Identification of functional sequences in the pregenomic RNA promoter of the Banana streak virus Cavendish strain (BSV-Cav)

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and regain-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-l-like element, the region around-141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease. (C) 2004 Elsevier B.V. All rights reserved.

Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the Goldfinger isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The selective-shotgun approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional linker-scanning deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.

Characterization of a strong, constitutive mung bean (Vigna radiata L.) promoter with a complex mode of regulation in planta

We report the cloning and characterization in tobacco and Arabidopsis of a Vigna radiata L. (mung bean) promoter that controls the expression of VR-ACS1, an auxin-inducible ACC synthase gene. The VR-ACS1 promoter exhibits a very unusual behavior when studied in plants different from its original host, mung bean. GUS and luciferase in situ assays of transgenic plants containing VR-ACS1 promoter fusions show strong constitutive reporter gene expression throughout tobacco and Arabidopsis development. In vitro quantitative analyses show that transgenic plants harboring VR-ACS1 promoter-reporter constructs have on average 4-6 fold higher protein and activity levels of both reporter genes than plants transformed with comparable CaMV 35S promoter fusions. Similar transcript levels are present in VR-ACS1 and CaMV 35S promoter lines, suggesting that the high levels of gene product observed for the VR-ACS1 promoter are the combined result of transcriptional and translational activation. All tested deletion constructs retaining the core promoter region can drive strong constitutive promoter activity in transgenic plants. This is in contrast to mung bean, where expression of the native VR-ACS1 gene is almost undetectable in plants grown under normal conditions, but is rapidly and highly induced by a variety of stimuli. The constitutive behavior of the VR-ACS1 promoter in heterologous hosts is surprising, suggesting that the control mechanisms active in mung bean are impaired in tobacco and Arabidopsis. The 'aberrant' behavior of the VR-ACS1 promoter is further emphasized by its failure to respond to auxin and cycloheximide in heterologous hosts. VR-ACS1 promoter regulatory mechanisms seem to be different from all previously characterized auxin-inducible promoters.

Characterization of the human sulfate anion transporter (hsat-1) protein and gene (SAT1; SLC26A1)

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.

Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data Suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation. (C) 2004 Elsevier Inc. All rights reserved.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappa B site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

E2F suppression and Sp1 overexpression are sufficient to induce the differentiation-specific marker, transglutaminase type 1, in a squamous cell carcinoma cell line

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.

PAX2 activates WNT4 expression during mammalian kidney development

The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the S-shaped body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5'-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.

The JNK are important for development and survival of macrophages

We report in, this study that activation of the JNK by the growth factor, CSF-1 is critical for macrophage development, proliferation, and survival. Inhibition of JNK with two distinct classes of inhibitors, the pharmacological agent SP600125, or the peptide D-JNKI1 resulted in cell cycle inhibition with an arrest at the G(2)/M transition and subsequent apoptosis. JNK inhibition resulted in decreased expression of CSF-1R (c-fins) and Bcl-x(L) mRNA in mature macrophages and repressed CSF-1-dependent differentiation of bone marrow cells to macrophages. Macrophage sensitivity to JNK inhibitors may be linked to phosphorylation of the PU.1 transcription factor. Inhibition of JNK disrupted PUA binding to an element in the c-fins gene promoter and decreased promoter activity. Promoter activity could be restored by overexpression of PUA. A comparison of expression profiles of macrophages with 22 other tissue types showed that genes that signal JNK activation downstream of tyrosine kinase receptors, such as focal adhesion kinase, Nck-interacting kinase, and Rac1 and scaffold proteins are highly expressed in macrophages relative to other tissues. This pattern of expression may underlie the novel role of JNK in macrophages.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Regulation of the murine TRACP gene promoter

The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the –1958 bp AS promoter linked to the β-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in –1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than –405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least –640 bp of the AS promoter but not those with –523 bp or smaller promoter fragments. Fusion of the –640 to –523 bp region to a –381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.