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PubMed2004

Deacetylase inhibitors and the viral transactivator TaxBLV synergistically activate bovine leukemia virus gene expression via a cAMP-responsive element- and cAMP-responsive element-binding protein-dependent mechanism.

Efficient bovine leukemia virus (BLV) transcription requires the virus-encoded transactivator Tax(BLV), which acts through three Tax(BLV)-responsive elements located in the 5' long terminal repeat. It has been proposed that the binding of the CRE-binding protein (CREB) and the activating transcription factor (ATF) to the three imperfect cAMP-responsive elements (CREs) located in each Tax(BLV)-responsive element mediates Tax(BLV) transactivation. Here we demonstrated that deacetylase inhibitors (HDACis) synergistically enhanced the transcriptional activation of the BLV promoter by Tax(BLV) in a CRE-dependent manner. Tax(BLV) was acetylated in vivo at its N(alpha) terminus but not at internal lysine residues. Rather, HDACi potentiation of Tax(BLV) transactivation was mediated by an HDACi indirect action that requires new protein synthesis. Mechanistically, using a dominant-negative form of CREB, we showed that Tax(BLV) and HDACi synergistically activated BLV gene expression via a CREB-dependent mechanism. Moreover, electrophoretic mobility shift assay and Western blot experiments revealed that HDACi increased the in vitro DNA binding activity of CREB/ATF but did not alter CREB/ATF intranuclear presence. Remarkably, chromatin immunoprecipitation assays demonstrated that HDACi treatment increased the level of CREB bound to the BLV promoter in vivo. Our results together suggest that an increase in CREB/ATF occupancy of the viral CREs in response to HDACi potentiates Tax(BLV) transactivation of the BLV promoter.

Animals↗
PubMed1989

Bovine leukemia virus trans-activator p38tax activates heterologous promoters with a common sequence known as a cAMP-responsive element or the binding site of a cellular transcription factor ATF.

The genome of bovine leukemia virus (BLV) encodes a transcriptional trans-activator p38tax (also referred to as pXBL-I) which amplifies the virus gene expression driven by its long terminal repeat (LTR). It was proposed that activation of cellular gene expression by p38tax might be involved in the mechanism of B-cell transformation caused in vivo by BLV infection. Here, we report that the U3 region of BLV LTR contains multiple regulatory elements responsive to p38tax. A core element composing the p38tax-inducible U3 structure is suggested to be a heptanucleotide motif of 5'TGACGTCA3', the consensus sequence proposed for a cAMP-responsive element (CRE) and for the binding sites of a cellular transcription factor (ATF). Adenovirus-5 E3 and E4, c-fos and somatostatin regulatory regions containing CRE/ATF-element exhibited responsiveness to p38tax in a chloramphenicol acetyltransferase transient expression assay. These suggest that in BLV-infected cells, cellular gene expression might be induced abnormally by the virus trans-activator through ATF or ATF-like factors.

Activating Transcription Factors↗
PubMed2004

Cyclic adenosine 5'-monophosphate response element modulator is responsible for the decreased expression of c-fos and activator protein-1 binding in T cells from patients with systemic lupus erythematosus.

T cells from patients with systemic lupus erythematosus express increased levels of the cAMP response element modulator (CREM) that has been shown to bind to the IL-2 promoter and suppress its activity. In this study, we demonstrate that CREM binds to the proximal promoter of the c-fos proto-oncogene in live systemic lupus erythematosus T cells and represses its expression following stimulation in vitro. Decreased levels of c-fos protein result in decreased AP-1 activity, as determined in shift assays. Blockade of the translation of CREM mRNA with an antisense CREM vector increases the expression of c-fos and the AP-1 activity. The levels of c-fos mRNA vary with disease activity. We conclude that CREM represses the expression of c-fos and limits the activity of the enhancer AP-1. Thus, CREM is involved indirectly in the modulation of transcriptional regulation of multiple genes including IL-2.

Adult↗
PubMed1996

Ethanol exposure alters the phosphorylation of cyclic AMP responsive element binding protein and cyclic AMP responsive element binding activity in rat cerebellum.

The purpose of the present study was to track the acute effects of ethanol on the cerebellar adenylyl cyclase cascade from membrane to nucleus and to determine how this important signaling pathway neuroadapts during chronic ethanol exposure. An acute ethanol challenge increased cyclic AMP content and protein kinase A activity by 80% compared to control rats. In the nucleus the phosphorylated form of cAMP responsive element binding protein (CREB) increased 500%. Gel retardation assays with an oligomer encoding the rat proenkephalin cyclic AMP (CRE)1 were performed. Assays from protein derived from rats acutely exposed to ethanol identified three CRE-protein complexes also observed in assays of protein from saline-treated animals. However, after acute ethanol exposure, the intensity of the upper and middle CRE-protein complexes increased by 3-fold (280 +/- 10 vs. 70 +/- 3 arbitrary units; P < .01) compared to the sham treatment. Intensity of complex formation was still elevated 1 and 6 hr after ethanol exposure compared to sham conditions. In contrast, chronic ethanol treatment as well as pair-fed treatment did not alter the phosphorylation state of CREB or the intensity of the specific CRE-protein complexes on gel retardation assays. In summary, acute ethanol exposure resulted in the activation of the adenylyl cyclase signal transduction cascade from membrane to nucleus. In contrast, chronic ethanol exposure did not alter the phosphorylation of CREB or CRE binding activity. The behavioral significance of these events remain unclear, but may be related to the development of ethanol-induced tolerance in specific cerebellar functions.

Adenylyl Cyclases↗
PubMed2006

Induction of AKR1C2 by phase II inducers: identification of a distal consensus antioxidant response element regulated by NRF2.

AKR1C2, also referred to as the human bile acid binder and 3alpha-hydroxysteroid dehydrogenase type III, is a multifunctional oxidoreductase able to stereoselectively reduce steroids as well as oxidize or reduce polyaromatic hydrocarbons. Previously, this same protein was also identified by its robust induction by phase II inducers in HT29 cells. In HepG2 cells, both AKR1C2 and AKR1C1 (97% sequence homology) were induced by phase II inducers but not the highly related AKR1C3 and AKR1C4 family members (84% sequence homology). We now report the initial characterization of the proximal promoter of AKR1C2 in HepG2 cell line and the identification of a potent enhancer-like element responsive to phase II inducers located approximately 5.5 kilobases upstream from the transcription start site. DNA sequence analysis of this enhancer element revealed that it contained a consensus antioxidant response element (ARE), which was confirmed by mutation analysis. Treatment with phase II inducers leads to increased accumulation of nuclear factor-erythroid 2 p45-related factor (NRF) 2 in the nucleus, which was associated with increased binding to this ARE as determined by electrophoretic mobility shift assay. Transient transfection with Nrf2 increased the transcriptional activity of the ARE of AKR1C2 comparable with that observed with phase II inducers. Chromatin immunoprecipitation (ChIP) analysis also confirmed increased NRF2 binding to the ARE after induction by a phase II inducer. The AKR1C1 promoter also harbored this same ARE element in a highly homologous region, which was also bound by NRF2 in a ChiP analysis. No induction of the ARE of AKR1C2 was detected in Nrf2-/- fibroblasts. The regulation of AKR1C2 by this distal ARE suggests that AKR1C2 detoxifies products of reactive oxidant injury, which has important implications for both hormone and xenobiotic metabolism.

Base Sequence↗
PubMed1993

A novel cAMP-dependent regulatory region including a sequence like the cAMP-responsive element, far upstream of the human CYP21A2 gene.

Deletion mutants in the 5' upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP-dependent expression of the gene in the transient expression system using Y-1 cells (mouse adrenocortical tumor cell line). This regulatory element (-2574 to -2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP-responsive element (CRE) and a CAAT-like sequence. The CRE-like sequence has a five-base motif (5'-TGACG-3') of the palindromic CRE consensus (TGACGTCA). Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT-like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue-specific enhancers (adrenal-specific protein factor and Ad4-like sequences) and their binding factors to express a high level of cAMP-responsive expression of the CYP21A2 gene.

Adrenal Cortex↗
PubMed1999

Transcription of the human thyrotropin-releasing hormone receptor gene-analysis of basal promoter elements and glucocorticoid response elements.

The gene for the human thyrotropin-releasing hormone receptor (TRHR) spans 35 kb and contains three exons and two introns (Matre et al. (1999) J. Neurochem. 72, 1-11). Despite a reported transcription start site (TSS) mapped to position -885 upstream of the translation initiation codon (Iwasaki et al. (1996) J. Biol. Chem. 271, 22183-8), we found cell type specific promoter activity directed by a fragment downstream of this site (-770 to +1). To elucidate the basis for this unexpected activity, we analyzed basal promoter elements in this region of the gene. One divergent TATA box, TTTAAA in position -759, was found by mutational analysis to be critical for promoter activity, providing a likely explanation for the basal activity observed. This proximal region apparently contains several promoter elements, including Pit-1 binding sequences within the first intron of the TRHR gene as previously reported. Here we describe the analysis of two putative glucocorticoid response elements (GREs) that we identified in this region, one (distal) half site overlapping the proposed TSS at -885 and one (proximal) full site within the first intron at position -624. Accordingly, stimulation of rat pituitary GH3 and GH4C1 cells with dexamethasone strongly enhanced transcription activity of a reporter construct containing the distal GRE half site and the proximal GRE site. Both sites bound the glucocorticoid receptor (GR) in a specific manner. Deletion of the distal GRE half site abolished the dexamethasone induction of CAT transcription, as did mutations in the proximal site. We therefore conclude that both sites are necessary for regulation of the TRHR gene transcription by glucocorticoids.

Animals↗
PubMed2002

Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter.

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.

Binding Sites↗
PubMed2001

Angiotensin II-responsive element is the insulin-responsive element in the adipocyte fatty acid synthase gene: role of adipocyte determination and differentiation factor 1/sterol-regulatory-element-binding protein 1c.

We have previously shown that angiotensin II (Ang II) increases the expression of the gene encoding adipocyte fatty acid synthase (FAS). Here we investigate the mechanism responsible for increased FAS gene transcription by Ang II. We demonstrate that Ang II increased luciferase activity by 3-fold in 3T3-L1 adipocytes transfected with fusion constructs linking the FAS promoter to the luciferase reporter gene. Interestingly, we mapped the Ang II regulatory sequences to the insulin-responsive region (E box) in the proximal FAS promoter. The E box alone was able to mediate Ang II responsiveness when linked to a heterologous promoter. However, this response was lost when mutations that abolished the binding of the E box to its transcription factors were introduced. Using adenoviral overexpression of a dominant-negative form of adipocyte determination and differentiation factor 1 (ADD1), a transcription factor that binds to the insulin-responsive E box, we demonstrated that ADD1 was required for Ang II regulation of the FAS gene in 3T3-L1 adipocytes. Furthermore, ADD1 expression was also up-regulated by Ang II. With the use of transfections as well as glucose transport assays, we further demonstrated that Ang II stimulation of the FAS gene was dependent on glucose. In conclusion, this is the first report that Ang II regulates adipocyte FAS gene transcription via insulin response sequences in a glucose-dependent manner and that this regulation is mediated at least in part via the ADD1 transcription factor.

3T3 Cells↗
PubMed1991

A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter.

Genomic and cDNA sequences for the mouse cellular retinol binding protein I (mCRBPI) are presented. A specific cis-acting element responsible for retinoic acid (RA) inducibility of the mCRBPI promoter was identified and characterized. Deletion mapping of a CRBPI promoter--chloramphenicol acetyltransferase reporter gene construct localized this element to a 259 bp restriction fragment located approximately 1 kb upstream from the transcription start-site. A sequence closely resembling the previously characterized RA response element (RARE) of the RA receptor beta 2 (RAR-beta 2) promoter, and consisting of a direct repeat of the motif 5'-GGTCA-3' separated by three nucleotides, was found within this restriction fragment. Mutation of these 5'-GGTCA-3' motifs to GGAGC and GGGGC abolished RA-inducible transcription whereas a mutation to a direct repeat of the GTTCA motif found in the RARE of the RAR-beta 2 promoter resulted in enhanced inducibility. Oligonucleotides containing the direct repeat of the GGTCA motif were able to confer RA-dependent transcriptional enhancement to the herpes simplex thymidine kinase promoter, as well as to bind directly all three retinoic acid receptors (RARs) alpha, beta and gamma, as determined by gel retardation/shift assays. The control of CRBPI gene transcription by RA-RAR complexes interacting with the RARE characterized here may correspond to a feedback mechanism important in regulating retinoid metabolism and action.

Amino Acid Sequence↗
PubMed2000

The murine IL-2 promoter contains distal regulatory elements responsive to the Ah receptor, a member of the evolutionarily conserved bHLH-PAS transcription factor family.

Signaling through the TCR and costimulatory signals primarily control transcription of the IL-2 gene in naive T cells. The minimal promoter necessary for this expression lies proximal, between -300 and the transcription start site. We had previously shown that activation of the arylhydrocarbon receptor (AHR), a member of the bHLH-PAS family of transcription factors, leads to increased mRNA expression of IL-2 in murine fetal thymocytes. The AHR is abundant in the thymus and may play a role for the development of the immune system. Moreover, its overactivation by chemicals such as dioxins leads to immunosuppression and thymic involution. Binding motifs for the liganded AHR can be identified in the distal region -1300 to -800 of the mouse IL-2 promoter. We show here that these DNA motifs, the so-called dioxin response elements, after binding to the liganded AHR are sufficient to transactivate luciferase expression in a reporter gene system. The IL-2 gene can be induced by the AHR also in thymocytes in vivo after injection of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, a potent ligand of the AHR. The AHR mediates the IL-2 induction as shown with AHR-deficient mice. However, in spleen cells in vitro costimulation via the TCR is necessary for optimal IL-2 gene induction. Thus, the IL-2 promoter region contains novel distal regulatory elements that can be addressed by the AHR to induce IL-2 and can cooperate with the proximal promoter in this.

Animals↗
PubMed2000

Ability of the glucocorticoid modulatory element to modify glucocorticoid receptor transactivation indicates parallel pathways for the expression of glucocorticoid modulatory element and glucocorticoid response element activities.

The glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is located at -3.6 kb and 1 kb upstream of the glucocorticoid response elements (GREs). The GME has the unique transcriptional properties of modulating both the dose-response curve of agonists bound to the glucocorticoid receptor (GR) and the residual agonist activity of GR-bound antisteroids. The expression of GME activity involves the binding of two novel proteins (GMEB-1 and GMEB-2) that we have recently cloned. However, the mechanistic details are limited. The DNA sequence requirements for GME activity (CGTC) also remain poorly defined, which restricts efforts to identify other GME modulated genes. To help understand the mechanism for the unusual activities of the GME and to identify permissive gene environments for GME activity, we compared the changes in GME activity and GRE action (i.e. the fold induction by GR) caused by modifying several parameters. Phasing between the GME and downstream tandem GREs was unimportant, in contrast to other cis-acting elements like the GRE, while GME activity decreased rapidly when placed at increasingly larger distances 3' to a tandem GRE. A minimal promoter was less effective in supporting GME than GRE activity. Although CREB binds to the GME, overexpression of CREB reduced GRE, but not GME, activity and a CRE was inactive when substituted for the GME. No effect of the GME was observed on the binding of GRs to a single GRE. However, the GME upstream of a single GRE was also unable to produce a left shift in the Dex dose-response curve under conditions where the GME was active with two GREs. In the absence of any GREs, the GME displayed intrinsic activity by elevating basal level expression. Collectively, these results indicate that an optimal position for a functional GME is within 250 bp upstream of a tandem GRE driving a complex promoter. Furthermore, as the changes in GME activity did not correlate with those for fold induction from the GRE, the mechanisms for expression of GME and GRE activities appear to utilize parallel, as opposed to common pathways.

Animals↗
PubMed2004

Synergistic signaling by corticotropin-releasing hormone and leukemia inhibitory factor bridged by phosphorylated 3',5'-cyclic adenosine monophosphate response element binding protein at the Nur response element (NurRE)-signal transducers and activators of transcription (STAT) element of the proopiomelanocortin promoter.

Leukemia inhibitory factor (LIF) cooperates with CRH at the pituitary level to induce POMC gene transcription, resulting in activation of the pituitary-adrenal axis. However, the underlying molecular mechanisms remain elusive. Here, we show that the NurRE-signal transducers and activators of transcription (STAT) composite element of the POMC promoter was the predominant target of the LIF-CRH synergy. Whereas NurRE or STAT sites alone conferred synergy, the maximal response was found with the NurRE-STAT reporter, suggesting that direct DNA binding of both transcription factors is required for an optimal synergy. During LIF-CRH stimulation, Nur77 and activated STAT1-3 were bound to the composite element, and the binding of each factor was abolished by appropriate mutations. CREB was also detected in this complex in a stimulation-dependent and DNA binding-independent manner. Nur77 and STAT1-3 bound to the NurRE-STAT site were each sufficient for CREB recruitment. Recombinant CREB directly interacted with recombinant Nur77 or STAT1-3. Moreover, CREB-Nur77 interaction was increased by CREB phosphorylation at Ser-133 and the dominant-negative mutant CREB-M1 efficiently inhibited the synergistic LIF-CRH response. This synergism was also inhibited after transfection of CREB-small interfering RNA. We conclude that both CREB phosphorylation at Ser-133 and level of CREB expression are crucial in LIF-CRH synergism where CREB, without direct DNA binding, could improve the stability of Nur77 and STAT1-3 binding to POMC promoter and facilitate the recruitment of coactivators. This novel intrapituitary signaling mechanism may have more general implications in cross talks between cAMP-protein kinase A and Janus kinase-STAT pathways.

Animals↗
PubMed1996

A CD30 responsive element in the germline epsilon promoter that is distinct from and inhibitory to the CD40 response element.

Germline epsilon (I epsilon) transcription is requisite for IgE switch recombination. I epsilon transcription is markedly increased by ligation of CD40 and/or by IL-4 stimulation. By contrast, we found previously that stimulation through CD30 inhibits I epsilon transcription in EBV-transformed B cell lines. To characterize the molecular mechanisms involved in these contradictory events, the promoter elements that are responsible for I epsilon transcriptional regulation were determined using stable CAT reporter gene constructs. The results define a 95 bp CD30 responsive element (CD30RE) located 5' of the previously defined CD40 responsive element (CD40RE) that resides within the same 95 bp fragment as the IL-4RE and ablates CD40L induced I epsilon promoter activity. However, IL-4 overrides the inhibitory effect of CD30L. These results define a CD30RE and provide further evidence for the complex regulation of I epsilon transcription by various members of the CD40L/TNF alpha family of molecules.

Base Sequence↗
PubMed2005

Mechanism of binding of serum response factor to serum response element.

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124-245 fragment of serum response factor) with specific oligonucleotides from c-fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core-SRF labeled with bis-ANS showed that the protein is monomeric at low concentration (10(-7) m). The titration curves for the fluorescence anisotropy of fluorescein-labeled oligonucleotide revealed that under equilibrium conditions, the core-SRF monomers were bound sequentially to SRE at very low concentration (10(-9) m). Curve-fitting data showed also major differences between the wild-type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core-SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core-SRF was bound as a dimer to the wild-type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far-UV CD spectra indicated that the flexibility of core-SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein-labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA-protein interaction.

Amino Acid Sequence↗
PubMed1987

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.

Acetyltransferases↗
PubMed1991

Sugar response element enhances wound response of potato proteinase inhibitor II promoter in transgenic tobacco.

The promoter region of the potato proteinase inhibitor II (PI-II) gene was studied to identify cis-acting regulatory sequences involved in sugar response using transgenic tobacco plants. The 5' control region covering an 892 nucleotide sequence upstream from the cap site and a 32 nucleotide untranslated region of the PI-II promoter was able to activate a reporter chloramphenicol acetyltransferase (cat) gene by wounding or by incubating in a sugar-free medium. This wound response was further enhanced by sugar. Hexoses, disaccharides, and some trisaccharides were strong inducers whereas pentoses, deoxy sugars, sugar acids, TCA cycle intermediates, amino acids, and other carbohydrates had little effect on the promoter activity. Deletion of the sequence between -892 and -573 abolished the wound response but not the sugar response. An additional 5' deletion to -453 removed the sugar inducibility. Locations of the cis-acting regulatory elements were further elucidated by 3' deletion analysis. Deletion of the downstream region from -520 did not affect the wound or sugar response of the promoter. However, 3' deletion mutant -574 was unable to respond to sugar but did respond weakly to wounding. Further deletion to -624 abolished both responses. Therefore, it can be concluded that a wound response element is located in between -624 and -574 and that the response is further enhanced by a sugar response element located in the sequence between -573 and -520.

Agrobacterium tumefaciens↗