Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.

Pea nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor that specifically interacts with regulatory DNA sequences upstream of the pea rbcS-3A-gene. This factor, designated GT-1, binds to two short sequences (boxes II and III) in the -150 region that are known to function as light-responsive elements (LREs) in transgenic tobacco. Binding of GT-1 to homologous sequences further upstream (boxes II and III in the -220 region) indicates that these boxes comprise the redundant LRE that functions in vivo when boxes II and III are deleted. In both box II and box II, methylation interference experiments demonstrate that two adjacent G residues are critical for GT-binding. Single Gs present in boxes III and III are also important. Since GT-1 is present in nuclear extracts from leaves of light-grown and dark-adapted pea plants, its regulatory role does not depend on de novo synthesis. Thus if GT-1 binds differentially in vivo it must be postranslationally modified or sterically blocked from binding by another factor in response to light.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5 to 3) in the 5 region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter--glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position-173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the-326 to-173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position-173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.     

Binding sites for two novel phosphoproteins, 3AF5 and 3AF3, are required for rbcS-3A expression.

Previous studies of boxes II (-151 to -138) and III (-125 to -114), binding sites for the nuclear factor GT-1 within the -166 deleted promoter of the ribulose-1,5-bisphosphate carboxylase-3A (rbcS-3A) gene, suggested that GT-1 might act in concert with an additional protein to confer light-responsive rbcS-3A expression. In this work, S1 analysis of RNA isolated from transgenic tobacco plants carrying mutant rbcS-3A constructs led to the identification of two short sequences located at the 5' and 3' ends of box III that are required for expression. These two sequences serve as binding sites for two novel proteins, 3AF5 and 3AF3. Gel shift studies using tetramerized binding sites for both 3AF5 and 3AF3 showed that complexes with faster mobilities were formed using nuclear extracts prepared from dark-adapted plants compared with those from light-grown tobacco plants. Phosphatase treatment of extracts from light-grown plants resulted in the formation of complexes with faster mobility. Although the binding of 3AF3 to its target site is dependent upon phosphorylation, the binding of 3AF5 does not appear to be affected by its phosphorylation state. These results suggest that the phosphorylated forms of both 3AF5 and 3AF3 are required for -166 rbcS-3A expression but that the mechanisms differ by which phosphorylation regulates the activities of 3AF5 and 3AF3.     

Molecular dissection of GT-1 from Arabidopsis.

We isolated and characterized an Arabidopsis cDNA encoding the DNA binding protein GT-1. This protein factor, which contains 406 amino acids, is highly homologous to the previously described tobacco DNA binding protein GT-1a/B2F but is 26 amino acids longer. Recombinant Arabidopsis GT-1, which was obtained from in vitro translation, bound to probes consisting of four copies of pea small subunit of ribulose bisphosphate carboxylase rbcS-3A box II and required the same GGTTAA core binding site as the binding activity of an Arabidopsis nuclear protein preparation. However, unlike the truncated tobacco GT-1a prepared from Escherichia coli extracts, the full-length Arabidopsis GT-1 bound to pea rbcS-3A box III and Arabidopsis chlorophyll a/b binding protein CAB2 light-responsive elements, both of which contain GATA motifs. Deletion and mutational analyses suggested that the predicted trihelix region of GT-1 is essential for DNA binding. Moreover, GT-1 binds to target DNA as a dimer, and its C-terminal region contains a putative dimerization domain that enhances the binding activity. Transient expression of the GT-1::beta-glucuronidase fusion protein in onion cells revealed the presence of a nuclear localization signal(s) within the first 215 amino acids of GT-1.     

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.Abbreviations CAT chloramphenicol acetyltransferase - CHS chalcone synthase (EC 2.3.1.74) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.     

The GATA-binding protein CGF-1 is closely related to GT-1

Many light-regulated genes contain a conserved GATA motif in their 5-upstream region. We have characterized in detail the GATA-binding factor, CGF-1, which binds within a 73 bp TATA-proximal light/circadian regulatory element in the Arabidopsis cab2 promoter and to two more sites farther upstream. CGF-1 was found to be distinct from other metal-dependent GATA-binding factors, but to have the same sequence requirements for binding and similar physical and chemical properties as GT-1, a factor required for light regulation of the tobacco rbcS-3A gene. CGF-1 was found to be constitutively present in extracts and was shown to be immunologically related to GT-1. The close similarity between CGF-1 and GT-1 suggests that a GT-1-like factor is involved in the phytochrome/circadian regulation of the cab2 gene. CGF-1 and GT-1 were also found to have similar sequence specificities to another constitutively-regulated GATA factor, IBF-2b, which binds the I box region of the tomato nitrate reductase gene. Of three complexes detected using an IBF-2b-specific probe, only one was identical to CGF-1/GT-1. The other two were similar to IBF-2b, demonstrating that CGF-1/GT-1, although very similar, are actually distinct from IBF-2b. These data indicate that more than one factor can bind to the same short sequence and may indicate how constitutively present factors like GT-1 can play a role in light regulation.     

Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver-specific enhancer for the human alpha-1-microglobulin/bikunin precursor.

Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4.     

Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.     

Molecular structure of the chicken vitamin D-induced calbindin-D28K gene reveals eleven exons, six Ca2+-binding domains, and numerous promoter regulatory elements

The seco-steroid hormone 1,25-dihydroxyvitamin D3 is known to induce the expression of a calcium binding protein termed calbindin-D28K in a variety of target tissues. In order to comprehend the mechanism of induction we have cloned and sequenced the chicken calbindin-D28K gene. The gene spans some 18.5 kilobases (kb) of chromosomal DNA from the putative Cap site to the polyadenylation site of the 2.8 kb mRNA. It is split into 11 coding exons by 10 intervening sequences. The promoter region of this gene is markedly G + C-rich (60-80%) extending from -225 to +400. Within this region we find 70 CpG dinucleotides, four G-C boxes, and numerous known promoter regulatory signals. These putative regulatory signals include a TATA box (ATAAATA) at -30 and a CAT box (CCAAT) at -326. Ten additional variant CAT boxes are found in the upstream promoter region (-218 to -770) of this gene. Furthermore we have identified a glucocorticoid-like responsive element at -410 (TCTACACACTGTTCC) and this element overlaps a metal responsive element (TGCACTC) and a variant CAT box (CCAAAT) and juxtaposes an enhancer-like core element (AAATGGT) on its 3'-side. In addition, the calbindin-D28K promoter is composed of a variety of simple repeated sequences, some of which are components of putative regulatory signals. All splice junctions were found to conform to the GT-AG rule. A consensus sequence of the 5'-splice junction reads AG/GTAAG-TTATA. A consensus sequence of the 3'-splice site consists of two elements: a pyrimidine track (mainly T) followed by ACAG/G-T. A two-dimensional model of calbindin-D28K was constructed which projects the existence of 6 alpha-helix-loop-alpha-helix regions characteristic of calcium binding domains. The 3'-end of the gene consists of a single large (2039 base pair) uninterrupted exon, an organizational feature common to other members of the calcium binding protein gene family which include calmodulin, parvalbumin, Spec I, myosin light chains, etc. Another feature common to the gene family is the presence of the repeated sequence ATTT or TTTA located in the 3'-untranslated exons. These simple repeat sequences could be involved in regulating mRNA degradation by serving as a ribonuclease recognition signal.