cancer patients with lower expression levels of either PRR11 or SKA2, along with wild type p53, exhibited better disease-free survival compared to others with p53 mutations and/or higher expression levels of either PRR11 or SKA2. Collectively, our study indicates that the PRR11 and SKA2 transcription unit might be an oncogenic contributor and might serve as a novel diagnostic and therapeutic target in breast cancer.

Abstract B36: FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer

Abstract Purpose: FOXM1 is located at 12p13.33, which shows copy number gains in the majority of high-grade serous ovarian cancer (HGSC) cases. The 12p13.33 amplicon contains 33 genes, some of which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 overexpression is associated with genomic instability in cancer, whether FOXM1 contributes directly to this phenotype is unclear. We hypothesized that FOXM1 induces DNA replication stress (DRS), resulting in ATR-CHK1 activation. Intriguingly, we noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 activation, is contained within the 12p13.33 amplicon and is arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1/RHNO1 bidirectional promoter, FOXM1/RHNO1 expression, and determined the role of FOXM1/RHNO1 in DRS, the DRS response, and clonogenic growth of HGSC cells. Methods: We used reporter assays to characterize the FOXM1/RHNO1 bidirectional promoter and FOXM1 transcriptional activity. We analyzed copy number, mRNA, and protein expression datasets in TCGA, CCLE, and GTEX. We measured FOXM1/RHNO1 expression in HGSC cell lines and tumors. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to DRS and DNA damage. We knocked down RHNO1 in HGSC cells to determine its contribution to the DRS response and clonogenic growth. We performed Western blot analyses to measure markers of DRS and DNA damage. We characterized FOXM1-induced DNA damage at single-cell resolution using Comet assays and flow cytometry measurement of γ-H2AX. Results: FOXM1/RHNO1 showed copy number gains or amplifications in 58% of TCGA HGSC tumors, and FOXM1/RHNO1 mRNA expression significantly correlated in TCGA HGSC tumors, CCLE HGSC lines, and GTEX normal tissues. The FOXM1/RHNO1 bidirectional promoter showed balanced activity in each direction in FTE and HGSC cells, and bidirectional promoter activity correlated with endogenous mRNA expression. TCGA HGSC data indicated that FOXM1 expression associates with markers of DNA replication, global copy number alterations, and CHK1-Ser345-P, a canonical marker of DRS. In agreement, FOXM1 overexpression in FTE cells induced phosphorylation of ATR-Thr1989, CHK1-Ser317/345, H2AX-Ser139, and the proportion of DNA in Comet tails. In contrast, a DNA-binding domain mutant of FOXM1 did not induce DRS or DNA damage. RHNO1 knockdown attenuated CHK1-Ser345-P in response to DRS and RHNO1 shRNA knockdown or CRISPR-Cas9 knockout severely reduced HGSC cell clonogenic growth. Importantly, the effects of RHNO1 disruption were rescued by WT RHNO1 expression, but not by a RHNO1 mutant defective for interaction with the 9-1-1 complex. Conclusion: Our data reveal that FOXM1/RHNO1 share a bidirectional promoter and that this may account for their frequent coexpression in HGSC. FOXM1 induces DRS, and this is dependent on its ability to bind DNA. Conversely, RHNO1 contributes to ATR-CHK1 signaling and promotes the clonogenic growth of HGSC cells. We hypothesize that balanced FOXM1/RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1/RHNO1 on DRS and genomic instability in model systems. Finally, our data suggest that the ATR-CHK1 pathway is a synthetic lethal vulnerability in HGSC tumors that overexpress FOXM1. Citation Format: Carter J. Barger, Linda Chee, Connor Branick, Kunle Odunsi, Ronny Drapkin, Adam R. Karpf. FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B36.

UHRF1 regulates CDH1 via promoter associated non-coding RNAs in prostate cancer cells

Non-coding RNAs (ncRNAs) transcribed from the promoter and the downstream region can affect the expression of the corresponding coding genes. It has been shown that sense-directed ncRNAs arising from the promoter region of the E-cadherin gene (CDH1) mediate its repression. Here, we show that an antisense-directed ncRNA (paRCDH1-AS) transcribed from the CDH1 promoter is necessary for its expression. paRCDH1-AS acts as a hooking scaffold by recruiting the epigenetic regulators, UHRF1, DNMT3A, SUV39H1 and SUZ12, involved in CDH1 repression. The binding of epigenetic regulators to paCRDH1-AS, indeed, prevents their localization to the chromatin on CDH1 promoter. Moreover, paRCDH1-AS silencing induces CDH1 repression and a switch of the epigenetic profile on the promoter towards a more closed chromatin. Using bioinformatic and experimental approaches we defined that the promoter of the paRCDH1-AS is shared with the E-cadherin gene, showing a bidirectional promoter activity. We found that UHRF1 controls both CDH1 and paRCDH1-AS by directly binding this bidirectional promoter region. Our study provides evidences, for the first time, that UHRF1 recruitment can be affected by promoter-associated non-coding RNAs, opening new perspective regarding the role of UHRF1 in these complex regulatory networks.

Analysis of Promoters of Arabidopsis thaliana Divergent Gene Pair SERAT3;2 and IDH-III Shows SERAT3;2 Promoter is Nested Within the IDH-III Promoter.

Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161bp shown in TAIR annotation to 512bp. GUS analysis of transgenic A. thaliana plants carrying the 691bp fragment (512bp intergenic region plus 5' UTR of both the genes) linked to uidA gene revealed that SERAT3;2 promoter drives gene expression in the tapetum, whereas IDH-III promoter functions specifically in microspores/pollen. Serial 5' deletion of the 691bp fragment showed SERAT3;2 promoter extends up to -355 position, whereas IDH-III promoter encompasses the 512bp intergenic region. In transgenics, uidA transcript levels were lower than native SERAT3;2 and IDH-III transcripts indicating presence of additional cis regulatory elements beyond the 691bp fragment. The present study demonstrated for the first time occurrence of a nested promoter in plants and identified a novel bidirectional promoter capable of driving gene expression in tapetum and microspores/pollen.

Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression

ABSTRACTDifferentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study—owing to their myogenic DMRs—overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage.

INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes.

The conserved INO80 chromatin remodeling complex is involved in regulation of DNA damage repair, replication and transcription. It is commonly recruited to the transcription start region and contributes to the establishment of promoter-proximal nucleosomes. We find a substantial influence of INO80 on nucleosome dynamics and gene expression during stress induced transcription. Transcription induced by osmotic stress leads to genome-wide remodeling of promoter proximal nucleosomes. INO80 function is required for timely return of evicted nucleosomes to the 5΄ end of induced genes. Reduced INO80 function in Arp8-deficient cells leads to correlated prolonged transcription and nucleosome eviction. INO80 and the related complex SWR1 regulate incorporation of the H2A.Z isoform at promoter proximal nucleosomes. However, H2A.Z seems not to influence osmotic stress induced gene regulation. Furthermore, we show that high rates of transcription promote INO80 recruitment to promoter regions, suggesting a connection between active transcription and promoter proximal nucleosome remodeling. In addition, we find that absence of INO80 enhances bidirectional promoter activity at highly induced genes and expression of a number of stress induced transcripts. We suggest that INO80 has a direct repressive role via promoter proximal nucleosome remodeling to limit high levels of transcription in yeast.

A comprehensive epigenome map of Plasmodium falciparum reveals unique mechanisms of transcriptional regulation and identifies H3K36me2 as a global mark of gene suppression.

BackgroundRole of epigenetic mechanisms towards regulation of the complex life cycle/pathogenesis of Plasmodium falciparum, the causative agent of malaria, has been poorly understood. To elucidate stage-specific epigenetic regulation, we performed genome-wide mapping of multiple histone modifications of P. falciparum. Further to understand the differences in transcription regulation in P. falciparum and its host, human, we compared their histone modification profiles.ResultsOur comprehensive comparative analysis suggests distinct mode of transcriptional regulation in malaria parasite by virtue of poised genes and differential histone modifications. Furthermore, analysis of histone modification profiles predicted 562 genes producing anti-sense RNAs and 335 genes having bidirectional promoter activity, which raises the intriguing possibility of RNA-mediated regulation of transcription in P. falciparum. Interestingly, we found that H3K36me2 acts as a global repressive mark and gene regulation is fine tuned by the ratio of activation marks to H3K36me2 in P. falciparum. This novel mechanism of gene regulation is supported by the fact that knockout of SET genes (responsible for H3K36 methylation) leads to up-regulation of genes with highest occupancy of H3K36me2 in wild-type P. falciparum. Moreover, virulence (var) genes are mostly poised and marked by a unique set of activation (H4ac) and repression (H3K9me3) marks, which are mutually exclusive to other Plasmodium housekeeping genes.ConclusionsOur study reveals unique plasticity in the epigenetic regulation in P. falciparum which can influence parasite virulence and pathogenicity. The observed differences in the histone code and transcriptional regulation in P. falciparum and its host will open new avenues for epigenetic drug development against malaria parasite.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0029-1) contains supplementary material, which is available to authorized users.

A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells.

Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.

A novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities.

Long-range enhancers of transcription are a key component of the genomic regulatory architecture. Recent studies have identified bi-directionally transcribed RNAs emanating from these enhancers known as eRNAs. However, it remains unclear how tightly coupled eRNA production is with enhancer activity. Through our systematic search for long-range elements that interact with the interferon-β gene, a model system for studying inducible transcription, we have identified a novel enhancer, which we have named L2 that regulates the expression of interferon-β. We have demonstrated its virus-inducible enhancer activity by analyzing epigenomic profiles, transcription factor association, nascent RNA production and activity in reporter assays. This enhancer exhibits intimately linked virus-inducible enhancer and bidirectional promoter activity that is largely dependent on a conserved Interferon Stimulated Response Element and robustly generates virus inducible eRNAs. Notably, its enhancer and promoter activities are fully retained in reporter assays even upon a complete elimination of its associated eRNA sequences. Finally, we show that L2 regulates IFNB1 expression by siRNA knockdown of eRNAs, and the deletion of L2 in a BAC transfection assay. Thus, L2 is a novel enhancer that regulates IFNB1 and whose eRNAs exert significant activity in vivo that is distinct from those activities recapitulated in the luciferase reporter assays.

A Conserved Noncoding Sequence Can Function as a Spermatocyte-Specific Enhancer and a Bidirectional Promoter for a Ubiquitously Expressed Gene and a Testis-Specific Long Noncoding RNA

Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5′-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.

Construction of a reporter vector for analysis of bidirectional transcriptional activity of retrovirus LTR

To study the transcriptional activity of the HIV-1 LTR, we constructed a vector containing Renilla and Firefly luciferase genes under the control of the LTR (wild-type or mutated version) and oriented in a manner that allowed them to be transcribed in opposite directions. We found that the HIV-1 LTR acted as a bidirectional promoter, which activity was controlled by NF-κB- and Sp1-binding sites in both orientations. We next analyzed with this reporter vector the bidirectional promoter activity of the HTLV-1 LTR and showed that this LTR also possessed a bidirectional transcriptional activity. Interestingly, Sp1-binding elements were also involved in the control of HTLV-1 bidirectional transcription. Moreover, both retroviral trans-activators, Tat and Tax, could preferentially activate sense transcription with no or limited effect on the extent of antisense transcription. We also cloned into this plasmid the MLV LTR and found that the LTR of a simple retrovirus also possessed bidirectional transcriptional activity. This reporter vector represents a powerful tool to analyze the bidirectional transcriptional activity of retrovirus LTRs.

Bovine TWINKLE and mitochondrial ribosomal protein L43 genes are regulated by an evolutionary conserved bidirectional promoter

TWINKLE is a mitochondrial DNA helicase playing an important role in mitochondrial DNA replication. In human, mutations in this gene cause progressive external ophtalmoplegia and mitochondrial DNA depletion syndrome-7. TWINKLE is well conserved among multicellular eukaryotes and is believed to be a key regulator of mitochondrial DNA copy number in mammals.Despite its involvement in several diseases and its important function in mitochondrial DNA metabolism, nothing is known about the regulation of the expression of TWINKLE. We have analysed the 5′-flanking genomic region of the bovine TWINKLE gene and found it was localised adjacent to the MRPL43 gene in a head-to-head orientation, suggesting that both genes are regulated by a shared bidirectional promoter. The bovine 75-bp long intergenic region shows substantial homology across different species and contains several conserved putative transcription factor binding sites. A TATA box, however, was lacking.Using a dual fluorescent reporter system and transient transfection assays, we have analysed the bovine intergenic region between TWINKLE and MRPL43. This small genomic fragment showed a bidirectional promoter activity. As the TWINKLE/MRPL43 bidirectional promoter tested was highly conserved, it is likely that the results we obtained here in cattle may be extended to the other species.

Major cis-regulatory elements for rice bidirectional promoter activity reside in the 5′-untranslated regions

Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by <1kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5′-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond −461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

Identification and characterization of a noncoding RNA at the mouse Pcna locus.

AK007836 encodes a noncoding RNA (ncRNA) consisting of 2 exons. Since AK007836 is located just upstream of Pcna and transcribed in the opposite direction to that of Pcna, we analyzed its expression pattern. Both ncRNA and Pcna expressions were detected in in vitro and in vivo cells, showing a positive correlation. A 177 bp region separating the first exons of Pcna and AK007836 has a bidirectional promoter activity. When the expression of ncRNA was reduced by siRNA, Pcna expression was also reduced in normal cells, but not in cancer cells. These results suggest that the ncRNA is divergently transcribed from the bidirectional promoter, positively regulating the neighboring protein-coding Pcna gene transcription, and this regulatory function is somehow disrupted in cancer cells.