Positive Regulatory Elements Research Articles

Function identification of bovine Nramp1 promoter and intron 1

The Nramp1/Slc11a1 locus encodes a proton-coupled divalent cation transporter, expressed in late endosomes/lysosomes of macrophages, that constitutes a component of the innate immune response to combat intracellular pathogens and it was shown to play an important role in regulating inherent immunity. The previously identified Z-DNA forming polymorphic repeat(GT)n in the promoter region of the human Nramp1 gene does act as a functional polymorphism influencing gene expression. Research has shown that INF-γ, TNF-α, IL-1β and bacteria LPS increase the level of Nramp1 expression. However, the molecular mechanism for Nramp1 gene regulation is unclear. In this research, bovine Nramp1 5′-flanking region (−1748~+769) was cloned and analyzed by bioinformatics. Then to find the core promoter and the cis-acting elements, deletion analysis of promoter was performed using a set of luciferase reporter gene constructs containing successive deletions of the bovine Nramp1 5′-flanking regions. Promoter activity analysis by the dual luciferase reporter assay system showed that the core promoter of Nramp1 was located at +58~−89 bp. Some positive regulatory elements are located at −89~−205 bp and −278~−1495 bp. And the repressor elements were in region −205~−278 bp, intron1 and −1495~−1748 bp. LPS-responsive regions were located at −1495~−1748 bp and −278~−205 bp. The present study provides an initial effort to explore the molecular mechanism of transcriptional activation of the bovine Nramp1 gene and should facilitate further studies to decode the complex regulatory process and for molecular breeding for disease resistance in bovines.

Characterization of a novel positive transcription regulatory element that differentially regulates the alpha-2-macroglobulin gene in replicative senescence

Alpha-2-macroglobulin (α2M), a protease inhibitor, is implicated in Alzheimer's disease, atherosclerosis, and other age-related diseases. The elevated level of α2M mRNA has been described in replicative senescence and it could be used as a biomarker of the aging cells. However, the mechanism responsible for the up-regulation of its expression is still unclear. This report identified a novel transcriptional regulatory element, the α2M transcription enhancement element (ATEE), within the α2M promoter. This element differentially activates α2M expression in senescent versus young fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. The DNase I footprint revealed the protein-binding core sequence through which the protein binds the ATEE. Mutation within ATEE selectively abolished α2M promoter activity in senescent (but not young) cells. These results indicated the ATEE, as a positive transcription regulatory element, contributes to the up-regulation of α2M during replicative senescence.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5'-flanking sequence (5'FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5'FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5'FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5'FS are used as promoter, efficient transcription only occurs with 44 bp of 5'FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5'FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Azithromycin Inhibits Expression of the GacA-Dependent Small RNAs RsmY and RsmZ in Pseudomonas aeruginosa

Azithromycin at clinically relevant doses does not inhibit planktonic growth of the opportunistic pathogen Pseudomonas aeruginosa but causes markedly reduced formation of biofilms and quorum-sensing-regulated extracellular virulence factors. In the Gac/Rsm signal transduction pathway, which acts upstream of the quorum-sensing machinery in P. aeruginosa, the GacA-dependent untranslated small RNAs RsmY and RsmZ are key regulatory elements. As azithromycin treatment and mutational inactivation of gacA have strikingly similar phenotypic consequences, the effect of azithromycin on rsmY and rsmZ expression was investigated. In planktonically growing cells, the antibiotic strongly inhibited the expression of both small RNA genes but did not affect the expression of the housekeeping gene proC. The azithromycin treatment resulted in reduced expression of gacA and rsmA, which are known positive regulators of rsmY and rsmZ, and of the PA0588-PA0584 gene cluster, which was discovered as a novel positive regulatory element involved in rsmY and rsmZ expression. Deletion of this cluster resulted in diminished ability of P. aeruginosa to produce pyocyanin and to swarm. The results of this study indicate that azithromycin inhibits rsmY and rsmZ transcription indirectly by lowering the expression of positive regulators of these small RNA genes.

The ZIIR Element of the Epstein-Barr Virus BZLF1 Promoter Plays a Central Role in Establishment and Maintenance of Viral Latency

The Epstein-Barr virus (EBV) BZLF1 gene encodes the immediate-early (IE) protein Zta, which plays a central role in regulating the switch between viral latency and lytic replication. A silencing element, ZIIR, is located between the ZID and ZII positive regulatory elements in the BZLF1 promoter Zp. We report here the phenotypes of variants of EBV strain B95.8 containing base substitution mutations in this ZIIR element. HEK293 cells infected with ZIIR mutant (ZIIRmt) virus produced at least 20-fold more viral IE Zta and Rta and early (E) EAD protein than did cells infected with the parental wild-type (WT) virus, leading to viral DNA replication and production of infectious virus. However, ZIIR mutant virus was 1/10 as efficient as WT virus in establishing proliferating B-cell clones following infection of human primary blood B cells. The ZIIRmt-infected lymphoblastoid cell lines (LCLs) that did grow out exhibited a phenotype similar to the one observed in 293 cells, including marked overproduction of IE and E gene products relative to WT-infected LCLs and lytic replication of the viral genome. Incubation of the ZIIRmt-infected LCLs with the chemical inducer 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to much greater activation of Zp than did the same treatment of WT- or ZVmt-infected LCLs. Furthermore, a protein kinase C (PKC) inhibitor, bis-indolylmaleimide, eliminated this activation by TPA. Thus, we conclude that ZIIR is a potent silencing element of Zp; it plays a key role in establishment and maintenance of EBV latency by inhibiting activation of Zp through the PKC signal transduction pathway.

Cooperative action of multiple cis-acting elements is required for N-myc expression in branchial arches: specific contribution of GATA3.

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.

Lithium induces c-Ret expression in mouse inner medullary collecting duct cells

We found in our present study that lithium (Li+) induced the expression of endogenous c-Ret, a tyrosine kinase receptor, in murine inner medullary collecting duct (mIMCD-3) cells. Delineation of the promoter region required for the effect of Li+ identified a positive regulatory element within 180bp upstream of the transcription initiation site. This region contained three putative GC-rich Sp1 binding sites found to be essential for c-Ret induction by Li+. The effect of Li+ was mediated through glycogen synthase kinase 3β (GSK-3β) inhibition, although there was no biding site for T cell factor/lymphoid enhancer factor (TCF/LEF) in the 180bp. We found that Li+ activated the mammalian target of rapamycin (mTOR) pathway via GSK-3β in these cells, and the effect of Li+ to induce c-Ret was amenable to the inhibitory effect of the mTOR inhibitor, rapamycin. We also found that alterations in both cellular β-catenin levels and mTOR activities affected the effect of Li+ on c-Ret transcription in a cooperative manner. In summary, our results show that Li+ can induce c-Ret expression in mIMCD-3 cells through both β-catenin- and mTOR-dependent pathways downstream of GSK-3β inhibition, which act synergistically on the GC-rich Sp1 binding elements in the promoter region.

Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease

In Fabry disease a deficiency of α-galactosidase A (α-gal A) activity leads to accumulation of globotriaosylceramide (Gb3) in various tissues including the heart. A specific cardiac variant of Fabry disease has also been described. Previously we have demonstrated the feasibility of gene therapy for Fabry disease. Here, to provide efficient transfer and increased specificity of transgene expression, we synthesized lentiviral vectors (LVs) with myocardial-specific promoters including: α-myosin heavy chain (α-MHC), myosin light chain (MLC2v), and cardiac troponin T (cTnT). Initially, neonatal Balb/c mice were injected with such LV constructs engineering expression of luciferase. One month post-injection, we found specific expression of luciferase in hearts of recipient animals when compared with transgene expression driven by the standard EF1-α promoter. To examine the feasibility of long-term therapy specifically targeting the heart, recombinant LV/α-gal A therapeutic vectors with analogous cardiac promoters were generated and injected into numerous neonatal Fabry mice. No immune response against the corrective α-gal A hydrolase was observed in the treated mice. Serum α-gal A activity of 10-week-old Fabry mice was increased in LV/α-gal A-injected animals compared to controls. In 28-week-old Fabry mice we observed significantly decreased Gb3 accumulation. Neonatal injections with LVs harboring cardiac-specific promoters may thus be an effective long-term treatment strategy for heart manifestations and cardiac variant Fabry disease. These results can be also extended to other progressive pathologies of the heart.

Characterization of the Scutellaria barbata glycosyltransferase gene and its promoter

The conversion of flavonoid aglycones to their glycosides by plant glycosyltransferases may affect a wide range of outcomes, including stability, solubility and bioavailability. Scutellaria barbata, rich in flavonoid glycosides, is widely used as a traditional Chinese herbal medicine. In this study, a flavonoid glycosyltransferase cDNA (SbUGT) and its promoter from S. barbata were cloned and characterized as a flavonoid glycosyltransferase using whole-cell biotransformation. Fragments of different lengths of the 5'-flanking region of the SbUGT gene were fused to the beta-glucuronidase (GUS) gene and analyzed with transgenic Arabidopsis plants using histochemical and fluorometric assays. GUS activity in transgenic plants carrying the SbP-850U construct (-850 to +86 relative to the transcription start site) displayed the highest level and was enhanced by salt and methyl jasmonate, similar to the expression patterns of the endogenous SbUGT. GUS activity disappeared when the promoter was deleted to -98, and deletion analyses indicated the existence of positive and negative regulatory element(s). Unexpectedly, plants carrying the construct SbP-102U (-102 to +86) exhibited strong GUS activity exclusively in the roots. Our experiments revealed that the specific expression is mediated by different promoter regions and the unique region driving root-preferred expression can be used as a root-specific promoter.

Regulation of retinal homeobox gene transcription by cooperative activity among cis-elements

The retinal homeobox ( Rx/rax) gene is essential for the development of the eye. Rax is among the earliest genes expressed during eye development, beginning in the prospective eye fields in the anterior neural plate. Additionally Rax expression persists in retinal progenitor cells and in differentiated photoreceptors. We have isolated and characterized a 2.8 kb genomic DNA fragment that regulates expression of Rax in the developing and maturing retina. We have discovered and characterized cis-acting elements that function to specifically control spatial and temporal Rax expression during retinal development. We have found that the regulation of Rax2A promoter activity requires cooperative interactions between positive and negative regulatory elements. Further, a highly conserved genomic element containing SOX, OTX, and POU transcription factor binding sites is necessary but not sufficient for promoter activity in retinal progenitor or stem cells. Finally, a putative binding element for forkhead transcription factors is necessary for promoter activity and can cooperate with other cis-acting elements to drive Rax2A promoter activity.

Distinct regulatory mechanism of immunoglobulin gene transcription in epithelial cancer cells

The restriction of immunoglobulin (Ig) expression to B lymphocytes is well established. However, several reports have confirmed that the Ig gene can be expressed in many non-B cancer cells and/or some normal cells. Our aim is to determine whether the Ig gene promoter can be activated in non-B cancer cells and to identify the regulatory mechanism for Ig gene expression. Our results show that the Ig promoter of VH4-59 was activated in several non-B cancer cell lines. Moreover, two novel positive regulatory elements, an enhancer-like element at -800 to -610 bp and a copromoter-like element at -610 to -300 bp, were identified in two epithelial cancer cell lines, HeLa S3 and HT-29. The octamer element (5'-ATGCAAAT-3') located in the Ig promoter, a crucial element for B-cell-derived Ig gene transcription, was also very important for non-B-cell-derived Ig gene transcription. More importantly, we confirmed that octamer-related protein-1 (Oct-1), but not Oct-2, was a crucial transcriptional factor for Ig gene transcription due to its ability to bind to the octamer element of the Ig promoter in epithelial cancer cells. These results suggested the presence of a distinct regulatory mechanism for Ig gene expression in non-B cancer cells.

Regulation of a Bovine Nonclassical Major Histocompatibility Complex Class I Gene Promoter1

Studies have shown in humans and other species that the major histocompatibility complex class I (MHC-I) region is involved at a number of levels in the establishment and maintenance of pregnancy. The aim of this study was to characterize how a bovine nonclassical MHC-I gene (NC1) is regulated. Initial serial deletion experiments of a 2-kb fragment of the NC1 promoter identified regions with positive regulatory elements in the proximal promoter and evidence for a silencer module(s) further upstream that cooperatively contributed to constitutive NC1 expression. The cytokines interferon tau (IFNT), interferon gamma (IFNG), and interleukin 4 (IL4) significantly increased luciferase expression in NC1 promoter reporter constructs and endogenous NC1 mRNA levels in a bovine endometrial cell line. In addition, IFNG, IL3, IL4, and progesterone significantly increased Day 7 bovine blastocyst NC1 mRNA expression when supplemented during in vitro embryo culture. Site-directed mutagenesis analysis identified a STAT6 binding site that conferred IL4 responsiveness in the NC1 proximal promoter. Furthermore, methylation treatment of the proximal promoter, which contains a CpG island, completely abrogated constitutive NC1 expression. Overall, the findings presented here suggest that constitutive NC1 expression is regulated positively by elements in the proximal promoter, which are further controlled by upstream silencer modules. The promoter is responsive to IFNT, IFNG, and IL4, suggesting possible roles for these cytokines in bovine preimplantation embryo survival and/or maternal-fetal tolerance. Our studies also suggest that methylation of the proximal promoter, in particular, could play a significant role in regulating NC1 expression.

Functional Analysis of the Role of the TPMT Gene Promoter VNTR Polymorphism in TPMT Gene Transcription

Thiopurine S-methyltransferase (TPMT) activity is polymorphic, and a trimodal distribution has been demonstrated in Caucasians (low, intermediate and high methylator groups). The TPMT gene promoter contains a variable number of three GC-rich tandem repeats, namely A, B and C, ranging from three to nine in length in a A(n)B(m)C architecture. Here, we investigated the influence of number and type of TPMT gene promoter tandem repeats on human TPMT gene transcription in K562 cells transiently transfected with reporter constructs bearing various variable number of tandem repeats (VNTR) and addressed the interaction of transcription factor binding to the VNTRs by electrophoretic mobility shift assays. We found that the distribution patterns of VNTR alleles do not significantly differ among acute lymphoblastic leukemia patients, acute myeloid leukemia patients and normal individuals. We also demonstrated that the A repeat has a negative effect in TPMT gene transcription and that a positive regulatory element, identified immediately upstream to the VNTR region of the TPMT gene promoter, is indispensable for TPMT gene transcription. Our electrophoretic mobility shift assay analysis indicated that the Sp1 and Sp3 transcription factors bind to the VNTR repeats. Overall, our data underline that both the number and type of VNTRs, as well as the upstream regulatory region of the TPMT gene promoter, determine the overall level of TPMT gene transcription. It remains to be seen whether these VNTRs can be employed as pharmacogenetic markers to individualize thiopurine therapy.

Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter

SBgLR (Solanum tuberosum genomic lysine-rich) is a pollen-specific gene cloned from potato (Solanum tuberosum L.). The region from -269 to -9 (The A of translation start site "ATG" as +1) of the SBgLR promoter was identified as critical for gene specific expression in pollen grains. Sequence analysis indicates a palindromic sequence "TTTCTATTATAATAGAAA" in the -227 to -209 region, in which two pollen-specific motifs TTTCT and AGAAA surround a unique putative TATA box. Moreover, nine putative pollen-specific motifs are located in the region between the TATA box and ATG. We placed the -227 to -9 region (reserving the palindrome) and the -222 to -9 region (breaking the palindrome) downstream of the CaMV35S enhancer, respectively, to construct two fusion promoters. Histochemical assays in transgenic plants demonstrated that the region from -222 to -9 is necessary and sufficient for pollen-specific expression of the uidA gene. However, the region of -227 to -9 is incapable of driving GUS expression in pollen grains and parts of vegetative tissues. A series of 5' deletions from -269 to -9 of SBgLR promoter were constructed. A transient expression assay indicated that the region from the -227 to -9 suppressed gfp gene expression in pollen, and a positive regulatory element was present in the region of -253 to -227. The function of the palindromic sequence as a repressor inhibiting gene expression in pollen was further confirmed by the mutated promoter, breaking the palindrome by substituting its 3'-flanking five base pairs, which resumes the reporter gene expression in mature pollen.

The tissue expression pattern of the AtGRP5 regulatory region is controlled by a combination of positive and negative elements

The AtGRP5 gene from Arabidopsis thaliana encodes a glycine-rich protein which has a major activity in protoderm-derived cells and is expressed in cells that undergo the first anatomical modifications leading to somatic embryo development. It has been previously demonstrated that its minimum promoter is 316 bp long including the 5'UTR and presents three putative TATA-boxes sequences and several regions that are homologous to previous characterized cis-acting elements. In order to better characterize the AtGRP5 expression and to identify the promoter regions involved in its preferential epidermal expression, in situ hybridization and 5' promoter deletions were employed. In situ hybridization and GUS expression assays indicate that, besides being present during somatic embryogenesis, AtGRP5 is also expressed during the zygotic embryo development. The sequential 5' deletions indicate that multiple negative and positive regulatory elements are present in the AtGRP5 promoter and operate in order to confer its distinct expression pattern. A 44-bp region was shown to be essential for the epidermal expression of this gene in leaves, stems, flowers and fruits, and is also responsible for high activity of the AtGRP5 promoter in zygotic embryos. An element responsible for the phloem expression was also identified in a 35-bp region.

Repressive BMP2 gene regulatory elements near the BMP2 promoter

The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

A novel negative Fe-deficiency-responsive element and a TGGCA-type-like FeRE control the expression of FTR1 in Chlamydomonas reinhardtii.

We have reported three Fe-deficiency-responsive elements (FEREs), FOX1, ATX1, and FEA1, all of which are positive regulatory elements in response to iron deficiency in Chlamydomonas reinhardtii. Here we describe FTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs of FTR1 distinguish itself from other iron response elements by containing both negative and positive regulatory regions. In FTR1, the −291/−236 region from the transcriptional start site is necessary and sufficient for Fe-deficiency-inducible expression. This region contains two positive FeREs with a TGGCA-like core sequence: the FtrFeRE1 (ATGCAGGCT) at −287/−279 and the FtrFeRE2 (AAGCGATTGCCAGAGCGC) at −253/−236. Furthermore, we identified a novel FERE, FtrFeRE3 (AGTAACTGTTAAGCC) localized at −319/−292, which negatively influences the expression of FTR1.

Identification of important regulatory region of Th-POK

CD4(+) and CD8(+) T cells develop from CD4(+)CD8(+) thymocytes. Although it has been reported that expression of the transcription factor Th-POK is important for CD4(+) T cell development, the detailed mechanism regulating Th-POK expression is still obscure. By comparing the promoter regions of the Th-POK gene between human and mouse, I found that the region 3600 base pairs (bps) upstream from the transcription initiation site of the Th-POK gene was highly conserved. To identify the important element(s) regulating Th-POK expression in CD4(+) T cells, I investigated the promoter activity of this region using a luciferase assay in the human T cell line Jurkat. I identified a positive regulatory element in this region 22 bps in length located 600 bp upstream from the transcription initiation site. This 22 bp element had a consensus binding sequence for SAP-1, which is encoded by the Elk4 gene and is activated by the Erk pathway. These data suggest that the 22 bp element might positively regulate Th-POK expression through Erk-SAP-1 signaling.

B cell–specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers

Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch-inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5' promoter region. The 5' upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-kappaB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell-specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell-specific and stimulation-responsive elements and ubiquitous silencers.

Targeted Knockdown of EGR-1 Inhibits IL-8 Production and IL-8-mediated Invasion of Prostate Cancer Cells through Suppressing EGR-1/NF-κB Synergy

IL-8 produced by prostate cancer cells may be responsible for the androgen-independent growth of advanced prostate cancers. Accumulating evidence from microarray analyses and animal genetic models highlights the central involvement of the transcription factor early growth response-1 (EGR-1) in prostate carcinoma progression. It is unknown, however, whether knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated tumor metastasis. Here we show that EGR-1 knockdown by a specific shRNA-Egr1 inhibited gene transcription and production of IL-8 by the human prostate cancer cell line DU145. Conversely, enforced expression of EGR-1 in EGR-1-lacking PC3 prostate cancer cells markedly enhanced IL-8 transcription and secretion. By using wild type and a series of mutant IL-8 promoter luciferase constructs, we found that the NF-kappaB binding site is important for EGR-1 regulation of IL-8. Furthermore, silencing EGR-1 suppressed a synergistically functional interaction between EGR-1 and NF-kappaB. Consequently, knockdown of EGR-1 inhibited IL-8-mediated tumor colony formation and invasion. Thus, targeted knockdown of EGR-1 could be an effective therapeutic approach against prostate cancer.