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- W2052204004 abstract "Article1 April 1997free access The distal GATA sequences of the sid1 promoter of Ustilago maydis mediate iron repression of siderophore production and interact directly with Urbs1, a GATA family transcription factor Zhiqiang An Zhiqiang An Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Baigen Mei Baigen Mei Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Walter M. Yuan Walter M. Yuan Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Sally A. Leong Corresponding Author Sally A. Leong Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA USDA-ARS Plant Disease Resistance Research Unit, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Zhiqiang An Zhiqiang An Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Baigen Mei Baigen Mei Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Walter M. Yuan Walter M. Yuan Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Sally A. Leong Corresponding Author Sally A. Leong Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA USDA-ARS Plant Disease Resistance Research Unit, 1630 Linden Drive, Madison, WI, 53706 USA Search for more papers by this author Author Information Zhiqiang An1,2, Baigen Mei1,3, Walter M. Yuan1 and Sally A. Leong 1,4 1Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI, 53706 USA 2Department of Biological Resources Development, ChemGenics Pharmaceuticals Inc., Building 300, One Kendall Square, Cambridge, MA, 02139 USA 3Promega Corp., 2800 Woods Hollow Road, Madison, WI, 53711 USA 4USDA-ARS Plant Disease Resistance Research Unit, 1630 Linden Drive, Madison, WI, 53706 USA The EMBO Journal (1997)16:1742-1750https://doi.org/10.1093/emboj/16.7.1742 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The sid1 and urbs1 genes encode L-ornithine N5-oxygenase and a GATA family transcription regulator, respectively, for siderophore biosynthesis in Ustilago maydis. The basic promoter and iron-regulatory sequences of the U.maydis sid1 gene were defined by fusing restriction and Bal31 nuclease-generated deletion fragments of the promoter region with the Escherichia coli β-glucuronidase (GUS) reporter gene. Sequences required for basal expression of sid1 mapped within 1043 bp upstream of the translation start site and include the first untranslated exon and first intron. Sequences needed for iron-regulated expression of sid1 were localized to a 306 bp region mapping 2.3 and 2.6 kb upstream of the ATG. The 306 bp region contains two G/TGATAA sequences, consensus DNA binding sites of GATA family transcription factors. Deletion or site-directed mutation of either or both GATA sequences resulted in deregulated expression of sid1. In vitro DNA binding studies showed that Urbs1 binds to the 3′-GATA site in the 306 bp iron-responsive region. However, deletion of 1.1 kb between the distal GATA sites and the basal promoter region led to deregulated expression of GUS, indicating that these GATA sequences are by themselves insufficient to regulate sid1. In vitro DNA binding and in vivo reporter gene analysis revealed that siderophores are not co-repressors of Urbs1. Introduction Iron is one of the most abundant elements in nature, but its availability to living organisms is very limited due to the extremely low solubility of ferric ion ([Fe3+] = 10−18 M at pH 7) in aerobic environments (Griffiths, 1987; Williams, 1990). Ferrous ion under physiological conditions tends to oxidize, hydrolyze and polymerize, forming insoluble ferric hydroxide and oxyhydroxide polymers (Williams, 1990). On the other hand, too much cellular iron is harmful because iron possesses unfilled d atomic orbitals and is able to undergo changes in oxidation state involving one electron. This easy access to two oxidation states allows iron to react with oxygen and produce hydroxyl radicals (Halliwell and Gutteridge, 1984; Imlay and Linn, 1988). Hydroxyl radicals react at an extremely high rate with most organic molecules found in cells, in particular, attacking and destroying cell membranes and DNA (Halliwell and Gutteridge, 1984). To acquire iron from the environment and, at the same time, avoid toxicity from excess cellular iron, organisms have developed various elaborate biological systems to coordinate iron transport and homeostasis (Griffiths, 1987). In plants, iron is transported in the sap in the form of complexes with low molecular weight chelators, such as citrate and malate. In mammals, iron is mobilized to different tissues via the iron binding protein transferrin. In egg white, iron is stored in the iron binding protein ovotransferrin. Most microorganisms produce and secrete a class of low molecular weight ferric iron chelating agents termed siderophores to gather environmental iron (Bagg and Neilands, 1987; Mei and Leong, 1994). In general, siderophore biosynthetic genes are negatively regulated by iron (Bagg and Neilands, 1987; Mei and Leong, 1994). One of the few microorganisms which do not produce siderophores is the yeast Saccharomyces cerevisiae (Klausner and Dancis, 1994). In S.cerevisiae, several factors, including two membrane-bound ferric reductases FRE1 (Dancis et al., 1992) and FRE2 (Georgatsou and Alexandraki, 1994), a ferric ion transporter, FTR1 (Stearman et al., 1996) and a membrane-associated multicopper oxidase/ferroxidase FET3 (Askwith et al., 1994; DeSilva et al., 1995), work together to mediate iron uptake under iron starvation conditions. Like siderophore biosynthetic genes, FRE1, FRE2, FTR1 and FET3 are negatively regulated by iron (Dancis et al., 1992; Askwith et al., 1994; DeSilva et al., 1995). This regulation is mediated by AFT-1 (Yamaguchi-Iwai et al., 1996), a transcription activator that binds to the promoters of these genes in the absence of iron. Ustilago maydis produces two cyclic hexapeptide siderophores, ferrichrome and ferrichrome A (Budde and Leong, 1989). The first step in ferrichrome and ferrichrome A biosynthesis is catalyzed by ornithine N5-oxygenase (Wang et al., 1989; Mei et al., 1993). The gene encoding ornithine N5-oxygenase (sid1) has been characterized (Mei et al., 1993). Siderophore biosynthesis in U.maydis is negatively regulated by the iron concentration in the growth medium. Urbs1, a transcription regulator of siderophore biosynthesis in U.maydis, contains two finger motifs characteristic of the GATA family of transcription factors (Voisard et al., 1993). Disruption of urbs1 leads to constitutive expression of sid1 (Mei et al., 1993), suggesting that Urbs1 may directly interact with the sid1 promoter to repress biosynthesis of siderophores in U.maydis when cellular iron is not limiting. In this communication we describe the structural analysis of the sid1 promoter and document the specific interaction of Urbs1 with sequences in this promoter. Two GATA motifs 1.6 kb upstream of the transcription initiation site in the sid1 promoter are required for iron-regulated expression of sid1. In vitro electrophoretic gel mobility shift assay indicated that Urbs1 interacts directly with one of the GATA sequences. Finally, a model involving DNA looping for the regulatory action of Urbs1 on sid1 expression is discussed. Results sid1 promoter analysis To delineate the functional domains in the sid1 promoter region, seven convenient restriction fragments in the sid1 promoter region (pCG2.8, pCGC, pCG2.0, pCG1.6, pCGPN, pCGS1 and pCGPS) were fused with the GUS reporter gene (Gallagher, 1992), which encodes the E.coli β-glucuronidase in the U.maydis self-replicating vector pCM54 (Tsukuda et al., 1988; Figure 1A). Both wild-type UM001 (strain 518) and a urbs1 disruption mutant UMC015 were transformed with these constructs and β–glucuronidase activities of the transformants were determined on plates. The results of these experiments are summarized in Figure 1A. A 0.7 kb fragment between 2.3 (pCG2.0) and 3 (pCG2.8) kb upstream of the translation start site was required for iron-regulated expression of sid1. The basic promoter activity was mapped within 1.27 kb (pCGS1) upstream of the translation start site. Sequences containing the 654 bp intron which is located between the transcription and translation start sites of sid1 were also required for basal expression of the gene (pCGPN and pCGPS). Deletion of the −2285 to −1267 region (pCGC), which is located downstream of the 700 bp iron-responsive sequence and upstream of the basic promoter of sid1, abolished iron-regulated expression of sid1. Fusions between GUS and promoter fragments in the wrong orientation were constructed as controls. None of these constructs gave GUS activity (Figure 1A). Figure 1.sid1 promoter analysis. (A) Restriction fragments of the 2.9 kb SspI–PvuII sid1 promoter were fused to the GUS reporter gene carried on the U.maydis replicating vector pCM54 and transformed into U.maydis wild-type (528) and an urbs1 disruption mutant (UMC015). GUS activity was determined colorimetrically as described in Materials and methods, on E medium containing no added iron or 10 μM FeSO4. + indicates that strong activity (colonies turned dark blue) and − indicates that no activity (colonies remained white) was seen within 6 h after application of X-gluc to colonies. This assay is only a qualitative measure of promoter activity. (B) A set of progressive deletions of the 3 kb sid1 promoter region created by Bal31 nuclease treatment were fused to the GUS reporter gene. Transformation and GUS activity assay were the same as in (A). (C) Sequence of the iron-responsive region. (D) GUS activities of gene replacement strains carrying the wild-type and deleted sid1 promoters fused to GUS. β–Glucuronidase activities were determined fluorometrically as described in Materials and methods, in whole cell extracts obtained from UMS113, UMS115 and UMS117 cells cultured on minimal liquid medium containing no added iron or 10 μM FeSO4. Assays were done in triplicate on five independent gene replacement strains for each construct. Activities were corrected by substracting the fluorescence observed using an extract of the untransformed strain 518, which gave similar levels of background fluorescence at 0 and 30 min to that observed at time 0 for extracts of the gene replacement strains. The filled boxes for p1011 (A) and UMS113 (D) indicate the location of GATA sequences required for iron-regulated expression of GUS, and the GATA boxes are underlined in the sequence showed in (C). Download figure Download PowerPoint To further define the promoter region of sid1, a set of progressive deletions of the 3 kb sid1 promoter region was created by Bal31 nuclease treatment (p1007–p1044; Figure 1B). The sequence for basal expression of the gene was further mapped within a 228 bp fragment between −1271 (pCGS1) and −1043 bp (p1028) upstream of the translation start site (Figure 1A and B). Sequences required for iron-regulated expression of sid1 were localized to a 306 bp region mapping 2.3 (pCG2.0) and 2.6 (p1011) kb upstream of the ATG (Figure 1A and B). This 306 bp fragment contains two G/TGATAA sequences separated by 23 bp (Figure 1C). To eliminate copy number and position effects, three Bal31 nuclease-generated promoter–GUS constructs (p1010, p1013 and p1028; Figure 1B) were introduced into the sid1 locus of U.maydis by gene replacement. GUS activity of strain UMS113, which has the reporter gene under the control of the 2.7 kb sid1 promoter region, was repressed 16-fold in cells grown on high iron medium (Figure 1D). When a fragment containing the two upstream G/TGATAA sequences was removed from the sid1 promoter in strain UMS115, GUS activity became constitutive and was no longer regulated by iron concentration in the medium (Figure 1D). In strain UMS117, the 228 bp fragment required for basal expression of sid1 was deleted. As expected, no GUS activity was detected in this strain (Figure 1D). Results from these experiments confirmed the observations made with fusion constructs in the self-replicating vector and GUS plate assay. Epitope tagging of urbs1 and detection of Urbs1 protein in U.maydis To confirm that urbs1 encodes an expressed protein in U.maydis, the influenza hemagglutinin antigen (YPYDVPDYA)3 was translationally fused as an epitope at the C-terminus of Urbs1. The epitope-tagged Urbs1 was constructed in the U.maydis self-replicating vector pCM54 to give pAN15. Biological activity of the epitope-tagged Urbs1 was tested by its ability to complement the urbs1 gene disruption mutant UMC015. On high iron medium, UMC015 gives an orange colony phenotype due to the constitutive production of siderophore by this strain. In comparison, UMCO15/pAN15 transformants, which carry the epitope-tagged Urbs1, displayed a complemented wild-type white colony phenotype on high iron medium (data not shown). This indicates that the epitope-tagged Urbs1 is biologically active in U.maydis. pAN15 replicates as a multicopy plasmid; therefore, epitope-tagged Urbs1 is expressed in multiple copies in UMC015/pAN15 transformants. To avoid copy number effects, UMurbs1tag, in which the wild-type urbs1 allele was replaced by the epitope-tagged allele, was constructed. Siderophore production by UMurbs1tag in both low and high iron medium was first determined qualitatively using the color plate assay. Wild-type strain 518, siderophore constitutive production mutant UMC015 and an urbs1 NTG mutants UMC002 were included as controls. The results showed that siderophore production in 518 and UMurbs1tag is negatively regulated by iron, as indicated by their white colony phenotype on high iron medium (data not shown). In contrast, the two urbs1 mutants, UMC015 and UMC002, displayed an orange colony phenotype on high iron medium (data not shown). Siderophore production by these strains was also determined quantitatively in both low and high iron media (Table I). These studies further indicate that epitope-tagged Urbs1 was biologically functional in U.maydis. A protein of ∼110 kDa, the predicted molecular weight of epitope-tagged Urbs1, was detected in U.maydis expressing epitope-tagged Urbs1 (UMC015/pAN15) by immunoprecipitation/Western blotting using a monoclonal antibody (anti-Ha) to influenza hemagglutinin antigen and protein A–Sepharose (Figure 2). No signal was detected in cells expressing wild-type Urbs1 (UMC015/pSC3; Figure 2). Urbs1tag was detected at similar levels in both low-iron and high-iron growth conditions (Q.Zhao and S.A.Leong, unpublished findings). Figure 2.Detection of Urbs1 in U.maydis. Immunoprecipitation of epitope-tagged Urbs1 and detection of Urbs1 in U.maydis by Western blotting as described in Materials and methods. UMC015 is an urbs1 disruption mutant. pSC3 carries the wild-type urbs1 gene while pAN15 carries the tagged urbs1 gene on the U.maydis replicating vector pCM54. Download figure Download PowerPoint Table 1. Siderophore productiona by wild-type and urbs1 mutants Low iron High iron Low iron/high iron ratio 518 0.46 ± 0.02 0.03 ± 0.00 15 UMC015 0.88 ± 0.04 0.26 ± 0.02 3 UMC002 0.71 ± 0.09 0.26 ± 0.01 3 UMurbs1tag 0.25 ± 0.01 0.04 ± 0.01 7 Cells were grown for 48 h at 28°C in low iron medium with or without 10 μM FeSO4. Culture supernatants were treated with an equal volume of ferric perchlorate solution and the absorbance was read at 495 nm (Budde and Leong, 1989). Analyses were done in triplicate. Data were normalized to equal cell growth using an adsorbance at OD600 of 1.0. Evidence for direct interaction of Urbs1 with the sid1 promoter by electrophoretic gel mobility shift analysis Since the 306 bp DNA fragment containing the iron-responsive sequence(s) in the sid1 promoter region has two GATA sites and urbs1 encodes a GATA family transcription factor with two putative zinc finger domains, Urbs1 may act as a transcription repressor of sid1 by binding to GATA motifs in the sid1 promoter. In vitro DNA binding studies were conducted with the 306 bp GATA-containing DNA fragment and whole cell extracts of UMC015, a urbs1 disruption mutant, and UMC015, carrying a replicating plasmid expressing the wild-type Urbs1 (pSC3) or epitope-tagged Urbs1 (pAN15). To induce Urbs1 to be in an active conformation for siderophore gene repression, whole cell extracts were isolated from cultures grown in high iron medium (10 μM FeSO4). As shown in Figure 3, the 306 bp fragment was shifted to give one major band by extracts obtained from strains expressing urbs1 and epitope-tagged urbs1, but not by extracts from the urbs1 null mutant. To determine whether the shifted bands were due to Urbs1, a monoclonal antibody (anti-Ha) to the influenza hemagglutinin epitope was included in the gel shift reactions. As shown in Figure 4, the addition of increasing amounts of antibody reduced the level of the shifted bands in the assays containing the tagged Urbs1 but not in assays containing wild-type Urbs1, indicating that Urbs1 is associated with the gel shift complex. No supershifted band was observed under the conditions of electrophoresis employed. Figure 3.DNA binding assays of the 0.3 kb DNA fragment which contains the two distal GATA sites in the iron-responsive sequence of the sid1 promoter region with whole cell protein extracts of UMC015, UMCO15/pSC3 and UMC015/pAN15. UMC015 is an urbs1 disruption mutant. pAN15 carries the urbs1 tagged gene on the U.maydis replicating vector pCM54 and pSC3 carries wild-type urbs1 on the same vector. Electrophoretic gel mobility shift assays were done as described in Materials and methods. Download figure Download PowerPoint Figure 4.DNA binding assays of UMC015/pSC3 and UMC015/pAN15 whole cell protein extracts and the 0.3 kb sid1 promoter probe with increasing amounts of monoclonal antibody (anti-Ha) to the influenza hemagglutinin antigen as indicated. Strains and conditions were as described in Figure 3 and in Materials and methods. Download figure Download PowerPoint To determine whether the GATA boxes in the 306 bp fragment are the in vitro DNA binding sites for Urbs1, a 63 bp oligonucleotide (oligonucleotide 1 in Figure 5A) encompassing the two distal GATA sequences was synthesized and used for DNA–protein binding assays with whole cell extracts of UMC015 and UMC015/pSC3. The results showed that the 63 bp oligonucleotide was shifted by UMC015/pSC3 extract, which expresses wild-type urbs1, but not by extract from the urbs1 null mutant UMC015 (Figure 5B). The specificity of DNA binding was examined by including unlabeled oligonucleotides in the DNA–protein binding reactions. The binding signal between the labeled probe and Urbs1 was reduced by the addition of excess unlabeled probe in the reaction (Figure 5B). Figure 5.Electrophoretic gel mobility shift analysis of Urbs1 and target DNA. (A) Oligonucleotides used in gel shift analysis. Sequences of the 63 bp oligonucleotides containing the wild-type or mutated distal GATA sites. Numbers indicate the distance of the G residues from the translation start of sid1: 1, wild-type; 2, 5′-GATA mutant; 3, 3′-GATA mutant; 4, double GATA mutant. (B) Specificity of DNA binding of the wild-type 63mer [oligonucleotide 1 in (A)] with whole cell protein extract of UMCO15/pSC3. Different amounts of unlabeled oligomer (0, 10, 20 and 30 ng) were added as indicated. Strains and conditions as described in Figure 3 and in Materials and methods. (C) Gel shift assays of UMC015/pSC3 whole cell protein extracts with the 63 bp oligonucleotide and its mutant variants shown in (A). Lane 1, wild-type; lane 2, 5′-GATA mutant; lane 3, 3′-GATA mutant; lane 4, double GATA mutant. Strains and conditions were as described in Figure 3 and in Materials and methods. Download figure Download PowerPoint The role of the two GATA motifs in the 63 bp oligonucleotide probe was assessed by mutation of one or both sites in the probe (oligonucleotides 2, 3 and 4 in Figure 5A). Mutation of the 5′-GATA site had no apparent effect on the in vitro binding of Urbs1 to the probe, while mutation of the 3′-GATA site or mutation of both GATA sites eliminated binding (Figure 5C). To test the in vivo roles of the two GATA sites, three sid1 promoter–GUS constructs which carry mutations at the 5′-GATA (pAY2), 3′-GATA (pAY3) or both (pAY4) sites were introduced into the sid1 locus of U.maydis by gene replacement to give UMS202 (5′-GATA to CTGA), UMS203 (3′-GATA to CTGA) and UMS204 (double mutation). GUS activities expressed by these three strains were determined in both low and high iron growth conditions. UMS113, which has the reporter gene under the control of the wild-type sid1 promoter region, was included as a control. The results are shown in Table II. When the 5′-, 3′- or both GATA sites were mutated to CTGA in the sid1 promoter region in strains UMS202, UMS203 and UMS204, GUS activity was no longer tightly regulated by iron concentration in the medium, as indicated by the low −Fe:+Fe ratio (Table II). Mutation of a single GATA site led to partial deregulation of GUS activity, while mutation of both sites led to complete deregulation of GUS activity. In contrast, the GUS activity in strain UMS113, which has the reporter under the control of the wild-type sid1 promoter, was negatively regulated by iron concentration in the medium (Table II). Table 2. β-Glucuronidase activity produced from wild-type and mutant sid1 promoters fused to the GUS genea Low iron High iron Low iron/high iron ratio UMS113 15.7 ± 1.8 1.0 ± 0.04 16 UMS202 13.7 ± 3.3 4.9 ± 0.9 3 UMS203 17.9 ± 1.7 10.4 ± 1.6 2 UMS204 19.8 ± 3.0 18.8 ± 1.7 1 Wild-type sid1 promoter and sid1 promoters containing distal GATA mutations fused with GUS were introduced into the sid1 locus by gene replacement. The genotype of each strain is described in Table III. Assays were done in triplicate on three independent transformants and the experiment was repeated three times. Assay conditions for GUS activity are described in Figure 1 and Materials and methods. Discussion This study represents the first comprehensive analysis of a U.maydis gene promoter. The complexity of the sid1 promoter provides a unique opportunity to understand better how U.maydis promoters and transcription factors function. By using GUS as a reporter, three regions in the 3 kb promoter have been defined to be important for expression of sid1. The first region is a 306 bp fragment encompassing two GATA sequences and mapping 1.6 kb from the start of transcription. Deletion of the GATA sequences resulted in deregulated expression of sid1 by iron. This result indicated that these GATA sequences might be iron-responsive elements and the DNA binding sites for Urbs1. Since these two GATA motifs are distantly located from the transcription start site, we refer to them as the ‘distal GATA sites’. A second region of 228 bp located 211 bp upstream of the start of transcription is required for basal level expression of the gene. A CAAT box and pyrimidine-rich sequence are both located in this region (Mei et al., 1993). A third region encompassing the first exon and intron of sid1 is also required for basal level expression of the gene. This finding is not surprising, since introns contribute to basic promoter activity as well as transcription regulation in other genes (Crestani et al., 1993; Brown and Taylor, 1994; Corrochano et al., 1995). However, a definitive role for the intron in expression of sid1 will require further mutational studies. In vitro electrophoretic gel mobility shift analysis using synthetic oligonucleotides containing the wild-type and mutated distal GATA sites indicate that Urbs1 binds specifically to the distal 3′-GATA sequence located 1.6 kb upstream of the transcription start site of the sid1 promoter region. Site-directed mutagenesis indicated that not only the distal 3′-GATA site but also the distal 5′-GATA site in the iron-responsive region is required for iron-mediated transcription repression of sid1 in vivo. This is not surprising, as high affinity binding sites in promoters are not always correlated with the biological function of transcription factors (A.Laughon, personal communication). However, the possibility that mutation of the 5′-GATA site disrupts binding of a second factor required for iron regulation of sid1 cannot be discounted. DNase protection studies will shed further light on the extent of the region bound by Urbs1. The distal arrangement of the iron-responsive elements in sid1 suggests that Urbs1 may effect a novel mechanism of transcription repression in sid1. In addition to the two distal GATA sequences, there are additional GATA sequences in the sid1 promoter region. These include the two proximal GATA sites (Figure 6) and GATA sites in the first intron (5′-CTATCG–182 bp–CGATATCT-3′). Some of these GATA sites may also play a role in Urbs1-mediated regulation of sid1. Even though the two distal GATA sites are required for iron-regulated expression of sid1, the presence of the distal GATA sites is not sufficient to mediate repression of sid1, as deletion of the 1018 bp HindIII–SacI fragment in the promoter region resulted in deregulated expression of sid1 (Figures 1A and 6C). This suggests that spacing of the distal GATA sites relative to some other site(s) in the promoter is critical and/or that deletion of the HindIII–SacI fragment eliminated another sequence(s) that is essential for iron-mediated regulation of sid1. It is interesting to note that this region contains the palindromic GATA sequence TTTTATCAGAT, which matches 11 bp of the distal 5′-GATA site perfectly and might serve as a binding site for Urbs1. However, this site is isolated from other full GATA sites. The GATA family transcription factor Nit2 shows a clear preference for binding sites containing two closely apposed GATA elements (Xiao and Marzluf, 1996). Moreover, GATA-1 interactions with palindromic GATA sites are considerably stronger than with those having single GATA elements (Trainor et al., 1996); yet strong binding of Urbs1 to the 5′ distal GATA site was not observed. As a result, we believe that this GATA site may not play an essential role in regulation of sid1. Mutation of this site and analysis of its ability to interact specifically with Urbs1 are underway to confirm this interpretation. Figure 6.A proposed transcription ‘looping’ model for the regulatory action of Urbs1 on sid1 expression. (A) In the presence of iron, Urbs1 interacts with the two distal GATA sites and one or more of the downstream GATA sites (the two proximal GATA sites were used in the Figure) to form a loop in the sid1 promoter region. Deletion of the distal sites (B) or alteration of the spacing of the binding sites by deletion of the −2285 to −1267 region (C) prevents loop formation, leading to constitutive expression of sid1. The two open boxes located −2472 and −2443 upstream of the ATG are the two distal GATA sites (D). The two open boxes located −875 and −850 upstream of the ATG are the two proximal GATA sites (D). The filled boxes indicate a putative transcription factor binding site (D). Download figure Download PowerPoint Based on the results available, we propose a model predicting that Urbs1 mediates the formation of a DNA loop in the 1.6 kb promoter region of sid1 which involves the distal GATA and the proximal GATA sites or the two GATA sites in the intron (Figure 6A and D). DNA looping has been reported in the regulation of a large number of prokaryotic and eukaryotic genes (Bellomy and Record, 1990; Schleif, 1992). One of the best studied examples is the AraC-regulated BAD operon in E.coli (Dunn et al., 1984; Martin et al., 1986; Huo et al., 1988). In eukaryotic systems, DNA looping has been shown to play a role in silencing of the mating type genes in the yeast Saccharomyces cerevisiae (Hofmann et al., 1989) and enhancer-mediated control of the prolactin gene in the rat (Cullen et al., 1993). Sequences required for basal expression of sid1 are located between the upstream and downstream GATA sites (Figure 6). Formation of a loop between the upstream and downstream GATA sites could impede the activity of basic transcription factors either through steric hindrance or direct interaction of transcription factors with Urbs1 (Herschbach et al., 1994) or by forming a topologically isolated sequence that can no longer bind transcription factors. Mutational studies of the proximal and intronic GATA sites are in progress to assess their role in regulation of sid1. However, loop formation might not involve the interaction of Urbs1 bound to both downstream and distal GATA sites, but rather the interaction of Urbs1 bound to distal GATA sites with another factor bound to the downstream promoter region. In this regard, it is interesting to note that GATA-1 can interact physically with the transcription factors Sp1 and EKLF and that Sp1 is able in vivo to recruit GATA-1 to a promoter in the absence of GATA binding sites (Merika and Orkin, 1995). A compreh" @default.
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- W2052204004 title "The distal GATA sequences of the sid1 promoter of Ustilago maydis mediate iron repression of siderophore production and interact directly with Urbs1, a GATA family transcription factor" @default.
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