Functional Cis-acting Elements Research Articles

Identification of cis-acting elements upstream of regR gene in streptococcus pneumoniae

The identification and characterization of functional cis-acting elements is of fundamental importance for comprehending the regulatory mechanisms of gene transcription and bacterial pathogenesis. The transcription factor RegR has been demonstrated to control both competence and virulence in Streptococcus pneumoniae. Despite the clear contribution of RegR to these pathways, the mechanisms underlying its transcriptional regulation remain poorly understood. In this study, we conducted mutational analysis, gene dissection and luciferase activity assays to characterize the cis-elements situated upstream of the regR gene. Our findings revealed that a 311 bp 3′-terminal DNA sequence of the spd0300 gene represents a central region of the upstream cis-acting element of regR. Further investigations identified two structurally similar enhancer-like sequences within this region which feature prominently in the regulation of regR transcription. Furthermore, employing DNA pull-down assays allowed us to enrich the trans-acting factors with the potential to interact with these cis-acting elements. Notably, we found that the competence regulator ComE was implicated in the regulation of regR transcription, a finding which was corroborated by electrophoretic mobility shift assays (EMSA) and quantitative real-time PCR analyses (qRT-PCR). Taken together, our data thus provide fresh insight into the transcriptional regulation of regR.

AtENO2 functions in the development of male gametophytes in Arabidopsis thaliana

Pollen fertility is an important factor affecting the seed setting rate and seed yield of plants. The Arabidopsis thaliana enolase gene ENO2 (AtENO2) can affect the pollen morphology, germination, and pollen tube growth. AtENO2 encodes two proteins AtENO2 and AtMBP-1. To examine the effect of AtENO2 protein on pollen development, the 2nd ATG of the AtENO2 coding sequence for AtMBP-1 was mutated by site-directed mutagenesis, and transgenic plants expressing only AtENO2 but not AtMBP-1 were obtained. Phenotypic analysis indicated that AtENO2 was essential in the pollen development. The mechanisms of AtENO2 on pollen development were analyzed. AtENO2 can affect development of the pollen intine, and the mechanism may be that AtENO2 regulated the methyl esterification of pectin in pollen intine through ARF3 and AtPMEI-pi. The -734 ∼ -573 sequence of AtENO2 promoter is the main transcriptional regulatory region of AtENO2 affecting pollen development. The functional cis-acting element may be GTGANTG10(GTGA), and the trans-acting factors may be KAN, AS2 and ARF3/ETT. Moreover, the deletion of AtENO2 can cause significant difference in the expression of multiple genes related to pollen exine development. These results are useful for further studying the function of AtENO2 and exploring the mechanism of plant pollen development.

The RNA Architecture of the SARS-CoV-2 3'-Untranslated Region.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.

Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication.

There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5’ and 3’ untranslated regions and three open reading frames (ORF). HEV genome has 5’ cap and 3’ poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains cis-acting elements that can be recognized by the virally encoded polymerase as “self” for replication. To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific “signal” that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector.

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease.

A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-β pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D', 0.97; r(2), 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. The coronary artery disease-associated rs17293632C>T single nucleotide polymorphism represents a novel functional cis-acting element at the SMAD3 locus. The protective (T) allele of rs17293632 disrupts a consensus AP-1 binding site in a SMAD3 intron 1 enhancer, reduces enhancer activity and SMAD3 expression, altering human arterial smooth muscle cell proliferation.

Functional characterization and expression study of sugarcane MYB transcription factor gene PEaMYBAS1 promoter from Erianthus arundinaceus that confers abiotic stress tolerance in tobacco

This work provides a thorough understanding about the function ofcis-acting elements regarding drought, salt, cold and wounding stress.

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica.

Pollen-specific expression. Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5' deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5' promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504 bp), OSIPP3-∆2 (968 bp), OSIPP3-∆3 (388 bp) and OSIPP3-∆4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated that they were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

Isolation of the Chinese rose sHSP gene promoter and its differential regulation analysis in transgenic Arabidopsis plants

In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected under other abiotic stresses. Deletion analysis indicated that the region from -178 to -771 was essential for the promoter's response to high temperature.

Comparative analysis of LEA‐like 11‐24 gene expression and regulation in related plant species within the Linderniaceae that differ in desiccation tolerance

The resurrection plant Craterostigma plantagineum is able to withstand desiccation of its vegetative tissues and is found in areas with variable water availability. The closely related species Lindernia brevidens and Lindernia subracemosa are both endemic to montane rainforests of coastal Africa, but remarkably L. brevidens is tolerant to desiccation. We studied the regulation of the desiccation-related LEA-like 11-24 gene at multiple levels in closely related species in order to investigate the conservation of mechanisms involved in desiccation tolerance. The dehydration-responsive transcription of the LEA-like 11-24 gene is differentially regulated in these plants. Comparison of the LEA-like 11-24 core promoter regions revealed that promoters have different activities, but some functional cis-acting elements are conserved between species. Upon dehydration, LEA-like 11-24 proteins are phosphorylated at different levels and phosphorylation sites are not conserved among the three LEA-like 11-24 proteins. Differences in the regulation of the LEA-like 11-24 gene in the studied plant species appear to be the result of mutations that occurred during evolution. We postulate that L. brevidens will eventually lose the ability to survive vegetative desiccation, given that this trait appears not to be essential for survival.

Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

Cis-acting elements regulating the placenta-specific promoter of the bovine Cyp19 gene.

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

Characterization of an NF-1/CTF Family Member as a Functional Activator of the Mouse Mammary Tumor Virus Long Terminal Repeat 5′ Enhancer

The long terminal repeat of the mouse mammary tumor virus restricts virus expression primarily to the mammary epithelium. The extreme 5' end of the long terminal repeat contains an enhancer that has been associated with tissue-specific expression of the virus. A total of six functional cis-acting elements have been identified in the enhancer. Although proteins binding to these elements have been reported, only one has been identified; this factor, mp5, is identical or closely related to the transcription factor AP-2 (Mellentin-Michelotti, J., John, S., Pennie, W. D., Williams, T., and Hager, G. L. (1994) J. Biol. Chem. 269, 31983-31990). The other factors are hitherto unidentified and poorly described. We report here the characterization of another of the six elements, previously referred to as the F3 site (Mink, S., Hartig, E., Jennewein, P., Doppler, W., and Cato, A. C. (1992) Mol. Cell Biol. 12, 4906-4918). We show that the F3 binding activity and AP-2 act synergistically to enhance mouse mammary tumor virus-directed transcription, but only in the presence of glucocorticoid hormone. The F3 element has an NF-1-like half-site, but the activity recognizing this element has binding characteristics distinct from the NF-1/CTF family as well as the rest of the CCAAT-binding proteins. We conclude that the F3 activity represents a new member of the NF-1/CTF family.

Cell-specific function of cis-acting elements in the regulation of human alcohol dehydrogenase 5 gene expression and effect of the 5′-nontranslated region.

Cell-specific function of cis-acting elements in the regulation of human alcohol dehydrogenase 5 gene expression and effect of the 5′-nontranslated region.

Cell type-specific regulation of transcription by cyclic adenosine 3,'5'-monophosphate-responsive elements within the calcitonin promoter.

A cAMP-induced enhancer was previously mapped to nucleotides -255 to -85 of the calcitonin (CT) gene 5'-flanking DNA. To determine the functional cis-acting elements within this region, we transfected medullary thyroid carcinoma (MTC) cells with CT 5'-flanking DNA/GH fusion genes containing potential cAMP-responsive elements and assessed their transcriptional activities with and without cAMP. In CT-expressing MTC cells (the TT line), we identified by deletions and point mutations three transcriptionally active motifs: a cAMP-responsive element (CRE), TGACGTCA, at -253 to -246, and a hybrid site containing a CRE-like element (CREL; TGACCTCA, -169 to -162) adjacent to an equally transcriptionally active octamer (O) sequence (ATG-CAAAT, -161 to -154). These three motifs acted synergistically and their transcriptional activity was completely dependent on cAMP. In HeLa cells their synergistic activity was more constitutive than cAMP induced, whereas in CT-negative MTC cells (the RO-D81-1 line) these motifs were inactive. Gel mobility shift assays with antibodies against CRE-binding protein (CREB) and activating transcription factor 1 (ATF-1) showed that both CREB and ATF-1 interacted with the CRE in MTC cells, whereas in HeLa cells only ATF-1 bound to the CRE. Specific binding to the CREL/O motif was detectable in extracts from tumors induced by injection of TT cells but not in extracts from any of the three cultured cell lines. We conclude that cAMP-induced transcription of the CT gene is modulated in a cell-specific manner by the CRE and the CREL/O elements.

Functional interactions of peroxisome proliferator-activated receptor, retinoid-X receptor, and Sp1 in the transcriptional regulation of the acyl-coenzyme-A oxidase promoter.

Peroxisome proliferator-activated receptor (PPARs) are members of the nuclear receptor superfamily. For transcriptional activation of their target genes, PPARs heterodimerize with the retinoid-X receptor (RXR). The convergence of the PPAR and RXR signaling pathways has been shown to have an important function in lipid metabolism. The promoter of the gene encoding the acyl-coenzyme-A oxidase (ACO), the rate-limiting enzyme in peroxisomal beta-oxidation of fatty acids, is a target site of PPAR action. In this study, we examined the role and the contribution of both cis-and trans-acting factors in the transcriptional regulation of this gene using transient transfections in insect cells. We identified several functional cis-acting elements present in the promoter of the ACO gene and established that PPAR-dependent as well as PPAR-independent mechanisms can activate the ACO promoter in these cells. We show that the PPAR/RXR heterodimer exerts its effect through two response elements within the ACO promoter, in synergy with the transcription factor Sp1 via five Sp1-binding sites. Furthermore, this functional interaction also occurs when Sp1 is co-expressed with PPAR or RXR alone, indicating that activation can occur independently of PPAR/RXR heterodimers.

Novel cis-acting elements in the human platelet-derived growth factor B-chain core promoter that mediate gene expression in cultured vascular endothelial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant constitutively expressed by a variety of normal and transformed cells. Transient transfection and deletion analysis of the human c-sis proto-oncogene in cultured vascular endothelial cells revealed a minimal core promoter region extending 82 base pairs upstream from the TATA box. Two novel and functional cis-acting elements were identified within the core that share considerable sequence homology with consensus binding elements for transacting factors of the ETS class and those involved in AP-1 complexes. Deletion or mutation of either the ETS-like site or the AP-1-like site resulted in significant attenuation in the ability of the core to drive transcription. Electrophoretic mobility shift assays revealed that proteins from bovine aortic and human umbilical vein endothelial nuclear extracts bound to these elements in a specific manner and that both sites were essential for protein binding. Ferguson analysis predicted a combined molecular mass of 153 kDa for these proteins. In addition, transient transfection, gel shift, and DNase I footprint analysis were used to identify a functional Sp1 binding site downstream of these elements in the core promoter. By localizing the functional cis-acting elements in the PDGF-B promoter, it may be possible to elucidate the normal transcriptional control of the gene, as well as the mechanisms that activate it in pathologic settings.

Recruitment of enzymes and stress proteins as lens crystallins.

The major water-soluble proteins--or crystallins--of the eye lens are either identical to or derived from proteins with non-refractive functions in numerous tissues. In general, the recruitment of crystallins has come from metabolic enzymes (usually with detoxification functions) or stress proteins. Some crystallins have been recruited without duplication of the original gene (i.e., lactate dehydrogenase B and alpha-enolase), while others have incurred one (i.e., argininosuccinate lyase and a small heat shock protein) or several (i.e., glutathione S-transferase) gene duplications. Enzyme (or stress protein)-crystallins often maintain their non-refractive function in the lens and/or other tissues as well as their refractive role, a process we call gene sharing. alpha-Crystallin/small heat shock protein/molecular chaperone is of special interest since it is the major crystallin of humans. There are two alpha-crystallin genes (alpha A and alpha B), with alpha B retaining the full functions of a small heat shock protein. Here we describe recent evidence indicating that alpha A and alpha B have kinase activity, which would make them members of the enzyme-crystallins. We also describe various regulatory elements of the mouse alpha-crystallin genes responsible for their expression in the lens and, for alpha B, in skeletal muscle. Delineating the control elements for gene expression of these multifunctional protective proteins provides the foundations for their eventual use in gene therapy. Finally, comparison of the mouse and chicken alpha A-crystallin genes reveals similarities and differences in their functional cis-acting elements, indicative of evolution at the level of gene regulation.

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5' deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between -326 and -130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions -326 and -130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.